LH and FSH receptors belong to a sub-family of G protein-coupled receptors with seven transmembrane regions and a large extracellular domain containing leucine-rich repeats. Our analysis of human gonadotropin receptors has allowed characterization of gain- and loss-of- function mutations in patients with familial male precocious puberty and Leydig cell hypoplasia, respectively. Also, coexpression of two receptors defective in either ligand binding or signal transduction restores ligand signaling. The human receptors, unlike their rodent counterparts. have unique species-specific ligand recognition properties. Earlier expression of extracellular regions of gonadotropin receptors indicated that this domain retains ligand binding ability, but remains trapped inside transfected cells. This phenomenon hampers analysis of their role in signal transduction and prevents the we of them as hormone-specific binding proteins. We recently demonstrated that the extracellular region of LH and FSH receptors can be anchored to the plasma membrane with retention of ligand binding capacity. We now propose to characterize the anchored gonadotropin receptors and the mechanisms underlying receptor activation following co-transfection of anchored receptors with mutant receptors containing the signal transduction domain. Using anchored receptors, we will determine minimal peptide sequences and N-linked carbohydrates needed for ligand recognition. We further inserted a thrombin cleavage site in anchored receptors to allow specific enzyme cleavage and generation of soluble receptor fragments capable of competition in the radioligand receptor assay. We will test the ability of the soluble, ligand binding domain of these receptors to specifically neutralize LH or FSH actions in vitro. Using a Baculovirus expression system, we propose to obtain large amounts-of soluble LH and FSH receptor fragments for testing as receptor antagonists or binding proteins in vivo. The present proposal will further our understanding of the mechanism by which gonadotropins activate their receptors and should yield truncated receptors capable of selectively antagonizing the actions of circulating LH and FSH in animals and patients.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
2R37HD023273-11
Application #
2471427
Study Section
Reproductive Endocrinology Study Section (REN)
Project Start
1997-04-01
Project End
2002-03-31
Budget Start
1997-04-01
Budget End
1998-03-31
Support Year
11
Fiscal Year
1997
Total Cost
Indirect Cost
Name
Stanford University
Department
Obstetrics & Gynecology
Type
Schools of Medicine
DUNS #
800771545
City
Stanford
State
CA
Country
United States
Zip Code
94305
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Ben-Shlomo, Izhar; Hsueh, Aaron J W (2005) Three's company: two or more unrelated receptors pair with the same ligand. Mol Endocrinol 19:1097-109
Luo, Ching-Wei; Pisarska, Margareta D; Hsueh, Aaron J W (2005) Identification of a stanniocalcin paralog, stanniocalcin-2, in fish and the paracrine actions of stanniocalcin-2 in the mammalian ovary. Endocrinology 146:469-76
Sudo, Satoko; Kuwabara, Yoshimitsu; Park, Jae-Il et al. (2005) Heterodimeric fly glycoprotein hormone-alpha2 (GPA2) and glycoprotein hormone-beta5 (GPB5) activate fly leucine-rich repeat-containing G protein-coupled receptor-1 (DLGR1) and stimulation of human thyrotropin receptors by chimeric fly GPA2 and human GPB5. Endocrinology 146:3596-604
Morita, Hiroki; Mazerbourg, Sabine; Bouley, Donna M et al. (2004) Neonatal lethality of LGR5 null mice is associated with ankyloglossia and gastrointestinal distension. Mol Cell Biol 24:9736-43
Luo, Ching-Wei; Kawamura, Kazuhiro; Klein, Cynthia et al. (2004) Paracrine regulation of ovarian granulosa cell differentiation by stanniocalcin (STC) 1: mediation through specific STC1 receptors. Mol Endocrinol 18:2085-96
Mazerbourg, Sabine; Bouley, Donna M; Sudo, Satoko et al. (2004) Leucine-rich repeat-containing, G protein-coupled receptor 4 null mice exhibit intrauterine growth retardation associated with embryonic and perinatal lethality. Mol Endocrinol 18:2241-54
Hsu, Sheau Yu Teddy; Nakabayashi, Koji; Nishi, Shinya et al. (2003) Relaxin signaling in reproductive tissues. Mol Cell Endocrinol 202:165-70
Sudo, Satoko; Kumagai, Jin; Nishi, Shinya et al. (2003) H3 relaxin is a specific ligand for LGR7 and activates the receptor by interacting with both the ectodomain and the exoloop 2. J Biol Chem 278:7855-62
Nakabayashi, Koji; Matsumi, Hirotaka; Bhalla, Alka et al. (2002) Thyrostimulin, a heterodimer of two new human glycoprotein hormone subunits, activates the thyroid-stimulating hormone receptor. J Clin Invest 109:1445-52

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