Four naturally occurring mutations in the human GnRH receptor affect ligand binding and receptor function

Abstract

In the present study, we performed functional analyses of four mutations in the human GnRH receptor (GnRHR) gene, identified in patients with idiopathic hypogonadotropic hypogonadism. These mutations result in amino acid substitutions in the extracellular N-terminal domain (Thr32Ile), second extracellular loop (Cys200Tyr), third intracellular loop (Leu266Arg) and sixth transmembrane helix (Cys279Tyr). Immunocytochemical analysis of cells transfected with HA-tagged GnRHR constructs revealed that all four mutant receptors were present on the cell surface. However, all four mutant receptors failed to exhibit measurable specific GnRH binding and, except for Thr32Ile, any significant inositol phosphate accumulation after GnRH stimulation. In addition, Leu266Arg and Cys279Tyr receptors were unable to stimulate gonadotropin subunit or GnRHR gene promoter activity in response to GnRH. Interestingly, the Cys200Tyr mutant was able to stimulate gonadotropin subunit and GnRHR promoter activity, albeit with a higher EC₅₀ and a markedly reduced maximal response compared to wild type receptor. The Thr32Ile mutant was also able to stimulate gonadotropin subunit and GnRHR promoters, but with a further significant increase in EC₅₀. Similarly, this mutant partially retained the ability to activate extracellular signal-regulated kinase 1 and stimulate CRE-luciferase activity with an identical shift in EC₅₀. Taken together, the studies suggest that the Thr32Ile mutation reduces hGnRHR function primarily by reducing ligand binding affinity, and the Cys200Tyr mutation reduces cell surface receptor expression. All four amino acid substitutions interfered with ligand binding, and affected signal transduction and stimulation of gonadotropin and GnRHR gene expression in response to GnRH.

Introduction

GnRH, a hypothalamic decapeptide, plays a central role in the neuroendocrine control of reproductive function. At the level of the anterior pituitary, GnRH binds to specific high affinity receptors (GnRHR) on the cell surface of pituitary gonadotropes, to effect synthesis and secretion of the gonadotropins, LH and FSH, which in turn regulate gonadal maturation and function. Sequence analysis of the GnRHR gene predicts a G-protein coupled receptor of the rhodopsin super-family with an extracellular N-terminal domain, seven transmembrane domains, and three extra- and three intracellular loops (Stojilkovic et al., 1994). From the time that the amino acid sequence of GnRH was first determined (Matsuo et al., 1971, Burgus et al., 1972), much effort has focused on elucidating the structural details of interaction of GnRH with its receptor, and the precise intracellular pathways by which GnRHR activation leads to the stimulation of gonadotropin synthesis and release (Stojilkovic et al., 1994, Kaiser et al., 1997, Sealfon et al., 1997).

Due to the central role of GnRH action in reproductive function, the GnRHR gene appeared to be a good candidate for mutations associated with reproductive defects. Indeed, several naturally occurring mutations in the human GnRHR (hGnRHR) identified in patients with idiopathic hypogonadotropic hypogonadism (IHH) have been reported (De Roux et al., 1997, Layman et al., 1998, Caron et al., 1999, De Roux et al., 1999, Pralong et al., 1999, Kottler et al., 2000, Beranova et al., 2001, Costa et al., 2001, Pitteloud et al., 2001, Soderlund et al., 2001). These patients have presented with a broad spectrum of phenotypes, ranging from partial IHH with delayed puberty or reduced fertility, to complete IHH with sexual infantilism, primary amenorrhea or cryptorchidism. The first patient reported was a compound heterozygote, with a mutation in one allele resulting in a Gln106Arg substitution in the first extracellular loop of the receptor, and a mutation in the other allele resulting in an Arg262Gln substitution in the third intracellular loop (De Roux et al., 1997). Functional analysis revealed that GnRH binding was reduced by the Gln106Arg mutation, whereas binding was normal but activation of phospholipase C (PLC) in response to GnRH was reduced by the Arg262Gln mutation. Subsequently, several additional naturally occurring mutations resulting in impaired cellular expression, ligand binding, and/or signal transduction have been described (Layman et al., 1998, Caron et al., 1999, De Roux et al., 1999, Pralong et al., 1999, Kottler et al., 2000, Beranova et al., 2001, Costa et al., 2001, Pitteloud et al., 2001, Soderlund et al., 2001, Maya-Nunez et al., 2002). The identification of these mutations provides an avenue for characterization of functional and physiologically important domains of the receptor. Furthermore, studies of these mutant receptors can be used to dissect the signal transduction pathways that mediate GnRH regulation of gonadotropin synthesis and release.

In a recent study of a population of patients with IHH, four novel mutations of the hGnRHR were identified (Beranova et al., 2001). These mutations each result in an amino acid substitution in a different domain of the receptor: in the extracellular N-terminal domain (Thr32Ile), second extracellular loop (Cys200Tyr), third intracellular loop (Leu266Arg) and sixth transmembrane helix (Cys279Tyr). We previously demonstrated that these mutations impair stimulation of cellular inositol phosphate (IP) production in response to GnRH (Beranova et al., 2001). However, the mechanisms of this impairment and the effects on gonadotropin production were not addressed. In the present study, we have performed more detailed analyses of the effects of these four mutations on hGnRHR function. We have measured cellular expression, binding capacity and affinity, and activation of several distinct signal transduction pathways. Furthermore, we have correlated these results with effects on GnRH stimulation of gonadotropin subunit and GnRHR gene promoter activity, to confirm in vitro that the mutations impair GnRH responsiveness and are the likely cause of the phenotypic manifestations observed in vivo, and to elucidate the mechanisms underlying the defects in receptor function.