1975 — 1979 |
Ji, Tae |
N/AActivity Code Description: No activity code was retrieved: click on the grant title for more information |
Interaction and Organization of Glycoproteins of Cell Membranes |
1 |
1985 — 1986 |
Ji, Tae H. |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Nature of Follitropin Binding and Its Receptor
Long term goals of this proposed study concerns the biochemical endocrinology of gonadotropins. The immediate objective is to determine which subunit of follitropin binds to the hormone receptor and identify and characterize the follitropin receptor in the following steps. 1) To prepare photoaffinity derivatives fo follitropin. 2) To test the binding activity and steroidogenesis of the follitropin derivatives. 3) To covalently label the hormone receptor with the follitropin derivatives. 4) To analyze photoaffinity labeled receptor-follitropin complexes by SDS-PAGE, gel permeation and centrifugation. 5) Characterize receptor components of the complexes by proteolytic digestions, immunoprecipitation.
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0.958 |
1985 — 1987 |
Ji, Tae H. |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
The Nature of Thyrotropin Binding and Its Receptor
The overall goal of this research program concerns the biochemical endocrinology of thyrotropin (TSH). The immediate objectives are to determine a) the structure of the TSH receptor and b) which subunit(s) of the TSH interacts with the receptor. This will be attempted primarily by photoaffinity-labeling and immunoprecipitation of the receptor, employing radioactive photoactivable heterobifunctional reagents (cleavable or noncleavable), cleavable homobifunctional reagents and monoclonal anti TSH receptor antibodies as follows: 1. Preparation of various photoaffinity derivatives of bovine TSH. 2. Characterization of the derivatives. 3. Photoaffinity-labeling or crosslinking of the receptor. 4. Characterization of the labeled receptor. 5. Preparation of monoclonal anti TSH receptor antibodies. 6. Immunoprecipitation of the TSH receptor.
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0.958 |
1985 — 1991 |
Ji, Tae H. |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
The Structure and Function of the Lutropin Receptor
Synthesis and characterization of cleavable as well as noncleavable photosensitive heterobifunctional reagents of maximum linking length up to 25 A; the photosensitive group will be phenylazides and N-hydroxysuccinimide or imidate will be the conventional functional groups. Several sulfhydryl specific reagents are also planned. Cleavable reagents will be prepared initially with a disulfide bond, and its position in reagents will be varied in order to increase its stability against potential disulfide exchanges. To synthesize cleavable photosensitive heterobifunctional reagents carrying radioisotopes, in particular at the photosensitive half of the reagents, and to establish ways to label surface receptors with or without permanently crosslinking hormones and receptors. To attach reagents to luteinizing hormone and to characterize activated molecules. To bind the activated hormone to granulosa cells and to identify binding molecules. To determine if binding molecules are receptors.
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0.958 |
1987 |
Ji, Tae H. |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
The Structure and Functions of the Lutropin Receptor
The goal is to determine the structure of the LH/hCG receptor and the nature of its interactions with the hormone and associated molecules including adenylate cyclase. We will pursue the following specific objectives: 1. Micropurification and characterization of receptor subunits (peptide maps, finger-prints, amino acid sequence and ADP-ribosylation). 2. Determination of receptor subunit organization (cytoplasmic and extracellular domains, neighboring subunits, location of subunit crosslinks, phosporylation, fatty acylation and the presence of carbohydrates). 3. Study of hormone-receptor interactions; sites in the hormone crosslinked to the receptor, the preference and the order of the affinity labeling of receptor subunits by each of the hormone (LH, hCG and deglycosylated hCG) subunits. 4. Study of interactions of the receptor with the adenylate cyclase system. 5. Further preparation of monoclaonal antibodies to the receptor.
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0.958 |
1991 — 1993 |
Ji, Tae H. |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Identification of Lh-Receptor Gene Regulatory Sequences
The long term goal of this proposed research is to elucidate the regulation of the rat LH receptor gene. The immediate objectives are to define the promoter region and to identify regulatory elements in this gene. Evidence for the critical roles of LH, hCG and their receptor in female reproductive physiology is compelling. A failure in expression of these hormones or the receptor will results in no ovulation and no pregnancy, leading to infertility. Transcription of the LH receptor gene can be induced by FSH or cAMP/estrogen and the expressed receptor can be suppressed by LH/hCG. Genes that are not only inducible but also suppressible are interesting as is the LH receptor gene. because they are likely to be involved in the switching mechanisms of important biological processes. Studies on these hormone-dependent inducibility and suppressibility could reveal novel enhancer and suppressor elements and will serve as a model for other protein hormone receptors. Understanding transcriptional control necessitates the identification of regulatory elements. Our preliminary study suggests that [1] a single gene encodes the rat LH receptor, [2] 11 exons are present In the coding region, [3] there are a sequence similar to the ERE. an exact and several homologous AP-2, and a G-rich CRE. but no consensus CRE, and [4] there are variant receptors that appear to be produced by alternate splicing. These results serve as the basis for proposed studies concerning the following major goals. 1. We will determine the size and restriction map of the entire gene, the transcription start-sites and the boundaries of an apparent intron in the 5'UTR. 2. The cAMP- and estradiol-responsive enhancer elements and LH-responsive suppressor elements in the rat LH-R gene will be identified and verified. 3. We would like to determine whether FSH- or cAMP/estradiol-induction of LH receptor transcription is direct or requires de novo protein synthesis. 4. We plan to characterize variant mRNAs which expect to produce a soluble form of receptors without a transmembrane domain. These soluble receptors may play important roles in the regulation of the hormone concentration in serum.
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0.958 |
1992 — 1996 |
Ji, Tae H. |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Interactions of the Lutropin Receptor
The LH receptor (LH-R) is a member of the G-protein-coupled receptor family and comprises an extracellular N-terminal half and a membrane- associated C-terminal half of a similar size The long-term goal of this project is to define the structural basis of hormone-dependent LH receptor-modulation and subsequent G-protein-activation. Hormonal responses are observed only after the hormone interacts with the receptor. Understanding how the receptor-modulation induces signal transduction necessitates the elucidation of hormone-receptor interactions and the determination of contact sites between the hormone and the receptor. The immediate objective is to determine these contact sites and to understand hCG-receptor interactions. The results of our studies suggest the presence of one or more hormone- receptor contact sites in each of hCGalpha, hCGbeta, the N-terminal half of LH-R and the C-terminal half of LH-R. In the HCGALPHA subunit, the C- terminal 4AAs (-Tyr-His-Lys-Ser) are essential for receptor-binding. AlphaPeptide (-Tyr-His-Lys-Ser) binds to the C-terminal half and stimulates CAMP synthesis and, particularly, AlphaLys91 is important for CAMP inducibility. This 4 amino acid peptide appears to have two sites, one for a receptor-contacting and the other for stimulation of CAMP synthesis. These and other contacts sites between HCG and LH-R will be determined in this study. To facilitate this study, we have prepared the wild type and various hCGs, LH-Rs, the C-terminal half of LH-R and the N-terminal half of LH-R (some naturally occurring soluble receptors). These hormones and receptors are expressed in stably transfected cell lines. For large quantity preparations, these CDNAS have been constructed in Vaculovirus and will be expressed in new more efficient SF21 cells. In addition, a number of HCG alpha peptides and LH-R peptides are available and antibodies against these peptides are being prepared. By using hormone binding and CAMP assays as well as affinity-labeling, we are at a breakthrough point to determine hormone-receptor contact sites and to identify specific amino acid residues in these sites. In addition, we hope to define the relationship of the high affinity HCG site in the N- terminal half and the low affinity HCG site in the C-terminal half of LH-R as well as the molecular mechanisms of HCG-dependent receptor- modulation to activate G-proteins. These results will serve as a model for not only other glycoproteins and their receptors but also bioactive peptides such as substance P and their receptors.
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0.958 |
1996 — 1997 |
Johnson, Jerry Ji, Tae Roth, Don (co-PI) [⬀] Petersen, Nancy Thorsness, Peter (co-PI) [⬀] |
N/AActivity Code Description: No activity code was retrieved: click on the grant title for more information |
A Phosphorimager For Molecular Biology Research and Teaching
This request is for funds to purchase a Molecular Dynamics PhosphorImager for use in research and teaching in molecular biology. This instrument will be critical for research applications of at least eight faculty as well as in training undergraduate and graduate students in molecular biology. Autoradiography using storage phosphor technology offers several advantages over traditional detection methods. Quantitation of amounts of radioactive protein or DNA on gels or filters has traditionally been done using X- ray film and densitometry or using scintillation counters. Storage phosphors are up to 100 times more sensitive than X-ray film, enabling measurements well below levels of detection for film. Also, screens are quantitatively accurate over five orders of magnitude as compared to X-ray film which has a linear range over only two orders of magnitude. The new technology is also environmentally advantageous because it eliminates the heavy metal waste disposal problems associated with film processing, and when it replaces scintillation counting it reduces mixed organic and radiochemical waste disposal problems. This technology will enable new experimental approaches, improve quantitation, enhance productivity, and it will afford our students state of the art training.
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1 |
1996 — 2003 |
Ji, Tae H. |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Interactions and Activation of the Fsh Receptor
The follicle stimulating hormone receptor (FSHR) and FSH play crucial roles in reproduction of mammal. Mutant FSHR and FSH are involved in fertility disorders, while FSHR and FSH are thought to be associated with ovarian and prostate cancers. Our long term goal is to understand the molecular mechanisms of FSHR's activities, which necessitates the study of interaction between FSHR and FSH and subsequent generation of signals for second messengers, cAMP and inositol phosphates. FSHR and other glycoprotein hormone receptors belong to a structurally unique subfamily of G protein-coupled receptors. They consist of the extracellular N-terminal half with approximately 350 amino acids (exodomain) and the membrane associated C-terminal half with an equal number of amino acids (endodomain). Since we and others reported, nearly ten years ago, that the exodomain is capable of high affinity hormone binding and signal generation, that the exodomain is the only high affinity binding site, and that the hormone/exodomain complex undergoes a conformation adjustment. Based on these observations we proposed that the hormone/exodomain complex interacts with the endodomain and this secondary interaction is responsible for signal generation. Lately, many of these have been verified and adopted by others. During the past grant period, we have reported a number of novel observations that hormone binding and signal generation of FSHR are distinct events, that exoloops in the endodomain constrain the FISH binding to the exodomain, and that FSH binding and cAMP induction work against each other and are compromised to maintain both activities at reasonable levels. Our preliminary findings suggest that the signals for cAMP and inositol phosphates are distinct, that the signals for inositol monophosphate, inositol biophosphate, and inositol triphosphate are also distinct, and that the putative Leu Rich Repeat motif in the exodomain is in need involved in FSH binding. Finally, we have found that the exodomain of one FSHR intermolecularly interacts with the endodomain of another FSHR, which has a significant implication in the understanding for the mechanism of multiple signal generation by not only FSHR but also other G protein coupled receptors. We propose to extend our observations (1) to determine the roles of individual amino acids in exoloops on hormone binding and induction of cAMP and three IP species, (2) to identify the contact points among the hormone, exodomain and endodomain, and (3) to conclusively demonstrate the intermolecular interaction of exodomain and endodomain and to examine the mechanisms. When successfully carried out, these studies will have far reaching consequences in understanding the underlying mechanisms of multiple signal generations by a FSHR as well as other receptors have significant implications in clinical and industrial applications.
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0.958 |
1997 — 2005 |
Ji, Tae H. |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Interaction and Activation of the Lh Receptor
DESCRIPTION (adapted from applicant's abstract): This application seeks to continue dissection of structure-function relationships of the interaction between LH/hCG and the LH/hCG receptor. Aim 1 concerns analysis of sites on the hormone that contact and activate the receptor, while Aim 2 examines the receptor for sites that contact the hormone. Aim 3 deals with analysis of receptor domains responsible for delivery of the activation signal to either cAMP or IP signalling pathways. The goal is to define, at the amino acid level, the chemical processes by which hCG binds and activates its receptor, leading to activation of two distinct pathways.
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0.958 |
2005 — 2006 |
Ji, Tae H. |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Intermolecular Activation of Gpcr
DESCRIPTION (provided by applicant): G protein coupled receptors (GPCRs) are the major hormone receptors, constituting approximately 3 percent of the human genome. It has been a dogma that, when a hormone1 binds to a GPCR, the liganded receptor activates itself to generate hormone signals (cis-activation). In contrast to this dogmatic cis-activation, the evidence is emerging that a liganded receptor molecule can intermolecularly activate nonliganded receptor molecules (trans-activation). There are only several reports on trans-activation of GPCRs: two on the thrombin receptors and our reports on the LH receptor (LHR). LHR consists of two equal halves, the extracellular N-terminal exo-domain and the membrane associated heptahelical endo-domain. Hormone binds to the exo-domain, whereas the endo-domain generates two distinct hormone signals: one for adenylyl cyclase to produce cAMP (cAMP signal) and the other for phospholipase cbeta to produce diacylglycerol and inositol phosphate (IP signal). Trans-activation is difficult to test, because it has to be differentiated from cis-activation and hormone binding needs to be distinguished from signal generation. LHR meets these requirements and offers a unique model to test trans-activation. We have established a large library of two mutant groups, one deficient in hormone binding and the other deficient in signal generation. Co-expression of a binding deficient mutant and a signal deficient mutant in a cell rescues signal generation. Our preliminary data show that the trans-activation generates either the cAMP signal or IP signal, but not both. Aim 1 will test the hypotheses that trans-activation occurs not only to the mutant receptor pairs but also to the wild type receptor and that the signal selection is specific. Aim 2 will define the mechanistics of the trans-activation and signal selection. These new paradigms will have broad implications on the mechanisms of GPCR signal generation and development of new receptor therapeutics, particularly to control a specific signal without invoking other signals. Receptors generate multiple signals, which is a source of undesirable/toxic side effects of hormone therapeutics.
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0.922 |
2007 — 2008 |
Ji, Tae H. |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Intermolecular Activation of G Protein Coupled Receptors
DESCRIPTION (provided by applicant): G protein coupled receptors (GPCRs) are the major hormone receptors, constituting approximately 3 percent of the human genome. It has been a dogma that, when a hormone1 binds to a GPCR, the liganded receptor activates itself to generate hormone signals (cis-activation). In contrast to this dogmatic cis-activation, the evidence is emerging that a liganded receptor molecule can intermolecularly activate nonliganded receptor molecules (trans-activation). There are only several reports on trans-activation of GPCRs: two on the thrombin receptors and our reports on the LH receptor (LHR). LHR consists of two equal halves, the extracellular N-terminal exo-domain and the membrane associated heptahelical endo-domain. Hormone binds to the exo-domain, whereas the endo-domain generates two distinct hormone signals: one for adenylyl cyclase to produce cAMP (cAMP signal) and the other for phospholipase cbeta to produce diacylglycerol and inositol phosphate (IP signal). Trans-activation is difficult to test, because it has to be differentiated from cis-activation and hormone binding needs to be distinguished from signal generation. LHR meets these requirements and offers a unique model to test trans-activation. We have established a large library of two mutant groups, one deficient in hormone binding and the other deficient in signal generation. Co-expression of a binding deficient mutant and a signal deficient mutant in a cell rescues signal generation. Our preliminary data show that the trans-activation generates either the cAMP signal or IP signal, but not both. Aim 1 will test the hypotheses that trans-activation occurs not only to the mutant receptor pairs but also to the wild type receptor and that the signal selection is specific. Aim 2 will define the mechanistics of the trans-activation and signal selection. These new paradigms will have broad implications on the mechanisms of GPCR signal generation and development of new receptor therapeutics, particularly to control a specific signal without invoking other signals. Receptors generate multiple signals, which is a source of undesirable/toxic side effects of hormone therapeutics.
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0.922 |