The overall goal of this project is to gain an understanding at the molecular level of the mechanisms by which LH and prostaglandins act in the ovary. Currently, it centers more of LH than prostaglandins, specifically on the elucidation of structure- function relationships of the receptors for LH and FSH. Using as a base a newly developed cloning technique that involves the construction of genomic libraries in an eukaryotic cell line (mouse L cells) followed by functional screening of stably transfected cells for appearance of LH and FSH receptors as seen by their ability to stimulate adenylyl cyclase, and using an already cloned Gs-stimulating receptor as a model system, we proposed: 1. To clone the cDNAs for the LH and FSH receptors, deduce from them the predicted amino acid sequence, and explore their various functional domains as seen: 1) on caparison with other non-peptide hormone receptors that stimulate Gs. 2) on molecular engineering and construction of chimeras between these receptors and receptors with other hormonal and/or G protein specificity, so as to deduce the exact domains responsible for ligand interaction, G protein interaction, desensitization and down regulation (internalization). 2. To study the genomic organization of the LH and FSH receptor genes, and determine for the LH receptor the molecular basis for the different behavior of follicular and luteal LH receptors: is it due to existence of two (or more) LH receptor genes?, to different post-translational modifications?, or to a change in the quality of the Gs protein that couples the receptor to the effector function(s)? 3. To determine the biological importance of LB receptor desensitization and down-regulation for the luteinizing and luteolytic effects of LH as studied in transgenic mice that express LH receptors engineered to be desensitization- and/or down-regula- tion-negative. Likewise, we propose to determine the molecular basis for the resistance of FSH receptors to FSH-induced down regulation, and whether these receptors have activities other than stimulation of Gs, e.g., increased phosphoinositide turnover.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Research Project (R01)
Project #
5R01HD009581-13
Application #
3311107
Study Section
Biochemical Endocrinology Study Section (BCE)
Project Start
1976-06-30
Project End
1992-03-31
Budget Start
1990-04-01
Budget End
1991-03-31
Support Year
13
Fiscal Year
1990
Total Cost
Indirect Cost
Name
Baylor College of Medicine
Department
Type
Schools of Medicine
DUNS #
074615394
City
Houston
State
TX
Country
United States
Zip Code
77030
Gudermann, T; Levy, F O; Birnbaumer, M et al. (1993) Human S31 serotonin receptor clone encodes a 5-hydroxytryptamine1E-like serotonin receptor. Mol Pharmacol 43:412-8
Zhu, X; Gudermann, T; Birnbaumer, M et al. (1993) A luteinizing hormone receptor with a severely truncated cytoplasmic tail (LHR-ct628) desensitizes to the same degree as the full-length receptor. J Biol Chem 268:1723-8
Themmen, A P; Hinrichs, V; Birnbaumer, M (1993) In situ assay of hormone-stimulated adenylyl cyclase in 96-well microtitration plates: an aide to rapid identification of transformed cell clones. J Recept Res 13:69-78
Levy, F O; Gudermann, T; Birnbaumer, M et al. (1992) Molecular cloning of a human gene (S31) encoding a novel serotonin receptor mediating inhibition of adenylyl cyclase. FEBS Lett 296:201-6
Graf, R; Mattera, R; Codina, J et al. (1992) A truncated recombinant alpha subunit of Gi3 with a reduced affinity for beta gamma dimers and altered guanosine 5'-3-O-(thio)triphosphate binding. J Biol Chem 267:24307-14
Levy, F O; Gudermann, T; Perez-Reyes, E et al. (1992) Molecular cloning of a human serotonin receptor (S12) with a pharmacological profile resembling that of the 5-HT1D subtype. J Biol Chem 267:7553-62
Gudermann, T; Birnbaumer, M; Birnbaumer, L (1992) Evidence for dual coupling of the murine luteinizing hormone receptor to adenylyl cyclase and phosphoinositide breakdown and Ca2+ mobilization. Studies with the cloned murine luteinizing hormone receptor expressed in L cells. J Biol Chem 267:4479-88
Graf, R; Mattera, R; Codina, J et al. (1992) Studies on the interaction of alpha subunits of GTP-binding proteins with beta gamma dimers. Eur J Biochem 210:609-19
Birnbaumer, L; Perez-Reyes, E; Bertrand, P et al. (1991) Molecular diversity and function of G proteins and calcium channels. Biol Reprod 44:207-24
Codina, J; Grenet, D; Chang, K J et al. (1991) Urea gradient/SDS-PAGE;a useful tool in the investigation of signal transducing G proteins. J Recept Res 11:587-601

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