Functional Importance of Transmembrane Helix 6 Trp²⁷⁹and Exoloop 3 Val²⁹⁹of Rat Gonadotropin-Releasing Hormone Receptor

Abstract

Previous studies have established that the interaction of gonadotropin-releasing hormone (GnRH) with its receptor (GnRHR) would require partial entry of the N- and C-terminal regions of ligand into the transmembrane core. The functional significance of the conserved aromatic residue Trp(279) present in the transmembrane helix 6, and Val(299) located in exoloop 3 of the rat GnRHR was investigated by mutagenesis followed by expression in Chinese hamster ovary-K1 cells. Compared with wild-type, substitution of Trp(279) with Ser or Arg resulted in a marked reduction or total abolition, respectively, of ligand binding and, in both cases, abrogation of GnRH-induced inositol phosphate production. A total absence of functionality was observed when Val(299) was simply replaced with Ala. Mention should be made that an expression of all mutated and wild-type receptor proteins was observed. Interestingly, the double mutant [Trp(279)Arg/Val(299)Ala]GnRHR restored B(max) to wild type (504 +/- 43 versus 541 +/- 41 fmol/mg protein), but with a diminished affinity (4.95 +/- 1.05 versus 0.94 +/- 0.35 nM), and GnRH failed to induce inositol phosphate. No influence of the mutations was seen on internalization of the receptor. The three-dimensional model of GnRH binding to the rat GnRHR was built predicting that Trp(279) is buried at 20 A in the transmembrane core of the receptor, directly in contact with Trp(3) of GnRH. In contrast, Val(299) is located in a region that cannot be precisely defined at the extracellular end of transmembrane helix 7. Although models cannot provide any clue concerning the observed interactivity between the two distal residues, altogether these data reveal the functional importance of both GnRHR Trp(279) and Val(299) and suggest that Trp(279), interacting with GnRH Trp(3), represents the bottom of the binding pocket.

Molecular Approximation between a Residue in the Amino-terminal Region of Calcitonin and the Third Extracellular Loop of the Class B G Protein-coupled Calcitonin Receptor

“…Most recently, we have shown that the first four or five secretin amino-terminal residues were positioned close to the third extracellular loop of the receptor in our secretin-bound receptor model, with His 1 of secretin positioned at the top of transmembrane helix six (38). Although this suggests a common mechanism for binding and activation of class B family G protein-coupled receptors, there has been a similar proposal for members of another family, such as receptors for follicle-stimulating hormone (56,57), luteinizing hormone/choriogonadotropin (52), and gonadotropin-releasing hormone (58).…”

“…The importance of the extracellular loop domains in ligand binding has been consistent for other members in the class B G protein-coupled receptor family, including receptors for secretin (14,15), vasoactive intestinal polypeptide (42,43), parathyroid hormone (44 -46), glucagon (47,48), growth hormone-releasing hormone (49), corticotropin-releasing factor (50), luteinizing hormone/choriogonadotropin (51-54), follicle-stimulating hormone (55)(56)(57), and gonadotropin-releasing hormone (58). In particular, the third extracellular loop has been shown to be important for high affinity binding for the parathyroid receptor (44,46).…”

“…This domain has also been shown to interact directly with the hormone ligand for another family of G protein-coupled receptors (i.e. receptors for follicle-stimulating hormone (56,57), luteinizing hormone/choriogonadotropin (52), and gonadotropin-releasing hormone (58)) to activate these receptors. It is noteworthy that the third extracellular loop of G protein-coupled receptors is near the sixth transmembrane domain, which has been shown to interact directly with the amino terminus of the peptide ligand for receptors for parathyroid hormone (23,24) and secretin (22).…”

Histidine7.36(305) in the conserved peptide receptor activation domain of the gonadotropin releasing hormone receptor couples peptide binding and receptor activation

“…In the absence of crystal structures of the GnRH receptor, computational models have been used to infer ligand binding interactions (Chauvin et al, 2000;Hovelmann et al, 2002;Li et al, 2005;Soderhall et al, 2005). However, only a few of the proposed contacts have been validated with appropriate ligand modifications Sealfon et al, 1997).…”

“…Other amino-terminal functional groups of GnRH may also induce changes in intramolecular receptor bonds that result in receptor activation. The Trp 3 residue of GnRH has been proposed to interact with receptor residues in the TM6 and second extracellular loop, but some of these are controversial (Chauvin et al, 2000;Coetsee et al, 2006;Forfar and Lu, 2011).…”