Journal - BMC Structural Biology

Abstract :

BackgroundSeveral Retinoic Acid Receptors (RAR) agonists have therapeutic activity against a variety of cancer types; however, unacceptable toxicity profiles have hindered the development of drugs. RAR agonists presenting novel structural and chemical features could therefore open new avenues for the discovery of leads against breast, lung and prostate cancer or leukemia.ResultsWe have analysed the induced fit of the active site residues upon binding of a known ligand. The derived binding site models were used to dock over 150,000 molecules in silico (or virtually) to the structure of the receptor with the Internal Coordinates Mechanics (ICM) program. Thirty ligand candidates were tested in vitro.ConclusionsTwo novel agonists resulting from the predicted receptor model were active at 50 nM. One of them displays novel structural features which may translate into the development of new ligands for cancer therapy.

Journal - Journal of Biological Chemistry

Abstract :

NRC/NCoA6 plays an important role in mediating the effects ofligand-bound nuclear hormone receptors as well as other transcriptionfactors. NRC interacting factor 1 (NIF-1) was cloned as a novelfactor that interacts in vivo with NRC. Although NIF-1 doesnot directly interact with nuclear hormone receptors, it enhancesactivation by nuclear hormone receptors presumably through itsinteraction with NRC. To further understand the cellular andbiological function of NIF-1, we identified NIF-1-associatedproteins by in-solution proteolysis followed by mass spectrometry.The identified components revealed factors involved in histonemethylation and cell cycle control and include Ash2L, RbBP5,WDR5, HCF-1, DBC-1, and EMSY. Although the NIF-1 complex containsAsh2L, RbBP5, and WDR5, suggesting that the complex might methylatehistone H3-Lys-4, we found that the complex contains a H3 methyltransferaseactivity that modifies a residue other than H3-Lys-4. The identifiedcomponents form at least two distinctly sized NIF-1 complexes.DBC-1 and EMSY were identified as integral components of anNIF-1 complex of 1.5 MDa and were found to play an importantrole in the regulation of nuclear receptor-mediated transcription.Stimulation of the Sox9 and HoxA1 genes by retinoic acid receptor-was found to require both DBC-1 and EMSY in addition to NIF-1for maximal transcriptional activation. Interestingly, NRC wasnot identified as a component of the NIF-1 complex, suggestingthat NIF-1 and NRC do not exist as stable in vitro purifiedcomplexes, although the separate NIF-1 and NRC complexes appearto functionally interact in the cell.* This work was supported, in whole or in part, by National Institutesof Health Grant DK16636 (to H. H. S.). This work was also supportedby an NIH Shared Instrumentation Grant 1S10 RR017990-01 andNCI Cancer Institute Core Grant P30CA016087-239025 (to T. A.N.). The costs of publication of this article were defrayedin part by the payment of page charges. This article must thereforebe hereby marked "advertisement" in accordance with 18 U.S.C.Section 1734 solely to indicate this fact.1 Present address: Constellation Pharmaceuticals, 148 Sidney St.,Cambridge, MA 02139.

Abstract :

DESCRIPTION (provided by applicant): Nuclear receptors (NRs) modulate transcription through interaction with co-activators and co-repressors. A wide body of research has been carried out with the DRIP/TRAPs and the p160/SRC family of co-activators, and with SMRT/N-CoR co-repressors. During the last granting period, our laboratory cloned and characterized four novel factors (NRC, NIF-1, NRIF3, and PSF-A) that appear to play an important role in mediating the activity of NRs and other transcription factors. NRC (Nuclear Receptor Co-regulation) is a novel 2063 amino acid nuclear protein with two LxxLL motifs. LxxLL-1 interacts with all NRs while LxxLL-2 interacts with ERbeta and LXRbeta. The binding of liganded-receptor with LxxLL-1 results in a conformational change in NRC leading to transcriptional activation. NRC interacts with CBP in vivo and also activates other transcription factors such as cFos and cJun. NIF-1 (NRC Interacting Factor-I) is a novel 1,342 amino acid nuclear protein that interacts in vivo and in vitro with NRC and enhances ligand-dependent transcriptional activation by NRs and cFos and cJun through association with NRC. NRIF3 (Nuclear Receptor Interacting Factor 3) is a 177 amino acid nuclear protein that only interacts with TRs and RXRs and contains both activation and repression domains. Repression is dependent on phosphorylation of Ser28. Thus, NRIF3 could act as an activator or repressor depending on the state of phosphorylation. In a number of breast cancer cells, NRIF3 is not an activator and its expression rapidly leads to caspase-2 mediated apoptosis. PSF-A is a 707 amino acid nuclear protein that associates with the DBD of many NRs and acts as a repressor through its association with Sin3 and HDAC1 and HDAC2. The focus of this proposal is to identify novel factors, through yeast two-hybrid screens and mass spectroscopy, that complex with and modulate the activity of NRC, NIF-1, and NRIF3. We also plan to further clarify the role of PSF on modulating gene expression by a variety of NRs. We developed NRIF3 -/- mice to study the physiological role of NRIF3. Although NRC +/- mice exhibit embryonic lethality, and NRC +/- mice are normal, we developed a strain of NRC +/- mice that exhibit interesting phenotypes related to NRC haploid deficiency. Identification of factors that associate with these regulators and study of these "knockout mice", should provide a comprehensive picture of how these factors modulate receptor function and other important biological processes in cells.

Abstract :

DESCRIPTION (provided by applicant): This application represents a new multi-disciplinary predoctoral training program in the Pharmacological Sciences at the New York University (NYU) School of Medicine. The Program is broad based and provides training in the area of molecular pharmacology, signaling, pharmacology of the nervous system, protein structure of receptors, and elements of environmental medicine/toxicology. To achieve this aim, the Program has included 25 faculty members from both basic science and clinical departments at the medical school, whose research interests lay in the field of Pharmacology. The training faculty members have many overlapping research interests, which ensure that predoctoral trainees are part of a strong intellectual environment larger than the individual laboratories. The program faculty are located in the Departments of Pharmacology, Cell Biology, Environmental Medicine/Toxicology, Psychiatry, Medicine, Microbiology, and, Physiology/Neuroscience. The laboratories draw students who directly apply to the Pharmacological Sciences Training Program and from an open graduate program operated by the Sackler Institute of Graduate Biomedical Sciences at the NYU School of Medicine. The entire faculty has attracted highly qualified students and postdoctoral fellows. The program faculty members all belong to the Sackler Institute. Trainees will participate in a number of core and advanced courses, weekly seminars, work-in-progress sessions, journal clubs and tutorials intended to ensure broad exposure to molecular pharmacology and the pharmacological sciences. Collaborations between trainees and participating faculty will be encouraged starting with initial laboratory rotations and continuing with mentoring of dissertation studies by individual faculty members. The goal of this program is to provide a strong, broad based training of predoctoral students who will develop into productive, competitive and creative scientists capable of making important contributions to the field of Pharmacology and its application to human disease.

Abstract :

DESCRIPTION (provided by applicant): This application represents a new multi-disciplinary predoctoral training program in the Pharmacological Sciences at the New York University (NYU) School of Medicine. The Program is broad based and provides training in the area of molecular pharmacology, signaling, pharmacology of the nervous system, protein structure of receptors, and elements of environmental medicine/toxicology. To achieve this aim, the Program has included 25 faculty members from both basic science and clinical departments at the medical school, whose research interests lay in the field of Pharmacology. The training faculty members have many overlapping research interests, which ensure that predoctoral trainees are part of a strong intellectual environment larger than the individual laboratories. The program faculty are located in the Departments of Pharmacology, Cell Biology, Environmental Medicine/Toxicology, Psychiatry, Medicine, Microbiology, and, Physiology/Neuroscience. The laboratories draw students who directly apply to the Pharmacological Sciences Training Program and from an open graduate program operated by the Sackler Institute of Graduate Biomedical Sciences at the NYU School of Medicine. The entire faculty has attracted highly qualified students and postdoctoral fellows. The program faculty members all belong to the Sackler Institute. Trainees will participate in a number of core and advanced courses, weekly seminars, work-in-progress sessions, journal clubs and tutorials intended to ensure broad exposure to molecular pharmacology and the pharmacological sciences. Collaborations between trainees and participating faculty will be encouraged starting with initial laboratory rotations and continuing with mentoring of dissertation studies by individual faculty members. The goal of this program is to provide a strong, broad based training of predoctoral students who will develop into productive, competitive and creative scientists capable of making important contributions to the field of Pharmacology and its application to human disease.

Abstract :

DESCRIPTION (provided by applicant): This application represents a new multi-disciplinary predoctoral training program in the Pharmacological Sciences at the New York University (NYU) School of Medicine. The Program is broad based and provides training in the area of molecular pharmacology, signaling, pharmacology of the nervous system, protein structure of receptors, and elements of environmental medicine/toxicology. To achieve this aim, the Program has included 25 faculty members from both basic science and clinical departments at the medical school, whose research interests lay in the field of Pharmacology. The training faculty members have many overlapping research interests, which ensure that predoctoral trainees are part of a strong intellectual environment larger than the individual laboratories. The program faculty are located in the Departments of Pharmacology, Cell Biology, Environmental Medicine/Toxicology, Psychiatry, Medicine, Microbiology, and, Physiology/Neuroscience. The laboratories draw students who directly apply to the Pharmacological Sciences Training Program and from an open graduate program operated by the Sackler Institute of Graduate Biomedical Sciences at the NYU School of Medicine. The entire faculty has attracted highly qualified students and postdoctoral fellows. The program faculty members all belong to the Sackler Institute. Trainees will participate in a number of core and advanced courses, weekly seminars, work-in-progress sessions, journal clubs and tutorials intended to ensure broad exposure to molecular pharmacology and the pharmacological sciences. Collaborations between trainees and participating faculty will be encouraged starting with initial laboratory rotations and continuing with mentoring of dissertation studies by individual faculty members. The goal of this program is to provide a strong, broad based training of predoctoral students who will develop into productive, competitive and creative scientists capable of making important contributions to the field of Pharmacology and its application to human disease.

Abstract :

DESCRIPTION (provided by applicant): This application represents a new multi-disciplinary predoctoral training program in the Pharmacological Sciences at the New York University (NYU) School of Medicine. The Program is broad based and provides training in the area of molecular pharmacology, signaling, pharmacology of the nervous system, protein structure of receptors, and elements of environmental medicine/toxicology. To achieve this aim, the Program has included 25 faculty members from both basic science and clinical departments at the medical school, whose research interests lay in the field of Pharmacology. The training faculty members have many overlapping research interests, which ensure that predoctoral trainees are part of a strong intellectual environment larger than the individual laboratories. The program faculty are located in the Departments of Pharmacology, Cell Biology, Environmental Medicine/Toxicology, Psychiatry, Medicine, Microbiology, and, Physiology/Neuroscience. The laboratories draw students who directly apply to the Pharmacological Sciences Training Program and from an open graduate program operated by the Sackler Institute of Graduate Biomedical Sciences at the NYU School of Medicine. The entire faculty has attracted highly qualified students and postdoctoral fellows. The program faculty members all belong to the Sackler Institute. Trainees will participate in a number of core and advanced courses, weekly seminars, work-in-progress sessions, journal clubs and tutorials intended to ensure broad exposure to molecular pharmacology and the pharmacological sciences. Collaborations between trainees and participating faculty will be encouraged starting with initial laboratory rotations and continuing with mentoring of dissertation studies by individual faculty members. The goal of this program is to provide a strong, broad based training of predoctoral students who will develop into productive, competitive and creative scientists capable of making important contributions to the field of Pharmacology and its application to human disease.

Abstract :

DESCRIPTION (provided by applicant): This application represents a new multi-disciplinary predoctoral training program in the Pharmacological Sciences at the New York University (NYU) School of Medicine. The Program is broad based and provides training in the area of molecular pharmacology, signaling, pharmacology of the nervous system, protein structure of receptors, and elements of environmental medicine/toxicology. To achieve this aim, the Program has included 25 faculty members from both basic science and clinical departments at the medical school, whose research interests lay in the field of Pharmacology. The training faculty members have many overlapping research interests, which ensure that predoctoral trainees are part of a strong intellectual environment larger than the individual laboratories. The program faculty are located in the Departments of Pharmacology, Cell Biology, Environmental Medicine/Toxicology, Psychiatry, Medicine, Microbiology, and, Physiology/Neuroscience. The laboratories draw students who directly apply to the Pharmacological Sciences Training Program and from an open graduate program operated by the Sackler Institute of Graduate Biomedical Sciences at the NYU School of Medicine. The entire faculty has attracted highly qualified students and postdoctoral fellows. The program faculty members all belong to the Sackler Institute. Trainees will participate in a number of core and advanced courses, weekly seminars, work-in-progress sessions, journal clubs and tutorials intended to ensure broad exposure to molecular pharmacology and the pharmacological sciences. Collaborations between trainees and participating faculty will be encouraged starting with initial laboratory rotations and continuing with mentoring of dissertation studies by individual faculty members. The goal of this program is to provide a strong, broad based training of predoctoral students who will develop into productive, competitive and creative scientists capable of making important contributions to the field of Pharmacology and its application to human disease.

Abstract :

DESCRIPTION (provided by applicant): Nuclear receptors (NRs) modulate transcription through interaction with co-activators and co-repressors. A wide body of research has been carried out with the DRIP/TRAPs and the p160/SRC family of co-activators, and with SMRT/N-CoR co-repressors. During the last granting period, our laboratory cloned and characterized four novel factors (NRC, NIF-1, NRIF3, and PSF-A) that appear to play an important role in mediating the activity of NRs and other transcription factors. NRC (Nuclear Receptor Co-regulation) is a novel 2063 amino acid nuclear protein with two LxxLL motifs. LxxLL-1 interacts with all NRs while LxxLL-2 interacts with ERbeta and LXRbeta. The binding of liganded-receptor with LxxLL-1 results in a conformational change in NRC leading to transcriptional activation. NRC interacts with CBP in vivo and also activates other transcription factors such as cFos and cJun. NIF-1 (NRC Interacting Factor-I) is a novel 1,342 amino acid nuclear protein that interacts in vivo and in vitro with NRC and enhances ligand-dependent transcriptional activation by NRs and cFos and cJun through association with NRC. NRIF3 (Nuclear Receptor Interacting Factor 3) is a 177 amino acid nuclear protein that only interacts with TRs and RXRs and contains both activation and repression domains. Repression is dependent on phosphorylation of Ser28. Thus, NRIF3 could act as an activator or repressor depending on the state of phosphorylation. In a number of breast cancer cells, NRIF3 is not an activator and its expression rapidly leads to caspase-2 mediated apoptosis. PSF-A is a 707 amino acid nuclear protein that associates with the DBD of many NRs and acts as a repressor through its association with Sin3 and HDAC1 and HDAC2. The focus of this proposal is to identify novel factors, through yeast two-hybrid screens and mass spectroscopy, that complex with and modulate the activity of NRC, NIF-1, and NRIF3. We also plan to further clarify the role of PSF on modulating gene expression by a variety of NRs. We developed NRIF3 -/- mice to study the physiological role of NRIF3. Although NRC +/- mice exhibit embryonic lethality, and NRC +/- mice are normal, we developed a strain of NRC +/- mice that exhibit interesting phenotypes related to NRC haploid deficiency. Identification of factors that associate with these regulators and study of these "knockout mice", should provide a comprehensive picture of how these factors modulate receptor function and other important biological processes in cells.

Abstract :

DESCRIPTION (provided by applicant): Nuclear receptors (NRs) modulate transcription through interaction with co-activators and co-repressors. A wide body of research has been carried out with the DRIP/TRAPs and the p160/SRC family of co-activators, and with SMRT/N-CoR co-repressors. During the last granting period, our laboratory cloned and characterized four novel factors (NRC, NIF-1, NRIF3, and PSF-A) that appear to play an important role in mediating the activity of NRs and other transcription factors. NRC (Nuclear Receptor Co-regulation) is a novel 2063 amino acid nuclear protein with two LxxLL motifs. LxxLL-1 interacts with all NRs while LxxLL-2 interacts with ERbeta and LXRbeta. The binding of liganded-receptor with LxxLL-1 results in a conformational change in NRC leading to transcriptional activation. NRC interacts with CBP in vivo and also activates other transcription factors such as cFos and cJun. NIF-1 (NRC Interacting Factor-I) is a novel 1,342 amino acid nuclear protein that interacts in vivo and in vitro with NRC and enhances ligand-dependent transcriptional activation by NRs and cFos and cJun through association with NRC. NRIF3 (Nuclear Receptor Interacting Factor 3) is a 177 amino acid nuclear protein that only interacts with TRs and RXRs and contains both activation and repression domains. Repression is dependent on phosphorylation of Ser28. Thus, NRIF3 could act as an activator or repressor depending on the state of phosphorylation. In a number of breast cancer cells, NRIF3 is not an activator and its expression rapidly leads to caspase-2 mediated apoptosis. PSF-A is a 707 amino acid nuclear protein that associates with the DBD of many NRs and acts as a repressor through its association with Sin3 and HDAC1 and HDAC2. The focus of this proposal is to identify novel factors, through yeast two-hybrid screens and mass spectroscopy, that complex with and modulate the activity of NRC, NIF-1, and NRIF3. We also plan to further clarify the role of PSF on modulating gene expression by a variety of NRs. We developed NRIF3 -/- mice to study the physiological role of NRIF3. Although NRC +/- mice exhibit embryonic lethality, and NRC +/- mice are normal, we developed a strain of NRC +/- mice that exhibit interesting phenotypes related to NRC haploid deficiency. Identification of factors that associate with these regulators and study of these "knockout mice", should provide a comprehensive picture of how these factors modulate receptor function and other important biological processes in cells.

Abstract :

DESCRIPTION (provided by applicant): Nuclear receptors (NRs) modulate transcription through interaction with co-activators and co-repressors. A wide body of research has been carried out with the DRIP/TRAPs and the p160/SRC family of co-activators, and with SMRT/N-CoR co-repressors. During the last granting period, our laboratory cloned and characterized four novel factors (NRC, NIF-1, NRIF3, and PSF-A) that appear to play an important role in mediating the activity of NRs and other transcription factors. NRC (Nuclear Receptor Co-regulation) is a novel 2063 amino acid nuclear protein with two LxxLL motifs. LxxLL-1 interacts with all NRs while LxxLL-2 interacts with ERbeta and LXRbeta. The binding of liganded-receptor with LxxLL-1 results in a conformational change in NRC leading to transcriptional activation. NRC interacts with CBP in vivo and also activates other transcription factors such as cFos and cJun. NIF-1 (NRC Interacting Factor-I) is a novel 1,342 amino acid nuclear protein that interacts in vivo and in vitro with NRC and enhances ligand-dependent transcriptional activation by NRs and cFos and cJun through association with NRC. NRIF3 (Nuclear Receptor Interacting Factor 3) is a 177 amino acid nuclear protein that only interacts with TRs and RXRs and contains both activation and repression domains. Repression is dependent on phosphorylation of Ser28. Thus, NRIF3 could act as an activator or repressor depending on the state of phosphorylation. In a number of breast cancer cells, NRIF3 is not an activator and its expression rapidly leads to caspase-2 mediated apoptosis. PSF-A is a 707 amino acid nuclear protein that associates with the DBD of many NRs and acts as a repressor through its association with Sin3 and HDAC1 and HDAC2. The focus of this proposal is to identify novel factors, through yeast two-hybrid screens and mass spectroscopy, that complex with and modulate the activity of NRC, NIF-1, and NRIF3. We also plan to further clarify the role of PSF on modulating gene expression by a variety of NRs. We developed NRIF3 -/- mice to study the physiological role of NRIF3. Although NRC +/- mice exhibit embryonic lethality, and NRC +/- mice are normal, we developed a strain of NRC +/- mice that exhibit interesting phenotypes related to NRC haploid deficiency. Identification of factors that associate with these regulators and study of these "knockout mice", should provide a comprehensive picture of how these factors modulate receptor function and other important biological processes in cells.

Abstract :

DESCRIPTION (provided by applicant): This request is for the purchase of an Amersham Biosciences Typhoon 9410. The Typhoon will serve as a core instrument for the Departments of Cell Biology, Microbiology, and Pharmacology with a user group of 29 investigators, nearly all with NIH funding, with a wide variety of research interests. In addition, any other investigator within the NYU School of Medicine who requires the use of the special capabilities of the Typhoon will be allowed to join the user group. No similar instrument currently exists in the entire School of Medicine. The Typhoon 9410 combines a phosphorimager, a fluorescence image scanner, and a luminescence reader in one machine. No company other than Amersham markets an instrument with the same capabilities. Moreover, the Typhoon will replace an obsolete Molecular Dynamics 425 phosphorimager currently in use. Since most of the laboratories in the user group already own Molecular Dynamics cassettes, which can only be read by the Amersham instrument, the purchase of the Typhoon is appropriate and cost effective. The Typhoon will also supplant an aging and limited Molecular Dynamics Fluorimeter 575 that is currently housed in a separate building. The Typhoon fluorimeter is much more sensitive than its predecessor, offers better resolution, and can detect a wider variety of fluorescent substrates. The detection capabilities of the Typhoon will not only provide improved fluorimetric measurements but also will enable quantitative imaging of Western and nucleic acid hybridization blots labeled with fluorescent probes, replace X-ray film for detection of blots using enhanced chemiluminescence, and allow visualization and quantitation of proteins stained with fluorescent dyes on one- and two-dimensional polyacrylamide gels. Funds are also requested for a software license to set up workstations for data analysis in the individual departments separate from the facility housing the instrument. A small committee of the user group will administer the instrument and design rules for its use. A trained technician will maintain and monitor the machine on a daily basis and educate new users. It is anticipated that the availability of the Typhoon will have great impact on the research of the user group and the institution as a whole.

Abstract :

Thyroid hormone receptors (c-erbAs) and retinoic acid receptors (RARs) are members of a subgroup of closely related nuclear receptor proteins. c-erbAs and RAR can each activate certain response elements and both receptors contain a highly conserved domain embedded within the ligand binding region containing a series of "leucine-zipper-like" hydrophobic heptad motifs. Functional studies suggest that the heptad repeat domain mediates homo- and heterodimeric interactions of c-erbA and RAR or interactions with other factors. Chick c-erbA-alpha and human RAR-alpha have been expressed in E. coli and purified to near homogeneity. These receptors bind ligand with appropriate affinity and form homo- and hetero-dimers on response elements which are permissive for dimerization. Dimer formation is enhanced by ligand suggesting that ligand mediates transcriptional activation by this mechanism. This application is a comprehensive proposal to define the functional domains involved in c-erbA, RAR, and related factors in transcriptional activation. For these studies we constructed a multifunctional bacterial/eucaryotic expression vector (pEXPRESS) which permits site directed mutagenesis and can be used to functionally analyze receptor in eucaryotic cells and to express receptor at high levels in E. coli. Gel shift studies using purified wild-type and mutant receptor proteins and a variety of native and synthetic response elements are proposed to elucidate the "rules" which govern how these receptors recognize functional response elements. These studies will also define receptor domains critical for homo- and hetero-dimer formation, for cooperative interactions between receptors and other factors, and for dominant negative activities. Possible differences in element recognition by c-erbA subtypes (alphal and betal) will also be examined along with studies to understand differences between v-erbA and c-erbA and how c-erbA-alpha2 functions as a dominant negative regulator. These studies will be complemented by analyzing receptor mutants derived from patients with the thyroid hormone resistance syndrome. Functional studies in mammalian cells will be extended to in vitro transcription with the goal of defining the protein and DNA requirements for transcriptional enhancement by these receptors. Finally, the availability of mg amounts of purified E. coli expressed wild-type c-erbA and its DNA binding domain will allow circular dichroism, fluorescence, and ultraviolet-visible spectroscopy (both proteins), and nuclear magnetic resonance studies (DNA binding domain) to provide structural information to elucidate the functional and physical properties of these proteins at the molecular level.

Abstract :

Thyroid hormone receptors (c-erbAs) and retinoic acid receptors (RARs) are members of a subgroup of closely related nuclear receptor proteins. c-erbAs and RAR can each activate certain response elements and both receptors contain a highly conserved domain embedded within the ligand binding region containing a series of "leucine-zipper-like" hydrophobic heptad motifs. Functional studies suggest that the heptad repeat domain mediates homo- and heterodimeric interactions of c-erbA and RAR or interactions with other factors. Chick c-erbA-alpha and human RAR-alpha have been expressed in E. coli and purified to near homogeneity. These receptors bind ligand with appropriate affinity and form homo- and hetero-dimers on response elements which are permissive for dimerization. Dimer formation is enhanced by ligand suggesting that ligand mediates transcriptional activation by this mechanism. This application is a comprehensive proposal to define the functional domains involved in c-erbA, RAR, and related factors in transcriptional activation. For these studies we constructed a multifunctional bacterial/eucaryotic expression vector (pEXPRESS) which permits site directed mutagenesis and can be used to functionally analyze receptor in eucaryotic cells and to express receptor at high levels in E. coli. Gel shift studies using purified wild-type and mutant receptor proteins and a variety of native and synthetic response elements are proposed to elucidate the "rules" which govern how these receptors recognize functional response elements. These studies will also define receptor domains critical for homo- and hetero-dimer formation, for cooperative interactions between receptors and other factors, and for dominant negative activities. Possible differences in element recognition by c-erbA subtypes (alphal and betal) will also be examined along with studies to understand differences between v-erbA and c-erbA and how c-erbA-alpha2 functions as a dominant negative regulator. These studies will be complemented by analyzing receptor mutants derived from patients with the thyroid hormone resistance syndrome. Functional studies in mammalian cells will be extended to in vitro transcription with the goal of defining the protein and DNA requirements for transcriptional enhancement by these receptors. Finally, the availability of mg amounts of purified E. coli expressed wild-type c-erbA and its DNA binding domain will allow circular dichroism, fluorescence, and ultraviolet-visible spectroscopy (both proteins), and nuclear magnetic resonance studies (DNA binding domain) to provide structural information to elucidate the functional and physical properties of these proteins at the molecular level.

Abstract :

Thyroid hormone receptors (c-erbAs) and retinoic acid receptors (RARs) are members of a subgroup of closely related nuclear receptor proteins. c-erbAs and RAR can each activate certain response elements and both receptors contain a highly conserved domain embedded within the ligand binding region containing a series of "leucine-zipper-like" hydrophobic heptad motifs. Functional studies suggest that the heptad repeat domain mediates homo- and heterodimeric interactions of c-erbA and RAR or interactions with other factors. Chick c-erbA-alpha and human RAR-alpha have been expressed in E. coli and purified to near homogeneity. These receptors bind ligand with appropriate affinity and form homo- and hetero-dimers on response elements which are permissive for dimerization. Dimer formation is enhanced by ligand suggesting that ligand mediates transcriptional activation by this mechanism. This application is a comprehensive proposal to define the functional domains involved in c-erbA, RAR, and related factors in transcriptional activation. For these studies we constructed a multifunctional bacterial/eucaryotic expression vector (pEXPRESS) which permits site directed mutagenesis and can be used to functionally analyze receptor in eucaryotic cells and to express receptor at high levels in E. coli. Gel shift studies using purified wild-type and mutant receptor proteins and a variety of native and synthetic response elements are proposed to elucidate the "rules" which govern how these receptors recognize functional response elements. These studies will also define receptor domains critical for homo- and hetero-dimer formation, for cooperative interactions between receptors and other factors, and for dominant negative activities. Possible differences in element recognition by c-erbA subtypes (alphal and betal) will also be examined along with studies to understand differences between v-erbA and c-erbA and how c-erbA-alpha2 functions as a dominant negative regulator. These studies will be complemented by analyzing receptor mutants derived from patients with the thyroid hormone resistance syndrome. Functional studies in mammalian cells will be extended to in vitro transcription with the goal of defining the protein and DNA requirements for transcriptional enhancement by these receptors. Finally, the availability of mg amounts of purified E. coli expressed wild-type c-erbA and its DNA binding domain will allow circular dichroism, fluorescence, and ultraviolet-visible spectroscopy (both proteins), and nuclear magnetic resonance studies (DNA binding domain) to provide structural information to elucidate the functional and physical properties of these proteins at the molecular level.

Abstract :

Thyroid hormone receptors (c-erbAs) and retinoic acid receptors (RARs) are members of a subgroup of closely related nuclear receptor proteins. c-erbAs and RAR can each activate certain response elements and both receptors contain a highly conserved domain embedded within the ligand binding region containing a series of "leucine-zipper-like" hydrophobic heptad motifs. Functional studies suggest that the heptad repeat domain mediates homo- and heterodimeric interactions of c-erbA and RAR or interactions with other factors. Chick c-erbA-alpha and human RAR-alpha have been expressed in E. coli and purified to near homogeneity. These receptors bind ligand with appropriate affinity and form homo- and hetero-dimers on response elements which are permissive for dimerization. Dimer formation is enhanced by ligand suggesting that ligand mediates transcriptional activation by this mechanism. This application is a comprehensive proposal to define the functional domains involved in c-erbA, RAR, and related factors in transcriptional activation. For these studies we constructed a multifunctional bacterial/eucaryotic expression vector (pEXPRESS) which permits site directed mutagenesis and can be used to functionally analyze receptor in eucaryotic cells and to express receptor at high levels in E. coli. Gel shift studies using purified wild-type and mutant receptor proteins and a variety of native and synthetic response elements are proposed to elucidate the "rules" which govern how these receptors recognize functional response elements. These studies will also define receptor domains critical for homo- and hetero-dimer formation, for cooperative interactions between receptors and other factors, and for dominant negative activities. Possible differences in element recognition by c-erbA subtypes (alphal and betal) will also be examined along with studies to understand differences between v-erbA and c-erbA and how c-erbA-alpha2 functions as a dominant negative regulator. These studies will be complemented by analyzing receptor mutants derived from patients with the thyroid hormone resistance syndrome. Functional studies in mammalian cells will be extended to in vitro transcription with the goal of defining the protein and DNA requirements for transcriptional enhancement by these receptors. Finally, the availability of mg amounts of purified E. coli expressed wild-type c-erbA and its DNA binding domain will allow circular dichroism, fluorescence, and ultraviolet-visible spectroscopy (both proteins), and nuclear magnetic resonance studies (DNA binding domain) to provide structural information to elucidate the functional and physical properties of these proteins at the molecular level.

Abstract :

Thyroid hormone receptors (c-erbAs) and retinoic acid receptors (RARs) are members of a subgroup of closely related nuclear receptor proteins. c-erbAs and RAR can each activate certain response elements and both receptors contain a highly conserved domain embedded within the ligand binding region containing a series of "leucine-zipper-like" hydrophobic heptad motifs. Functional studies suggest that the heptad repeat domain mediates homo- and heterodimeric interactions of c-erbA and RAR or interactions with other factors. Chick c-erbA-alpha and human RAR-alpha have been expressed in E. coli and purified to near homogeneity. These receptors bind ligand with appropriate affinity and form homo- and hetero-dimers on response elements which are permissive for dimerization. Dimer formation is enhanced by ligand suggesting that ligand mediates transcriptional activation by this mechanism. This application is a comprehensive proposal to define the functional domains involved in c-erbA, RAR, and related factors in transcriptional activation. For these studies we constructed a multifunctional bacterial/eucaryotic expression vector (pEXPRESS) which permits site directed mutagenesis and can be used to functionally analyze receptor in eucaryotic cells and to express receptor at high levels in E. coli. Gel shift studies using purified wild-type and mutant receptor proteins and a variety of native and synthetic response elements are proposed to elucidate the "rules" which govern how these receptors recognize functional response elements. These studies will also define receptor domains critical for homo- and hetero-dimer formation, for cooperative interactions between receptors and other factors, and for dominant negative activities. Possible differences in element recognition by c-erbA subtypes (alphal and betal) will also be examined along with studies to understand differences between v-erbA and c-erbA and how c-erbA-alpha2 functions as a dominant negative regulator. These studies will be complemented by analyzing receptor mutants derived from patients with the thyroid hormone resistance syndrome. Functional studies in mammalian cells will be extended to in vitro transcription with the goal of defining the protein and DNA requirements for transcriptional enhancement by these receptors. Finally, the availability of mg amounts of purified E. coli expressed wild-type c-erbA and its DNA binding domain will allow circular dichroism, fluorescence, and ultraviolet-visible spectroscopy (both proteins), and nuclear magnetic resonance studies (DNA binding domain) to provide structural information to elucidate the functional and physical properties of these proteins at the molecular level.

Abstract :

Thyroid hormone receptors (c-erbAs) and retinoic acid receptors (RARs) are members of a subgroup of closely related nuclear receptor proteins. c-erbAs and RAR can each activate certain response elements and both receptors contain a highly conserved domain embedded within the ligand binding region containing a series of "leucine-zipper-like" hydrophobic heptad motifs. Functional studies suggest that the heptad repeat domain mediates homo- and heterodimeric interactions of c-erbA and RAR or interactions with other factors. Chick c-erbA-alpha and human RAR-alpha have been expressed in E. coli and purified to near homogeneity. These receptors bind ligand with appropriate affinity and form homo- and hetero-dimers on response elements which are permissive for dimerization. Dimer formation is enhanced by ligand suggesting that ligand mediates transcriptional activation by this mechanism. This application is a comprehensive proposal to define the functional domains involved in c-erbA, RAR, and related factors in transcriptional activation. For these studies we constructed a multifunctional bacterial/eucaryotic expression vector (pEXPRESS) which permits site directed mutagenesis and can be used to functionally analyze receptor in eucaryotic cells and to express receptor at high levels in E. coli. Gel shift studies using purified wild-type and mutant receptor proteins and a variety of native and synthetic response elements are proposed to elucidate the "rules" which govern how these receptors recognize functional response elements. These studies will also define receptor domains critical for homo- and hetero-dimer formation, for cooperative interactions between receptors and other factors, and for dominant negative activities. Possible differences in element recognition by c-erbA subtypes (alphal and betal) will also be examined along with studies to understand differences between v-erbA and c-erbA and how c-erbA-alpha2 functions as a dominant negative regulator. These studies will be complemented by analyzing receptor mutants derived from patients with the thyroid hormone resistance syndrome. Functional studies in mammalian cells will be extended to in vitro transcription with the goal of defining the protein and DNA requirements for transcriptional enhancement by these receptors. Finally, the availability of mg amounts of purified E. coli expressed wild-type c-erbA and its DNA binding domain will allow circular dichroism, fluorescence, and ultraviolet-visible spectroscopy (both proteins), and nuclear magnetic resonance studies (DNA binding domain) to provide structural information to elucidate the functional and physical properties of these proteins at the molecular level.

Abstract :

Thyroid hormone receptors (c-erbAs) and retinoic acid receptors (RARs) are members of a subgroup of closely related nuclear receptor proteins. c-erbAs and RAR can each activate certain response elements and both receptors contain a highly conserved domain embedded within the ligand binding region containing a series of "leucine-zipper-like" hydrophobic heptad motifs. Functional studies suggest that the heptad repeat domain mediates homo- and heterodimeric interactions of c-erbA and RAR or interactions with other factors. Chick c-erbA-alpha and human RAR-alpha have been expressed in E. coli and purified to near homogeneity. These receptors bind ligand with appropriate affinity and form homo- and hetero-dimers on response elements which are permissive for dimerization. Dimer formation is enhanced by ligand suggesting that ligand mediates transcriptional activation by this mechanism. This application is a comprehensive proposal to define the functional domains involved in c-erbA, RAR, and related factors in transcriptional activation. For these studies we constructed a multifunctional bacterial/eucaryotic expression vector (pEXPRESS) which permits site directed mutagenesis and can be used to functionally analyze receptor in eucaryotic cells and to express receptor at high levels in E. coli. Gel shift studies using purified wild-type and mutant receptor proteins and a variety of native and synthetic response elements are proposed to elucidate the "rules" which govern how these receptors recognize functional response elements. These studies will also define receptor domains critical for homo- and hetero-dimer formation, for cooperative interactions between receptors and other factors, and for dominant negative activities. Possible differences in element recognition by c-erbA subtypes (alphal and betal) will also be examined along with studies to understand differences between v-erbA and c-erbA and how c-erbA-alpha2 functions as a dominant negative regulator. These studies will be complemented by analyzing receptor mutants derived from patients with the thyroid hormone resistance syndrome. Functional studies in mammalian cells will be extended to in vitro transcription with the goal of defining the protein and DNA requirements for transcriptional enhancement by these receptors. Finally, the availability of mg amounts of purified E. coli expressed wild-type c-erbA and its DNA binding domain will allow circular dichroism, fluorescence, and ultraviolet-visible spectroscopy (both proteins), and nuclear magnetic resonance studies (DNA binding domain) to provide structural information to elucidate the functional and physical properties of these proteins at the molecular level.

Abstract :

Thyroid hormone receptors (c-erbAs) and retinoic acid receptors (RARs) are members of a subgroup of closely related nuclear receptor proteins. c-erbAs and RAR can each activate certain response elements and both receptors contain a highly conserved domain embedded within the ligand binding region containing a series of "leucine-zipper-like" hydrophobic heptad motifs. Functional studies suggest that the heptad repeat domain mediates homo- and heterodimeric interactions of c-erbA and RAR or interactions with other factors. Chick c-erbA-alpha and human RAR-alpha have been expressed in E. coli and purified to near homogeneity. These receptors bind ligand with appropriate affinity and form homo- and hetero-dimers on response elements which are permissive for dimerization. Dimer formation is enhanced by ligand suggesting that ligand mediates transcriptional activation by this mechanism. This application is a comprehensive proposal to define the functional domains involved in c-erbA, RAR, and related factors in transcriptional activation. For these studies we constructed a multifunctional bacterial/eucaryotic expression vector (pEXPRESS) which permits site directed mutagenesis and can be used to functionally analyze receptor in eucaryotic cells and to express receptor at high levels in E. coli. Gel shift studies using purified wild-type and mutant receptor proteins and a variety of native and synthetic response elements are proposed to elucidate the "rules" which govern how these receptors recognize functional response elements. These studies will also define receptor domains critical for homo- and hetero-dimer formation, for cooperative interactions between receptors and other factors, and for dominant negative activities. Possible differences in element recognition by c-erbA subtypes (alphal and betal) will also be examined along with studies to understand differences between v-erbA and c-erbA and how c-erbA-alpha2 functions as a dominant negative regulator. These studies will be complemented by analyzing receptor mutants derived from patients with the thyroid hormone resistance syndrome. Functional studies in mammalian cells will be extended to in vitro transcription with the goal of defining the protein and DNA requirements for transcriptional enhancement by these receptors. Finally, the availability of mg amounts of purified E. coli expressed wild-type c-erbA and its DNA binding domain will allow circular dichroism, fluorescence, and ultraviolet-visible spectroscopy (both proteins), and nuclear magnetic resonance studies (DNA binding domain) to provide structural information to elucidate the functional and physical properties of these proteins at the molecular level.

Abstract :

Thyroid hormone receptors (c-erbAs) and retinoic acid receptors (RARs) are members of a subgroup of closely related nuclear receptor proteins. c-erbAs and RAR can each activate certain response elements and both receptors contain a highly conserved domain embedded within the ligand binding region containing a series of "leucine-zipper-like" hydrophobic heptad motifs. Functional studies suggest that the heptad repeat domain mediates homo- and heterodimeric interactions of c-erbA and RAR or interactions with other factors. Chick c-erbA-alpha and human RAR-alpha have been expressed in E. coli and purified to near homogeneity. These receptors bind ligand with appropriate affinity and form homo- and hetero-dimers on response elements which are permissive for dimerization. Dimer formation is enhanced by ligand suggesting that ligand mediates transcriptional activation by this mechanism. This application is a comprehensive proposal to define the functional domains involved in c-erbA, RAR, and related factors in transcriptional activation. For these studies we constructed a multifunctional bacterial/eucaryotic expression vector (pEXPRESS) which permits site directed mutagenesis and can be used to functionally analyze receptor in eucaryotic cells and to express receptor at high levels in E. coli. Gel shift studies using purified wild-type and mutant receptor proteins and a variety of native and synthetic response elements are proposed to elucidate the "rules" which govern how these receptors recognize functional response elements. These studies will also define receptor domains critical for homo- and hetero-dimer formation, for cooperative interactions between receptors and other factors, and for dominant negative activities. Possible differences in element recognition by c-erbA subtypes (alphal and betal) will also be examined along with studies to understand differences between v-erbA and c-erbA and how c-erbA-alpha2 functions as a dominant negative regulator. These studies will be complemented by analyzing receptor mutants derived from patients with the thyroid hormone resistance syndrome. Functional studies in mammalian cells will be extended to in vitro transcription with the goal of defining the protein and DNA requirements for transcriptional enhancement by these receptors. Finally, the availability of mg amounts of purified E. coli expressed wild-type c-erbA and its DNA binding domain will allow circular dichroism, fluorescence, and ultraviolet-visible spectroscopy (both proteins), and nuclear magnetic resonance studies (DNA binding domain) to provide structural information to elucidate the functional and physical properties of these proteins at the molecular level.

Abstract :

Thyroid hormone receptors (c-erbAs) and retinoic acid receptors (RARs) are members of a subgroup of closely related nuclear receptor proteins. c-erbAs and RAR can each activate certain response elements and both receptors contain a highly conserved domain embedded within the ligand binding region containing a series of "leucine-zipper-like" hydrophobic heptad motifs. Functional studies suggest that the heptad repeat domain mediates homo- and heterodimeric interactions of c-erbA and RAR or interactions with other factors. Chick c-erbA-alpha and human RAR-alpha have been expressed in E. coli and purified to near homogeneity. These receptors bind ligand with appropriate affinity and form homo- and hetero-dimers on response elements which are permissive for dimerization. Dimer formation is enhanced by ligand suggesting that ligand mediates transcriptional activation by this mechanism. This application is a comprehensive proposal to define the functional domains involved in c-erbA, RAR, and related factors in transcriptional activation. For these studies we constructed a multifunctional bacterial/eucaryotic expression vector (pEXPRESS) which permits site directed mutagenesis and can be used to functionally analyze receptor in eucaryotic cells and to express receptor at high levels in E. coli. Gel shift studies using purified wild-type and mutant receptor proteins and a variety of native and synthetic response elements are proposed to elucidate the "rules" which govern how these receptors recognize functional response elements. These studies will also define receptor domains critical for homo- and hetero-dimer formation, for cooperative interactions between receptors and other factors, and for dominant negative activities. Possible differences in element recognition by c-erbA subtypes (alphal and betal) will also be examined along with studies to understand differences between v-erbA and c-erbA and how c-erbA-alpha2 functions as a dominant negative regulator. These studies will be complemented by analyzing receptor mutants derived from patients with the thyroid hormone resistance syndrome. Functional studies in mammalian cells will be extended to in vitro transcription with the goal of defining the protein and DNA requirements for transcriptional enhancement by these receptors. Finally, the availability of mg amounts of purified E. coli expressed wild-type c-erbA and its DNA binding domain will allow circular dichroism, fluorescence, and ultraviolet-visible spectroscopy (both proteins), and nuclear magnetic resonance studies (DNA binding domain) to provide structural information to elucidate the functional and physical properties of these proteins at the molecular level.

Abstract :

Thyroid hormone receptors (c-erbAs) and retinoic acid receptors (RARs) are members of a subgroup of closely related nuclear receptor proteins. c-erbAs and RAR can each activate certain response elements and both receptors contain a highly conserved domain embedded within the ligand binding region containing a series of "leucine-zipper-like" hydrophobic heptad motifs. Functional studies suggest that the heptad repeat domain mediates homo- and heterodimeric interactions of c-erbA and RAR or interactions with other factors. Chick c-erbA-alpha and human RAR-alpha have been expressed in E. coli and purified to near homogeneity. These receptors bind ligand with appropriate affinity and form homo- and hetero-dimers on response elements which are permissive for dimerization. Dimer formation is enhanced by ligand suggesting that ligand mediates transcriptional activation by this mechanism. This application is a comprehensive proposal to define the functional domains involved in c-erbA, RAR, and related factors in transcriptional activation. For these studies we constructed a multifunctional bacterial/eucaryotic expression vector (pEXPRESS) which permits site directed mutagenesis and can be used to functionally analyze receptor in eucaryotic cells and to express receptor at high levels in E. coli. Gel shift studies using purified wild-type and mutant receptor proteins and a variety of native and synthetic response elements are proposed to elucidate the "rules" which govern how these receptors recognize functional response elements. These studies will also define receptor domains critical for homo- and hetero-dimer formation, for cooperative interactions between receptors and other factors, and for dominant negative activities. Possible differences in element recognition by c-erbA subtypes (alphal and betal) will also be examined along with studies to understand differences between v-erbA and c-erbA and how c-erbA-alpha2 functions as a dominant negative regulator. These studies will be complemented by analyzing receptor mutants derived from patients with the thyroid hormone resistance syndrome. Functional studies in mammalian cells will be extended to in vitro transcription with the goal of defining the protein and DNA requirements for transcriptional enhancement by these receptors. Finally, the availability of mg amounts of purified E. coli expressed wild-type c-erbA and its DNA binding domain will allow circular dichroism, fluorescence, and ultraviolet-visible spectroscopy (both proteins), and nuclear magnetic resonance studies (DNA binding domain) to provide structural information to elucidate the functional and physical properties of these proteins at the molecular level.

Abstract :

Thyroid hormone receptors (c-erbAs) and retinoic acid receptors (RARs) are members of a subgroup of closely related nuclear receptor proteins. c-erbAs and RAR can each activate certain response elements and both receptors contain a highly conserved domain embedded within the ligand binding region containing a series of "leucine-zipper-like" hydrophobic heptad motifs. Functional studies suggest that the heptad repeat domain mediates homo- and heterodimeric interactions of c-erbA and RAR or interactions with other factors. Chick c-erbA-alpha and human RAR-alpha have been expressed in E. coli and purified to near homogeneity. These receptors bind ligand with appropriate affinity and form homo- and hetero-dimers on response elements which are permissive for dimerization. Dimer formation is enhanced by ligand suggesting that ligand mediates transcriptional activation by this mechanism. This application is a comprehensive proposal to define the functional domains involved in c-erbA, RAR, and related factors in transcriptional activation. For these studies we constructed a multifunctional bacterial/eucaryotic expression vector (pEXPRESS) which permits site directed mutagenesis and can be used to functionally analyze receptor in eucaryotic cells and to express receptor at high levels in E. coli. Gel shift studies using purified wild-type and mutant receptor proteins and a variety of native and synthetic response elements are proposed to elucidate the "rules" which govern how these receptors recognize functional response elements. These studies will also define receptor domains critical for homo- and hetero-dimer formation, for cooperative interactions between receptors and other factors, and for dominant negative activities. Possible differences in element recognition by c-erbA subtypes (alphal and betal) will also be examined along with studies to understand differences between v-erbA and c-erbA and how c-erbA-alpha2 functions as a dominant negative regulator. These studies will be complemented by analyzing receptor mutants derived from patients with the thyroid hormone resistance syndrome. Functional studies in mammalian cells will be extended to in vitro transcription with the goal of defining the protein and DNA requirements for transcriptional enhancement by these receptors. Finally, the availability of mg amounts of purified E. coli expressed wild-type c-erbA and its DNA binding domain will allow circular dichroism, fluorescence, and ultraviolet-visible spectroscopy (both proteins), and nuclear magnetic resonance studies (DNA binding domain) to provide structural information to elucidate the functional and physical properties of these proteins at the molecular level.

Abstract :

Using cultured GH1 and GC cells, two growth hormone producing rat pituitary cell lines, we have shown that L-triiodothyronine (L-T3) stimulats an increase in growth hormone mRNA accumulation which results from stimulation of growth hormone gene transcription. In these cells glucocorticoid hormones also act synergistically with L-T3 to stimulate transcription of the growth hormone gene. A goal of this proposal is to map the DNA control elements involved in the hormonal regulation of growth hormone gene expression. We have constructed a chimeric gene (pGH-xgpt) consisting of 1.8 kb of the 5'-flanking region of the rat growth hormone gene which was ligated to bacterial DNA containing the structural gene which encodes for the enzyme, xanthine-guainine phosphoribosyl transferase (XGPT). In pGH-xgpt expression of SGPT is under regulation of any hormone control elements in the 5' region of the growth hormone gene. This construct yields stable transformants of GC cells with relatively high frequency in which the XGPT gene is highly regulated by L-T3. A library of 5' deletion mutants will be constructed to locate the position and boundaries of the DNA elements which mediate regulated expression by hormone. Whether DNA sequences 3' of the transcriptional start site of the growth hormone gene can mediate regulated expression will also be explored. Regions of interest will be subloned and further analyzed in detail using stable as well as transient expression. We will also assess whether the 5'-flanking region of the human growth hormone gene elicits regulation by hormone in GC cells to assess the possible homology of hormone control elements across species lines. Other studies will relate the location of hormone regulatory elements to nuclease hypersensitive sites of the rat growth hormone goene. Various approaches wil explore the interaction of the thyroid hormone receptor with defined growth hormone gene fragments. Using a photoaffinity label derivative of L-T3 (L-T3-PAL) we have identified two molecular weight (Mr) thyroid hormone receptor forms; 47,000 and 56,000. Photoaffinity labeling will be used to clarify the interrelationship of these Mr forms and their interaction with gene fragments and chromatin domains. Photoaffinity labeling will also be used to identify possible subtle alterations of thyroid hormone receptor in fibroblasts from patients with thyroid hormone resistance.

Abstract :

Using cultured GH1 and GC cells, two growth hormone producing rat pituitary cell lines, we have shown that L-triiodothyronine (L-T3) stimulats an increase in growth hormone mRNA accumulation which results from stimulation of growth hormone gene transcription. In these cells glucocorticoid hormones also act synergistically with L-T3 to stimulate transcription of the growth hormone gene. A goal of this proposal is to map the DNA control elements involved in the hormonal regulation of growth hormone gene expression. We have constructed a chimeric gene (pGH-xgpt) consisting of 1.8 kb of the 5'-flanking region of the rat growth hormone gene which was ligated to bacterial DNA containing the structural gene which encodes for the enzyme, xanthine-guainine phosphoribosyl transferase (XGPT). In pGH-xgpt expression of SGPT is under regulation of any hormone control elements in the 5' region of the growth hormone gene. This construct yields stable transformants of GC cells with relatively high frequency in which the XGPT gene is highly regulated by L-T3. A library of 5' deletion mutants will be constructed to locate the position and boundaries of the DNA elements which mediate regulated expression by hormone. Whether DNA sequences 3' of the transcriptional start site of the growth hormone gene can mediate regulated expression will also be explored. Regions of interest will be subloned and further analyzed in detail using stable as well as transient expression. We will also assess whether the 5'-flanking region of the human growth hormone gene elicits regulation by hormone in GC cells to assess the possible homology of hormone control elements across species lines. Other studies will relate the location of hormone regulatory elements to nuclease hypersensitive sites of the rat growth hormone goene. Various approaches wil explore the interaction of the thyroid hormone receptor with defined growth hormone gene fragments. Using a photoaffinity label derivative of L-T3 (L-T3-PAL) we have identified two molecular weight (Mr) thyroid hormone receptor forms; 47,000 and 56,000. Photoaffinity labeling will be used to clarify the interrelationship of these Mr forms and their interaction with gene fragments and chromatin domains. Photoaffinity labeling will also be used to identify possible subtle alterations of thyroid hormone receptor in fibroblasts from patients with thyroid hormone resistance.

Abstract :

Using cultured GH1 and GC cells, two growth hormone producing rat pituitary cell lines, we have shown that L-triiodothyronine (L-T3) stimulats an increase in growth hormone mRNA accumulation which results from stimulation of growth hormone gene transcription. In these cells glucocorticoid hormones also act synergistically with L-T3 to stimulate transcription of the growth hormone gene. A goal of this proposal is to map the DNA control elements involved in the hormonal regulation of growth hormone gene expression. We have constructed a chimeric gene (pGH-xgpt) consisting of 1.8 kb of the 5'-flanking region of the rat growth hormone gene which was ligated to bacterial DNA containing the structural gene which encodes for the enzyme, xanthine-guainine phosphoribosyl transferase (XGPT). In pGH-xgpt expression of SGPT is under regulation of any hormone control elements in the 5' region of the growth hormone gene. This construct yields stable transformants of GC cells with relatively high frequency in which the XGPT gene is highly regulated by L-T3. A library of 5' deletion mutants will be constructed to locate the position and boundaries of the DNA elements which mediate regulated expression by hormone. Whether DNA sequences 3' of the transcriptional start site of the growth hormone gene can mediate regulated expression will also be explored. Regions of interest will be subloned and further analyzed in detail using stable as well as transient expression. We will also assess whether the 5'-flanking region of the human growth hormone gene elicits regulation by hormone in GC cells to assess the possible homology of hormone control elements across species lines. Other studies will relate the location of hormone regulatory elements to nuclease hypersensitive sites of the rat growth hormone goene. Various approaches wil explore the interaction of the thyroid hormone receptor with defined growth hormone gene fragments. Using a photoaffinity label derivative of L-T3 (L-T3-PAL) we have identified two molecular weight (Mr) thyroid hormone receptor forms; 47,000 and 56,000. Photoaffinity labeling will be used to clarify the interrelationship of these Mr forms and their interaction with gene fragments and chromatin domains. Photoaffinity labeling will also be used to identify possible subtle alterations of thyroid hormone receptor in fibroblasts from patients with thyroid hormone resistance.

Abstract :

Using cultured GH1 and GC cells, two growth hormone producing rat pituitary cell lines, we have shown that L-triiodothyronine (L-T3) stimulats an increase in growth hormone mRNA accumulation which results from stimulation of growth hormone gene transcription. In these cells glucocorticoid hormones also act synergistically with L-T3 to stimulate transcription of the growth hormone gene. A goal of this proposal is to map the DNA control elements involved in the hormonal regulation of growth hormone gene expression. We have constructed a chimeric gene (pGH-xgpt) consisting of 1.8 kb of the 5'-flanking region of the rat growth hormone gene which was ligated to bacterial DNA containing the structural gene which encodes for the enzyme, xanthine-guainine phosphoribosyl transferase (XGPT). In pGH-xgpt expression of SGPT is under regulation of any hormone control elements in the 5' region of the growth hormone gene. This construct yields stable transformants of GC cells with relatively high frequency in which the XGPT gene is highly regulated by L-T3. A library of 5' deletion mutants will be constructed to locate the position and boundaries of the DNA elements which mediate regulated expression by hormone. Whether DNA sequences 3' of the transcriptional start site of the growth hormone gene can mediate regulated expression will also be explored. Regions of interest will be subloned and further analyzed in detail using stable as well as transient expression. We will also assess whether the 5'-flanking region of the human growth hormone gene elicits regulation by hormone in GC cells to assess the possible homology of hormone control elements across species lines. Other studies will relate the location of hormone regulatory elements to nuclease hypersensitive sites of the rat growth hormone goene. Various approaches wil explore the interaction of the thyroid hormone receptor with defined growth hormone gene fragments. Using a photoaffinity label derivative of L-T3 (L-T3-PAL) we have identified two molecular weight (Mr) thyroid hormone receptor forms; 47,000 and 56,000. Photoaffinity labeling will be used to clarify the interrelationship of these Mr forms and their interaction with gene fragments and chromatin domains. Photoaffinity labeling will also be used to identify possible subtle alterations of thyroid hormone receptor in fibroblasts from patients with thyroid hormone resistance.

Abstract :

Using cultured GH1 and GC cells, two growth hormone producing rat pituitary cell lines, we have shown that L-triiodothyronine (L-T3) stimulats an increase in growth hormone mRNA accumulation which results from stimulation of growth hormone gene transcription. In these cells glucocorticoid hormones also act synergistically with L-T3 to stimulate transcription of the growth hormone gene. A goal of this proposal is to map the DNA control elements involved in the hormonal regulation of growth hormone gene expression. We have constructed a chimeric gene (pGH-xgpt) consisting of 1.8 kb of the 5'-flanking region of the rat growth hormone gene which was ligated to bacterial DNA containing the structural gene which encodes for the enzyme, xanthine-guainine phosphoribosyl transferase (XGPT). In pGH-xgpt expression of SGPT is under regulation of any hormone control elements in the 5' region of the growth hormone gene. This construct yields stable transformants of GC cells with relatively high frequency in which the XGPT gene is highly regulated by L-T3. A library of 5' deletion mutants will be constructed to locate the position and boundaries of the DNA elements which mediate regulated expression by hormone. Whether DNA sequences 3' of the transcriptional start site of the growth hormone gene can mediate regulated expression will also be explored. Regions of interest will be subloned and further analyzed in detail using stable as well as transient expression. We will also assess whether the 5'-flanking region of the human growth hormone gene elicits regulation by hormone in GC cells to assess the possible homology of hormone control elements across species lines. Other studies will relate the location of hormone regulatory elements to nuclease hypersensitive sites of the rat growth hormone goene. Various approaches wil explore the interaction of the thyroid hormone receptor with defined growth hormone gene fragments. Using a photoaffinity label derivative of L-T3 (L-T3-PAL) we have identified two molecular weight (Mr) thyroid hormone receptor forms; 47,000 and 56,000. Photoaffinity labeling will be used to clarify the interrelationship of these Mr forms and their interaction with gene fragments and chromatin domains. Photoaffinity labeling will also be used to identify possible subtle alterations of thyroid hormone receptor in fibroblasts from patients with thyroid hormone resistance.

Abstract :

This study will determine if there is a significant heterogeneity in the clinical manifestations of patients in various families with generalized thyroid hormone resistance (GTHR). The investigators postulate that a variety of different thyroid hormone receptor defects may be responsible for this syndrome. The study will evaluate the response of these patients to L- triiodothyronine and will document the molecular defects in their thyroid hormone receptors. Family members of affected patients will be screened.

Abstract :

The proposed research involves defining the detailed mechanisms by which glucocorticoids and other hormones interact with thyroid hormone to control biological processes in mammalian cells. The cell culture systems to be investigated are GH1 cells, two clonal cells of rat pituitary cell lines which synthesize growth hormone. With these cells physiological concentrations of thyroid hormones increase growth hormone synthesis and the effect of glucocorticoids on regulating growth hormone synthesis is highly dependent on the action of thyroid hormone. These cells contain nuclear receptors for thyroid hormone and also contain glucocorticoid receptors and the regulation of the synthesis of growth hormone by thyroid hormone and glucocorticoid appears to be modulated by their respective regular receptors. In addition, GH1 cells and GC cells contain insulin receptors with an estimated Kd of 2-5 x 10 to the minus 10th power M with approximately 8,000 insulin binding sites per cell. In GC cells thyroid hormone and glucocorticoid induce increases in insulin receptor levels and the influence of glucocorticoid is additive but not synergistic with that of thyroid hormone differing from the regulation of the growth hormone response. Furthermore, thyroid hormone and glucocorticoid independently inhibit insulin degradation by these cells by a process which appears to involve a decrease in cell uptake of insulin and therefore the inhibition of insulin degradation appears to be modulated by the influence of thyroid hormone and or glucocorticoid on inhibiting insulin internalization. The mechanism of inhibition of insulin metabolism in GH1 cells will be investigated by assessing the kinetics of internalization of insulin and the kinetics of internalization of surface membrane insulin receptors. This will be determined from a biochemical point of view as well as by direct visualization using fluorescent insulin derivitives by Video Intensification Microscopy to monitor the kinetics of internalization. The influence of insuin action and internalization of these cells will be assessed in terms of the influence of insulin on controlling the growth hormone response in concert with thyroid and glucocorticoid hormones.

Abstract :

The proposed research involves defining the detailed mechanisms by which glucocorticoids and other hormones interact with thyroid hormone to control biological processes in mammalian cells. The cell culture systems to be investigated are GH1 cells, two clonal cells of rat pituitary cell lines which synthesize growth hormone. With these cells physiological concentrations of thyroid hormones increase growth hormone synthesis and the effect of glucocorticoids on regulating growth hormone synthesis is highly dependent on the action of thyroid hormone. These cells contain nuclear receptors for thyroid hormone and also contain glucocorticoid receptors and the regulation of the synthesis of growth hormone by thyroid hormone and glucocorticoid appears to be modulated by their respective regular receptors. In addition, GH1 cells and GC cells contain insulin receptors with an estimated Kd of 2-5 x 10 to the minus 10th power M with approximately 8,000 insulin binding sites per cell. In GC cells thyroid hormone and glucocorticoid induce increases in insulin receptor levels and the influence of glucocorticoid is additive but not synergistic with that of thyroid hormone differing from the regulation of the growth hormone response. Furthermore, thyroid hormone and glucocorticoid independently inhibit insulin degradation by these cells by a process which appears to involve a decrease in cell uptake of insulin and therefore the inhibition of insulin degradation appears to be modulated by the influence of thyroid hormone and or glucocorticoid on inhibiting insulin internalization. The mechanism of inhibition of insulin metabolism in GH1 cells will be investigated by assessing the kinetics of internalization of insulin and the kinetics of internalization of surface membrane insulin receptors. This will be determined from a biochemical point of view as well as by direct visualization using fluorescent insulin derivitives by Video Intensification Microscopy to monitor the kinetics of internalization. The influence of insuin action and internalization of these cells will be assessed in terms of the influence of insulin on controlling the growth hormone response in concert with thyroid and glucocorticoid hormones.

Abstract :

The proposed research involves defining the detailed mechanisms by which glucocorticoids and other hormones interact with thyroid hormone to control biological processes in mammalian cells. The cell culture systems to be investigated are GH1 cells, two clonal cells of rat pituitary cell lines which synthesize growth hormone. With these cells physiological concentrations of thyroid hormones increase growth hormone synthesis and the effect of glucocorticoids on regulating growth hormone synthesis is highly dependent on the action of thyroid hormone. These cells contain nuclear receptors for thyroid hormone and also contain glucocorticoid receptors and the regulation of the synthesis of growth hormone by thyroid hormone and glucocorticoid appears to be modulated by their respective regular receptors. In addition, GH1 cells and GC cells contain insulin receptors with an estimated Kd of 2-5 x 10 to the minus 10th power M with approximately 8,000 insulin binding sites per cell. In GC cells thyroid hormone and glucocorticoid induce increases in insulin receptor levels and the influence of glucocorticoid is additive but not synergistic with that of thyroid hormone differing from the regulation of the growth hormone response. Furthermore, thyroid hormone and glucocorticoid independently inhibit insulin degradation by these cells by a process which appears to involve a decrease in cell uptake of insulin and therefore the inhibition of insulin degradation appears to be modulated by the influence of thyroid hormone and or glucocorticoid on inhibiting insulin internalization. The mechanism of inhibition of insulin metabolism in GH1 cells will be investigated by assessing the kinetics of internalization of insulin and the kinetics of internalization of surface membrane insulin receptors. This will be determined from a biochemical point of view as well as by direct visualization using fluorescent insulin derivitives by Video Intensification Microscopy to monitor the kinetics of internalization. The influence of insuin action and internalization of these cells will be assessed in terms of the influence of insulin on controlling the growth hormone response in concert with thyroid and glucocorticoid hormones.

Abstract :

The proposed research involves defining the detailed mechanisms by which glucocorticoids and other hormones interact with thyroid hormone to control biological processes in mammalian cells. The cell culture systems to be investigated are GH1 cells, two clonal cells of rat pituitary cell lines which synthesize growth hormone. With these cells physiological concentrations of thyroid hormones increase growth hormone synthesis and the effect of glucocorticoids on regulating growth hormone synthesis is highly dependent on the action of thyroid hormone. These cells contain nuclear receptors for thyroid hormone and also contain glucocorticoid receptors and the regulation of the synthesis of growth hormone by thyroid hormone and glucocorticoid appears to be modulated by their respective regular receptors. In addition, GH1 cells and GC cells contain insulin receptors with an estimated Kd of 2-5 x 10 to the minus 10th power M with approximately 8,000 insulin binding sites per cell. In GC cells thyroid hormone and glucocorticoid induce increases in insulin receptor levels and the influence of glucocorticoid is additive but not synergistic with that of thyroid hormone differing from the regulation of the growth hormone response. Furthermore, thyroid hormone and glucocorticoid independently inhibit insulin degradation by these cells by a process which appears to involve a decrease in cell uptake of insulin and therefore the inhibition of insulin degradation appears to be modulated by the influence of thyroid hormone and or glucocorticoid on inhibiting insulin internalization. The mechanism of inhibition of insulin metabolism in GH1 cells will be investigated by assessing the kinetics of internalization of insulin and the kinetics of internalization of surface membrane insulin receptors. This will be determined from a biochemical point of view as well as by direct visualization using fluorescent insulin derivitives by Video Intensification Microscopy to monitor the kinetics of internalization. The influence of insuin action and internalization of these cells will be assessed in terms of the influence of insulin on controlling the growth hormone response in concert with thyroid and glucocorticoid hormones.

Abstract :

The proposed research involves defining the detailed mechanisms by which glucocorticoids and other hormones interact with thyroid hormone to control biological processes in mammalian cells. The cell culture systems to be investigated are GH1 cells, two clonal cells of rat pituitary cell lines which synthesize growth hormone. With these cells physiological concentrations of thyroid hormones increase growth hormone synthesis and the effect of glucocorticoids on regulating growth hormone synthesis is highly dependent on the action of thyroid hormone. These cells contain nuclear receptors for thyroid hormone and also contain glucocorticoid receptors and the regulation of the synthesis of growth hormone by thyroid hormone and glucocorticoid appears to be modulated by their respective regular receptors. In addition, GH1 cells and GC cells contain insulin receptors with an estimated Kd of 2-5 x 10 to the minus 10th power M with approximately 8,000 insulin binding sites per cell. In GC cells thyroid hormone and glucocorticoid induce increases in insulin receptor levels and the influence of glucocorticoid is additive but not synergistic with that of thyroid hormone differing from the regulation of the growth hormone response. Furthermore, thyroid hormone and glucocorticoid independently inhibit insulin degradation by these cells by a process which appears to involve a decrease in cell uptake of insulin and therefore the inhibition of insulin degradation appears to be modulated by the influence of thyroid hormone and or glucocorticoid on inhibiting insulin internalization. The mechanism of inhibition of insulin metabolism in GH1 cells will be investigated by assessing the kinetics of internalization of insulin and the kinetics of internalization of surface membrane insulin receptors. This will be determined from a biochemical point of view as well as by direct visualization using fluorescent insulin derivitives by Video Intensification Microscopy to monitor the kinetics of internalization. The influence of insuin action and internalization of these cells will be assessed in terms of the influence of insulin on controlling the growth hormone response in concert with thyroid and glucocorticoid hormones.

Abstract :

Thyroid hormone receptors are chromatin associated proteins which can be extracted with 0.4 M KCl, sediment at 3.8 S, and have an estimated molecular weight (Mr) of 54,000. In contrast, at low ionic strength micrococcal nuclease excises the receptors as a precominent 6.5 S form and as a less abundant 12.5 S species which appears to represent mononucleosome associated receptor. The 6.5 S form has an estimated M(r) of 149,000 and density estimates suggest that it is composed of 85% protein (Mr = 127,000) and 15% DNA (Mr = 22,000 or 35 base pairs). This suggests that the 6.5 S receptor is composed of the 54,000 Mr 3.8 S binding component in dimeric form alone or in monomeric form associated with other unique proteins bound to a DNA domain of approximately 35 base pairs. To further explore the interaction of the hormone binding component in chromatin we have synthesized a photoaffinity label derivitive of L-triiodothyronine which binds to nuclear receptors in intact cells (N-2-diazo, 3,3,3-trifluoropropionyl-L-triiodothyronine). UV irradiation at 254 nm followed by SDS-polyacrylamide gel electrophoresis demonstrates an abundant 47,000 Mr receptor form and a less abundant 57,000 Mr species. Both components yield identical 125I-peptide fragments with Staphlacocus aureus V8 protease or trypsin. Proteolytic conversion of the 57,000 to the 47,000 Mr form does not account for the two identified species. Several explanations for the two Mr receptor species are proposed. These include: (1) the UV protein-protein cross-linking of the receptor binding component to a 10,000 Mr component in chromatin; (2) a non-binding subunit which can be covalently linked to the 47,000 Mr species by protein-protein cross-linking; (3) specific conversion of one Mr form to the other either by post-synthetic modification or specific cleavage in the cell nucleus which results in receptor activation; (4) the two M(r) components may be derived from a heterodimer structure as has been reported for the progesterone receptor. Studies in this proposal will utilize the photoaffinity label derivative in conjunction with contact site cross-linking agents to probe the chromatin components with which the receptor interacts. These studies will be performed in cultured GH1 rat somatotroph cells as well as normal and thyroid hormone resistant fibroblasts which contain a normal complement of thyroid hormone nuclear receptors.

Abstract :

Thyroid hormone receptors are chromatin associated proteins which can be extracted with 0.4 M KCl, sediment at 3.8 S, and have an estimated molecular weight (Mr) of 54,000. In contrast, at low ionic strength micrococcal nuclease excises the receptors as a precominent 6.5 S form and as a less abundant 12.5 S species which appears to represent mononucleosome associated receptor. The 6.5 S form has an estimated M(r) of 149,000 and density estimates suggest that it is composed of 85% protein (Mr = 127,000) and 15% DNA (Mr = 22,000 or 35 base pairs). This suggests that the 6.5 S receptor is composed of the 54,000 Mr 3.8 S binding component in dimeric form alone or in monomeric form associated with other unique proteins bound to a DNA domain of approximately 35 base pairs. To further explore the interaction of the hormone binding component in chromatin we have synthesized a photoaffinity label derivitive of L-triiodothyronine which binds to nuclear receptors in intact cells (N-2-diazo, 3,3,3-trifluoropropionyl-L-triiodothyronine). UV irradiation at 254 nm followed by SDS-polyacrylamide gel electrophoresis demonstrates an abundant 47,000 Mr receptor form and a less abundant 57,000 Mr species. Both components yield identical 125I-peptide fragments with Staphlacocus aureus V8 protease or trypsin. Proteolytic conversion of the 57,000 to the 47,000 Mr form does not account for the two identified species. Several explanations for the two Mr receptor species are proposed. These include: (1) the UV protein-protein cross-linking of the receptor binding component to a 10,000 Mr component in chromatin; (2) a non-binding subunit which can be covalently linked to the 47,000 Mr species by protein-protein cross-linking; (3) specific conversion of one Mr form to the other either by post-synthetic modification or specific cleavage in the cell nucleus which results in receptor activation; (4) the two M(r) components may be derived from a heterodimer structure as has been reported for the progesterone receptor. Studies in this proposal will utilize the photoaffinity label derivative in conjunction with contact site cross-linking agents to probe the chromatin components with which the receptor interacts. These studies will be performed in cultured GH1 rat somatotroph cells as well as normal and thyroid hormone resistant fibroblasts which contain a normal complement of thyroid hormone nuclear receptors.

Abstract :

Thyroid hormone receptors are chromatin associated proteins which can be extracted with 0.4 M KCl, sediment at 3.8 S, and have an estimated molecular weight (Mr) of 54,000. In contrast, at low ionic strength micrococcal nuclease excises the receptors as a precominent 6.5 S form and as a less abundant 12.5 S species which appears to represent mononucleosome associated receptor. The 6.5 S form has an estimated M(r) of 149,000 and density estimates suggest that it is composed of 85% protein (Mr = 127,000) and 15% DNA (Mr = 22,000 or 35 base pairs). This suggests that the 6.5 S receptor is composed of the 54,000 Mr 3.8 S binding component in dimeric form alone or in monomeric form associated with other unique proteins bound to a DNA domain of approximately 35 base pairs. To further explore the interaction of the hormone binding component in chromatin we have synthesized a photoaffinity label derivitive of L-triiodothyronine which binds to nuclear receptors in intact cells (N-2-diazo, 3,3,3-trifluoropropionyl-L-triiodothyronine). UV irradiation at 254 nm followed by SDS-polyacrylamide gel electrophoresis demonstrates an abundant 47,000 Mr receptor form and a less abundant 57,000 Mr species. Both components yield identical 125I-peptide fragments with Staphlacocus aureus V8 protease or trypsin. Proteolytic conversion of the 57,000 to the 47,000 Mr form does not account for the two identified species. Several explanations for the two Mr receptor species are proposed. These include: (1) the UV protein-protein cross-linking of the receptor binding component to a 10,000 Mr component in chromatin; (2) a non-binding subunit which can be covalently linked to the 47,000 Mr species by protein-protein cross-linking; (3) specific conversion of one Mr form to the other either by post-synthetic modification or specific cleavage in the cell nucleus which results in receptor activation; (4) the two M(r) components may be derived from a heterodimer structure as has been reported for the progesterone receptor. Studies in this proposal will utilize the photoaffinity label derivative in conjunction with contact site cross-linking agents to probe the chromatin components with which the receptor interacts. These studies will be performed in cultured GH1 rat somatotroph cells as well as normal and thyroid hormone resistant fibroblasts which contain a normal complement of thyroid hormone nuclear receptors.

Abstract :

Using cultured GH1 cells, a growth hormone producing rat pituitary cell line, we have shown that L-triiodothyronine (T3) stimulates an increase in the rate of growth hormone synthesis and mRNA accumulation. This response appears to be mediated by a chromatin associated receptor which can be extracted from nuclei by 0.4 M KCl and sediments in sucrose gradients as a 3.8 S species. In contrast, micrococcal nuclease digestion excises the receptor as a major 6.5 S species and a less abundant 12.5 S form which sediments slightly more rapdily than the bulk of the mononucleosomes generated (11.5 S). In intact cells T3 elicits a time dependent decrease in the level of the receptor which is dependent on the ligand-receptor interaction. In addition, the level of nuclear bounds receptor is inversely related to the extent of acetylation of chromatin associated proteins. We have developed density labeling methods to quantitate nuclear receptor synthesis and degradation and we intend to examine the cellular mechanisms which control steady-state nuclear receptor levels. We also intend to identify whether the receptor exists as a cytoplasmic species which functions as a precursor to the nuclear bound form. We have synthesized an affinity label which covalently links to the receptor binding site. We will use this as an aid in receptor purification and in studying the primary structure of the receptor. Using these receptor preparations we propose to prepare monoclonal antibody using the hybridoma technique for use in further receptor purification and as an additional tool to study the interaction of the receptor in chromatin. We will examine the precursor-product relationship of formation of the 6.5S and 12.5 S receptor species from chromatin and determine whether they are derived from putative transcriptionally active regions of chromatin. Whether the receptor is associated with other proteins in chromatin will be assessed by chemical cross-linking studies. Finally using gene cloning techniques we will examine the mechanism by which T3 increases an accumulation of growth hormone mRNA in GH1 cells.

Abstract :

Using cultured GH1 cells, a growth hormone producing rat pituitary cell line, we have shown that L-triiodothyronine (T3) stimulates an increase in the rate of growth hormone synthesis and mRNA accumulation. This response appears to be mediated by a chromatin associated receptor which can be extracted from nuclei by 0.4 M KCl and sediments in sucrose gradients as a 3.8 S species. In contrast, micrococcal nuclease digestion excises the receptor as a major 6.5 S species and a less abundant 12.5 S form which sediments slightly more rapdily than the bulk of the mononucleosomes generated (11.5 S). In intact cells T3 elicits a time dependent decrease in the level of the receptor which is dependent on the ligand-receptor interaction. In addition, the level of nuclear bounds receptor is inversely related to the extent of acetylation of chromatin associated proteins. We have developed density labeling methods to quantitate nuclear receptor synthesis and degradation and we intend to examine the cellular mechanisms which control steady-state nuclear receptor levels. We also intend to identify whether the receptor exists as a cytoplasmic species which functions as a precursor to the nuclear bound form. We have synthesized an affinity label which covalently links to the receptor binding site. We will use this as an aid in receptor purification and in studying the primary structure of the receptor. Using these receptor preparations we propose to prepare monoclonal antibody using the hybridoma technique for use in further receptor purification and as an additional tool to study the interaction of the receptor in chromatin. We will examine the precursor-product relationship of formation of the 6.5S and 12.5 S receptor species from chromatin and determine whether they are derived from putative transcriptionally active regions of chromatin. Whether the receptor is associated with other proteins in chromatin will be assessed by chemical cross-linking studies. Finally using gene cloning techniques we will examine the mechanism by which T3 increases an accumulation of growth hormone mRNA in GH1 cells.

Abstract :

Using cultured GH1 cells, a growth hormone producing rat pituitary cell line, we have shown that L-triiodothyronine (T3) stimulates an increase in the rate of growth hormone synthesis and mRNA accumulation. This response appears to be mediated by a chromatin associated receptor which can be extracted from nuclei by 0.4 M KCl and sediments in sucrose gradients as a 3.8 S species. In contrast, micrococcal nuclease digestion excises the receptor as a major 6.5 S species and a less abundant 12.5 S form which sediments slightly more rapdily than the bulk of the mononucleosomes generated (11.5 S). In intact cells T3 elicits a time dependent decrease in the level of the receptor which is dependent on the ligand-receptor interaction. In addition, the level of nuclear bounds receptor is inversely related to the extent of acetylation of chromatin associated proteins. We have developed density labeling methods to quantitate nuclear receptor synthesis and degradation and we intend to examine the cellular mechanisms which control steady-state nuclear receptor levels. We also intend to identify whether the receptor exists as a cytoplasmic species which functions as a precursor to the nuclear bound form. We have synthesized an affinity label which covalently links to the receptor binding site. We will use this as an aid in receptor purification and in studying the primary structure of the receptor. Using these receptor preparations we propose to prepare monoclonal antibody using the hybridoma technique for use in further receptor purification and as an additional tool to study the interaction of the receptor in chromatin. We will examine the precursor-product relationship of formation of the 6.5S and 12.5 S receptor species from chromatin and determine whether they are derived from putative transcriptionally active regions of chromatin. Whether the receptor is associated with other proteins in chromatin will be assessed by chemical cross-linking studies. Finally using gene cloning techniques we will examine the mechanism by which T3 increases an accumulation of growth hormone mRNA in GH1 cells.

Abstract :

Using cultured GH1 cells, a growth hormone producing rat pituitary cell line, we have shown that L-triiodothyronine (T3) stimulates an increase in the rate of growth hormone synthesis and mRNA accumulation. This response appears to be mediated by a chromatin associated receptor which can be extracted from nuclei by 0.4 M KCl and sediments in sucrose gradients as a 3.8 S species. In contrast, micrococcal nuclease digestion excises the receptor as a major 6.5 S species and a less abundant 12.5 S form which sediments slightly more rapdily than the bulk of the mononucleosomes generated (11.5 S). In intact cells T3 elicits a time dependent decrease in the level of the receptor which is dependent on the ligand-receptor interaction. In addition, the level of nuclear bounds receptor is inversely related to the extent of acetylation of chromatin associated proteins. We have developed density labeling methods to quantitate nuclear receptor synthesis and degradation and we intend to examine the cellular mechanisms which control steady-state nuclear receptor levels. We also intend to identify whether the receptor exists as a cytoplasmic species which functions as a precursor to the nuclear bound form. We have synthesized an affinity label which covalently links to the receptor binding site. We will use this as an aid in receptor purification and in studying the primary structure of the receptor. Using these receptor preparations we propose to prepare monoclonal antibody using the hybridoma technique for use in further receptor purification and as an additional tool to study the interaction of the receptor in chromatin. We will examine the precursor-product relationship of formation of the 6.5S and 12.5 S receptor species from chromatin and determine whether they are derived from putative transcriptionally active regions of chromatin. Whether the receptor is associated with other proteins in chromatin will be assessed by chemical cross-linking studies. Finally using gene cloning techniques we will examine the mechanism by which T3 increases an accumulation of growth hormone mRNA in GH1 cells.

Abstract :

Using cultured GH1 cells, a growth hormone producing rat pituitary cell line, we have shown that L-triiodothyronine (T3) stimulates an increase in the rate of growth hormone synthesis and mRNA accumulation. This response appears to be mediated by a chromatin associated receptor which can be extracted from nuclei by 0.4 M KCl and sediments in sucrose gradients as a 3.8 S species. In contrast, micrococcal nuclease digestion excises the receptor as a major 6.5 S species and a less abundant 12.5 S form which sediments slightly more rapdily than the bulk of the mononucleosomes generated (11.5 S). In intact cells T3 elicits a time dependent decrease in the level of the receptor which is dependent on the ligand-receptor interaction. In addition, the level of nuclear bounds receptor is inversely related to the extent of acetylation of chromatin associated proteins. We have developed density labeling methods to quantitate nuclear receptor synthesis and degradation and we intend to examine the cellular mechanisms which control steady-state nuclear receptor levels. We also intend to identify whether the receptor exists as a cytoplasmic species which functions as a precursor to the nuclear bound form. We have synthesized an affinity label which covalently links to the receptor binding site. We will use this as an aid in receptor purification and in studying the primary structure of the receptor. Using these receptor preparations we propose to prepare monoclonal antibody using the hybridoma technique for use in further receptor purification and as an additional tool to study the interaction of the receptor in chromatin. We will examine the precursor-product relationship of formation of the 6.5S and 12.5 S receptor species from chromatin and determine whether they are derived from putative transcriptionally active regions of chromatin. Whether the receptor is associated with other proteins in chromatin will be assessed by chemical cross-linking studies. Finally using gene cloning techniques we will examine the mechanism by which T3 increases an accumulation of growth hormone mRNA in GH1 cells.

Abstract :

The proposed research involves defining the detailed mechanisms by which glucocorticoids and other hormones interact with thyroid hormone to control biological processes in mammalian cells. The cell culture systems to be investigated are GH1 cells, two clonal cells of rat pituitary cell lines which synthesize growth hormone. With these cells physiological concentrations of thyroid hormones increase growth hormone synthesis and the effect of glucocorticoids on regulating growth hormone synthesis is highly dependent on the action of thyroid hormone. These cells contain nuclear receptors for thyroid hormone and also contain glucocorticoid receptors and the regulation of the synthesis of growth hormone by thyroid hormone and glucocorticoid appears to be modulated by their respective regular receptors. In addition, GH1 cells and GC cells contain insulin receptors with an estimated Kd of 2-5 x 10 to the minus 10th power M with approximately 8,000 insulin binding sites per cell. In GC cells thyroid hormone and glucocorticoid induce increases in insulin receptor levels and the influence of glucocorticoid is additive but not synergistic with that of thyroid hormone differing from the regulation of the growth hormone response. Furthermore, thyroid hormone and glucocorticoid independently inhibit insulin degradation by these cells by a process which appears to involve a decrease in cell uptake of insulin and therefore the inhibition of insulin degradation appears to be modulated by the influence of thyroid hormone and or glucocorticoid on inhibiting insulin internalization. The mechanism of inhibition of insulin metabolism in GH1 cells will be investigated by assessing the kinetics of internalization of insulin and the kinetics of internalization of surface membrane insulin receptors. This will be determined from a biochemical point of view as well as by direct visualization using fluorescent insulin derivitives by Video Intensification Microscopy to monitor the kinetics of internalization. The influence of insuin action and internalization of these cells will be assessed in terms of the influence of insulin on controlling the growth hormone response in concert with thyroid and glucocorticoid hormones.

Abstract :

The proposed research involves defining the detailed mechanisms by which thyroid and glucocorticoid hormones interrelate to control biologic processes in mammalian cells. A cell culture system has been developed which is responsive to physiological concentrations of thyroid hormones. The cells are GH1 cells, a rat growth hormone and prolactin producing pituitary tumor. Physiological concentrations of thyroid hormones increase cell replication rates and independent of cell growth stimulate a 4-6 fold increase in the rate of growth hormone synthesis. Glucocorticoid hormones only minimal control growth hormone synthesis in the absence of thyroid hormone response. Under these conditions growth hormone represents 2-30% of total cell protein synthesis. These responses appear to be mediated by the thyroid hormone nuclear receptor and the glucocorticoid receptor. The mechanism by which thyroid and glucocorticoid hormone control the same biologic response occurs independently of one hormone modulating the cellular concentration or distribution of the other. The mechanism by which both hormones interact to control the same biologic response will be evaluated by determining how both hormone modulate growth hormone mRNA levels in the cells. In addition, we will characterize the glucocorticoid receptor in GH1 cells and also examine the influence of one hormone on controlling the distribution of the receptor for the other in the fraction of chromatin active in transcription. In addition, we intend to isolate and purify the growth hormone mRNA and develop a cDNA probe to further clarify the detailed mechanism of the induction process by the hormones. It is hoped that these studies will help further clarify the mechanism of thyroid and glucocorticoid hormone action and provide information of multihormonal interrelationships in the regulation of normal and neoplastic mammalian cells.

Abstract :

The proposed research involves defining the detailed mechanisms by which glucocorticoids and other hormones interact with thyroid hormone to control biological processes in mammalian cells. The cell culture systems to be investigated are GH1 cells, two clonal cells of rat pituitary cell lines which synthesize growth hormone. With these cells physiological concentrations of thyroid hormones increase growth hormone synthesis and the effect of glucocorticoids on regulating growth hormone synthesis is highly dependent on the action of thyroid hormone. These cells contain nuclear receptors for thyroid hormone and also contain glucocorticoid receptors and the regulation of the synthesis of growth hormone by thyroid hormone and glucocorticoid appears to be modulated by their respective regular receptors. In addition, GH1 cells and GC cells contain insulin receptors with an estimated Kd of 2-5 x 10 to the minus 10th power M with approximately 8,000 insulin binding sites per cell. In GC cells thyroid hormone and glucocorticoid induce increases in insulin receptor levels and the influence of glucocorticoid is additive but not synergistic with that of thyroid hormone differing from the regulation of the growth hormone response. Furthermore, thyroid hormone and glucocorticoid independently inhibit insulin degradation by these cells by a process which appears to involve a decrease in cell uptake of insulin and therefore the inhibition of insulin degradation appears to be modulated by the influence of thyroid hormone and or glucocorticoid on inhibiting insulin internalization. The mechanism of inhibition of insulin metabolism in GH1 cells will be investigated by assessing the kinetics of internalization of insulin and the kinetics of internalization of surface membrane insulin receptors. This will be determined from a biochemical point of view as well as by direct visualization using fluorescent insulin derivitives by Video Intensification Microscopy to monitor the kinetics of internalization. The influence of insuin action and internalization of these cells will be assessed in terms of the influence of insulin on controlling the growth hormone response in concert with thyroid and glucocorticoid hormones.

Abstract :

The proposed research involves defining the detailed mechanisms by which thyroid hormones regulate biologic processes in mammalian cells. A cell culture system has been developed which is responsive to physiologic concentrations of the thyroid hormones. In this system thyroid hormones induce a three-fold increase in the rate of cell growth, glucose utilization and growth hormone secretion. In addition at identical physiological hormone concentrations prolactin secretion is inhibited in GH1 cell cultures. Studies on the early effects of thyroid hormones on cultured GH1 cells suggest an early effect at the level of DNA transcription. Nuclear receptor sites which appear to mediate the action of the thyroid hormones have been characterized in intact cells, isolated nuclei, and in solubilized nuclear extracts. The nuclear binding activity has characteristics of a non-histone protein and is relatively stable after extracted from nuclei in a soluble form. The initial events in the action of the thyroid hormones will be characterized with respect to early observed changes on nuclear template activity and RNA polymerase. The effect of thyroid hormone with respect to synthesis on transfer RNA, ribosomal RNA and total poly-A rich messenger RNA and hetergenous nuclear RNA will be defined. In addition we hope to purify the nuclear receptor for thyroid hormones and by reconstitution studies with chromatin constituents in vitro attempt to define the role of the receptor in initiating the cellular action of thyroid hormone.

Abstract :

The proposed research involves defining the detailed mechanisms by which thyroid hormones regulate biologic processes in mammalian cells. A Cell Culture system has been developed which is responsive to physiologic concentrations of the thyroid hormones. In this system thyroid hormones induce a three-fold increase in the rate of cell growth, glucose utilization and growth hormone secretion. In addition at identical physiological hormone concentrations prolactin secretion is inhibited in GH1 cell cultures. Studies on the early effects of thyroid hormones on cultured GH1 cells suggest an early effect at the level of DNA transcription. Nuclear receptor sites which appear to mediate the action of the thyroid hormones have been characterized in intact cells, isolated nuclei, and in solubilized nuclear extracts. The nuclear binding activity has characteristics of a non-histone protein and is relatively stable after extracted from nuclei in a soluble form. The initial events in the action of the thyroid hormones will be characterized with respect to early observed changes on nuclear template activity and RNA polymerase. The effect of thyroid hormone with resppct to synthesis on transfer RNA, ribosomal RNA and poly-A rich messenger RNA and hetergenous nuclear RNA will be defined. In addition we hope to purify the nuclear receptor for the thyroid hormones and by reconstitution studies in vitro attempt to define the role of the receptor in regulating nuclear activity. Also with respect to the regulation of a final gene product we also propose to clarify the mechanisms by which thyroid homones regulate growth hormone and prolactin secretion with respect to synthetic and secretory rates and possible alterations in the turnover of these polypeptide hormones. It is hoped that these studies will have the dual effect of clarifying the molecular mechanisms of thyroid hormone action as well as provide basic information concerning the regulatory processes of cell growth and differentiation of normal and neoplastic mammalian cells.

Abstract :

The thyroid hormone receptor is a chromatin associated protein which appears to mediate the action of thyroid hormone in mammalian cells. In cultured GH1 cells, a rat pituitary cell line, the chromatin associated receptor appears to represent a steady state value which is dependent upon receptor synthesis and receptor turnover. In these cells, L-T3, L-T4, and other thyroid hormone analogues elicit a time and dose dependent reduction in nuclear receptor levels which appear to be directly related to their relative affinity for the receptor binding site. In addition, sodium n-butyrate and other aliphatic carboxylic acids elicit a reduction in thyroid hormone nuclear receptor levels without altering total cell protein synthetic rates. This reduction appears to be secondary to an effect of the aliphatic carboxylic acids on inhibiting chromatin associated deacetylases which is reflected as an increase in acetylation of histones H3 and H4 of the nucleosome core. Micrococcal nuclease and DNase 1 digestion studies indicate that the receptor is released as a 6.5 S form which is significantly larger than the form which can be extracted from nuclei by 0.4 M KC1 (3.8 S). Preliminary studies indicate that the 6.5 S is converted to a 3.8 S form by raising the salt concentration but goes through an intermediate 5.0 S form suggesting that there is another protein component(s) with which the receptor associates. Furthermore, unlike the 3.8 S form, the 6.5 S form does not appear to bind to DNA suggesting that the 6.5 S form also reflects receptor associated with the small DNA fragment. In this study we propose to study the biochemical characteristics of the 6.5 S receptor complex released by chromatin digestion to define whether it is associated with the DNA fragment which may reflect with the component with which the receptor normally associates with nuclei and to determine whether other regulatory proteins remain associated with this complex.

Abstract :

The proposed research involves defining the detailed mechanisms by which thyroid hormones regulate biologic processes in mammalian cells. A cell culture system has been developed which is responsive to physiologic concentrations of the thyroid hormones. In this system thyroid hormones induce a three-fold increase in the rate of cell growth, glucose utilization and growth hormone secretion. In addition at identical physiological hormone concentrations prolactin secretion is inhibited in GH l cell cultures. Studies on the early effects of thyroid hormones on cultured GH 1 cells suggest an early effect at the level of DNA transcription. Nuclear receptor sites which appear to mediate the action of the thyroid hormones have been characterized in intact cells, isolated nuclei, and in solubilized nuclear extracts. The nuclear binding activity has characteristics of a non-histone protein and is relatively stable after extracted from nuclei in a soluble form. The initial events in the action of the thyroid hormones will be characterized with respect to early observed changes on nuclear template activity and RNA polymerase. The effect of thyroid hormone with respect to synthesis on transfer RNA, ribosomal RNA and poly-A rich messenger RNA and hetergenous nuclear RNA will be defined. In addition we hope to purify the nuclear receptor for the thyroid hormones and by reconstitution studies in vitro attempt to define the role of the receptor in regulating nuclear activity. Also with respect to the regulation of a final gene product we also propose to clarify the mechanisms by which thyroid hormones regulate growth hormone and prolactin secretion with respect to synthetic and secretory rates and possible alterations in the turnover of these polypeptide hormones. It is hoped that these studies will have the dual effect of clarifying the molecular mechanisms of thyroid hormone action as well as provide basic information concerning the regulatory processes of cell growth and differentiation of normal and neoplastic mammalian cells.

Abstract :

The proposed research involves defining the detailed mechanisms by which thyroid hormones regulate biologic processes in mammalian cells. A cell culture system has been developed which is responsive to physiologic concentrations of the thyroid hormones. In this system thyroid hormones induce a three-fold increase in the rate of cell growth, glucose utilization and growth hormone secretion. In addition at identical physiological hormone concentrations prolactin secretion is inhibited in GH1 cell cultures. Studies on the early effects of thyroid hormones on cultured GH1 cells suggest an early effect at the level of DNA transcription. Nuclear receptor sites which appear to mediate the action of the thyroid hormones have been characterized in intact cells, isolated nuclei, and in solubilized nuclear extracts. The nuclear binding activity has characteristics of a non-histone protein and is relatively stable after extracted from nuclei in a soluble form. The initial events in the action of the thyroid hormones will be characterized with respect to early observed changes on nuclear template activity and RNA polymerase. The effect of thyroid hormone with respect to synthesis on transfer RNA, ribosomal RNA and total poly-A rich messenger RNA and hetergenous nuclear RNA will be defined. In addition we hope to purify the nuclear receptor for thyroid hormones and by reconstitution studies with chromatin constituents in vitro attempt to define the role of the receptor in initiating the cellular action of thyroid hormone. BIBLIOGRAPHIC REFERENCE: Samuels, H.H., j. Casanova, R. Cintron, L. Shapiro, F. Stanley and J. Tsai. Molecular Biology of Thyroid Hormone Action. Excerpta Medica. V International Congress of Endocrinology, Hamburg, Germany, In Press. 1977.

Abstract :

The proposed research involves defining the detailed mechanisms by which thyroid hormones regulate biologic processes in mammalian cells. A cell culture system has been developed which is responsive to physiologic concentrations of the thyroid hormones. In this system thyroid hormones induce a three-fold increase in the rate of cell growth, glucose utilization and growth hormone secretion. In addition at identical physiological hormone concentrations prolactin secretion is inhibited in GH1 cell cultures. Studies on the early effects of thyroid hormones on cultured GH1 cells suggest an early effect at the level of DNA transcription. Nuclear receptor sites which appear to mediate the action of the thyroid hormones have been characterized in intact cells, isolated nuclei, and in solubilized nuclear extracts. The nuclear binding activity has characteristics of a non-histone protein and is relatively stable after extracted from nuclei in a soluble form. The initial events in the action of the thyroid hormones will be characterized with respect to early observed changes on nuclear template activity and RNA polymerase. The effect of thyroid hormone with respect to synthesis on transfer RNA, ribosomal RNA and poly-A rich messenger RNA and heterogenous nuclear RNA will be defined. In addition we hope to purify the nuclear receptor for the thyroid hormones and by reconstitution studies in vitro attempt to define the role of the receptor in regulating nuclear activity. Also with respect to the regulation of a final gene product we also propose to clarify the mechanisms by which thyroid hormones regulate growth hormone and prolactin secretion with respect to synthetic and secretory rates and possible alterations in the turnover of these polypeptide hormones. It is hoped that these studies will have the dual effect of clarifying the molecular mechanisms of thyroid hormone action as well as provide basis information concerning the nature of regulatory processes in mammalian cells.

Abstract :

This proposed research involves a long term study of the metabolic effects of the thyroid hormones on cells in culture. A cell culture system has been developed which is sensitive to the physiologic and pathologic concentrations of thyroxine (T4) and triiodothyronine (T3) determined in man. Iodothyronines inactive in animals and man are also inactive in this system. In the cell culture T4 and T3 induce a maximal 3 to 4-fold increase in glucose utilization, oxygen consumption, carbon dioxide production and cell growth. Associated with these findings is an increase in uridine transport and/or phosphorylation which can be demonstrated within several hours after the addition of T4 or T3. The mechanism of action of the thyroid hormones will be studied with regard to the temporal relation of these observations with particular respect to transcriptional and translational control mechanisms. This research will also include a study of the role of specific hormone receptor sites, as well as the necessity of the conversion of T4 to T3 as a requirement for the biologic action of T4. In addition, these changes will be investigated with regard to a possible role of 3', 5': cyclicAMP. It is hoped that these studies will have the dual effect of clarifying the molecular mechanism involved in the action of the thyroid hormones as well as providing basic information concerning the nature of the regulatory mechanisms involved in cell growth.