A single amino acid substitution in transmembrane helix VI results in overexpression of the human GnRH receptor

Abstract

Objective: Construction of constitutively active mutants of the GnRH receptor, a member of the Gprotein coupled receptor superfamily, would facilitate investigation of the mechanism of receptor activation. Design: Point mutations were introduced in the human GnRH receptor in positions corresponding to those which caused constitutive activity in other G-protein coupled receptors. The effects of these mutations on ligand binding, receptor intracellular signaling and receptor expression were determined. Methods: Wild type and mutated receptor cDNAs were expressed in COS-1 cells. Basal and agoniststimulated inositol phosphate production and ligand binding were determined. In addition, receptor mRNA levels, cell surface receptor stability and rate of internalization were measured. Results and conclusions: Although none of the mutant receptors exhibited constitutive activity, mutation of Phe-272 in transmembrane helix VI to Leu increased cell surface receptor numbers, with unchanged affinities for radiolabeled agonist, superagonist and antagonist peptides compared with wild type receptor. The cell surface receptor stability and rate of internalization were similar for wild type and F272L GnRH receptors. Thus a single amino acid mutation in transmembrane helix VI causes an increase in cell surface receptor numbers, which appears to result from an increased rate of receptor protein translation, processing or insertion into membranes.

Mutations Remote from the Human Gonadotropin-releasing Hormone (GnRH) Receptor-binding Sites Specifically Increase Binding Affinity for GnRH II but Not GnRH I

“…However, no constitutive activation of PhI turnover was observed in any of the mutant GnRH receptors, suggesting that the function of these residues in the human GnRH receptor may be not identical to that of other GPCRs, in which mutations do lead to constitutive activation. The human GnRH receptor may also be strongly constrained in the inactive state (27). Disruption of part of the intramolecular constraint network may not be sufficient to cause a significant agonist-independent shift of the receptor conformational equilibria to the active state that couples to G q , but it could nevertheless influence ligand-induced receptor conformational changes (Fig.…”

“…In contrast, none of the mutation sites that convey constitutive activity in a large number of GPCRs induced this effect in the GnRH receptor. These included mutations of Phe 272 (6.40) and Phe 276 (6.44) in the human GnRH receptor (27). However, there was increased receptor expression of the F272L mutant indicating a subtle conformational change of the receptor.…”

Twinning and heteropaternity in chimpanzees (Pan troglodytes)

“…Genetic differences in hormones, receptors, or patterns of gene expression (Arora et al, 1999;Liu et al, 2001;Myburgh et al, 1998;Karaman et al, 2003) could underlie species differences in twinning rates, could have farreaching effects in chimpanzee biology, and possibly played a role in the divergence of chimpanzees and humans from their common ancestor. The tenets that twinning is restricted to only a few pedigrees (Geissman, 1990) or is an artifact of captivity (Peacock and Rogers, 1959) are not supported by the evidence.…”

Complementary Deoxyribonucleic Acid Cloning, Gene Expression, and Ligand Selectivity of a Novel Gonadotropin-Releasing Hormone Receptor Expressed in the Pituitary and Midbrain of Xenopus laevis*

“…This motif and the conserved isoleucine residue that occurs two amino acid residues further downstream (Ile 143 in the human GnRH receptor and Ile 136 in XLGnRH-R I) have been found to be important in ligand-induced receptor activation and G protein coupling (44,45). Ala 261 in intracellular loop III of the human GnRH receptor, important for G protein coupling and receptor internalization is also conserved (Ala 247 in XLGnRH-R I) (47). Ala 261 in intracellular loop III of the human GnRH receptor, important for G protein coupling and receptor internalization is also conserved (Ala 247 in XLGnRH-R I) (47).…”