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.
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