The Functional Microdomain in Transmembrane Helices 2 and 7 Regulates Expression, Activation, and Coupling Pathways of the Gonadotropin-releasing Hormone Receptor

Abstract

Structural microdomains of G protein-coupled receptors (GPCRs) consist of spatially related side chains that mediate discrete functions. The conserved helix 2/helix 7 microdomain was identified because the gonadotropin-releasing hormone (GnRH) receptor appears to have interchanged the Asp 2.50 and Asn 7.49 residues which are conserved in transmembrane helices 2 and 7 of rhodopsin-like GPCRs. We now demonstrate that different side chains of this microdomain contribute specifically to receptor expression, heterotrimeric G protein-, and small G protein-mediated signaling. An Asn residue is required in position 2.50(87) for expression of the GnRH receptor at the cell surface, most likely through an interaction with the conserved Asn 1.50 (53) residue, which we also find is required for receptor expression. Most GPCRs require an Asp side chain at either the helix 2 or helix 7 locus of the microdomain for coupling to heterotrimeric G proteins, but the GnRH receptor has transferred the requirement for an acidic residue from helix 2 to 7. However, the presence of Asp at the helix 7 locus precludes small G protein-dependent coupling to phospholipase D. These results implicate specific components of the helix 2/helix 7 microdomain in receptor expression and in determining the ability of the receptor to adopt distinct activated conformations that are optimal for interaction with heterotrimeric and small G proteins.The gonadotropin-releasing hormone (GnRH) 1 receptor belongs to the rhodopsin-like family of G protein-coupled receptors (GPCR) (1). This family includes the light-sensitive opsins, protease-activated receptors, and receptors for neurotransmitters, peptides, and glycoproteins. High resolution structural data have not yet been obtained for any GPCR. However, projection maps of rhodopsin, amino acid sequence alignment, and computational modeling indicate that GPCRs have 7 membrane-spanning ␣-helices (2-6). There is a high degree of homology within the transmembrane helices and certain amino acids are highly conserved throughout the family (2, 3, 7). This diverse family shares the common function of propagating a signal across lipid membranes and the amino acid side chains which are conserved among the GPCRs are likely to constitute key structural motifs which subserve this universal GPCR function. Several models of GPCRs, including the GnRH receptor (4, 8), have been constructed as aids for investigating receptor structure-function relations. Molecular models of GPCRs can be used to integrate experimental observations and generate structural hypotheses. However, the complexity of these structures and the limited number of experimentally determined constraints can lead to inconsistent behavior of the models (4, 7). To overcome these limitations, we have pursued the approach of identifying discrete structural motifs within receptor models, which might constitute functional microdomains. The microdomains are characterized in detail and subsequently incorporated into whole receptor models. In the GnRH receptor, for exa...

Functional Analysis of Transmembrane Domain 2 of the M1 Muscarinic Acetylcholine Receptor

“…The total abolition of signaling that follows Ala substitution of either of these residues implies that their interaction may be critical for the stabilization of the activated state. This would be consistent with experiments on a number of different receptor types in which signaling activity abolished by a D2.50N mutation has been restored, but usually at a lower level, by making the complementary N7.49D mutant (17)(18)(19)(20)(21)(22). In the homologous group of G q -coupled mAChRs, including the M 1 and M 3 , the reciprocal mutant may actually have low activity, because it was not among those recovered in a comprehensive genetic screen for mutations of the M 3 mAChR that restored the signaling activity of the D2.50N mutant (15).…”

“…In the M 1 mAChR, Asn substitution first established the critical role of Asp 71 in signal transduction (13), a finding subsequently confirmed in the M 2 (14) and M 3 (15) subtypes as well as a host of other receptor types (16). Reciprocal mutagenesis experiments on a number of receptors (17)(18)(19)(20)(21)(22) have suggested that pairing between positions 2.50 and 7.49 is important for receptor activation.…”

A Chicken Gonadotropin-releasing Hormone Receptor That Confers Agonist Activity to Mammalian Antagonists

“…The GnRH receptor is a rhodopsin-like G protein-coupled receptor (GPCR) (4). Progress has been made in defining the structure of mammalian GnRH receptors (5)(6)(7)(8) and in identifying amino acid residues that are important for ligand binding (9 -12) and coupling to G proteins (13,14). Some insight has also been obtained into the molecular entities involved in receptor activation.…”

“…Some insight has also been obtained into the molecular entities involved in receptor activation. The interaction of Asn 2.50(87) , in transmembrane domain 2 (TM2), with Asp 7.49(318) in TM7 of the mouse GnRH receptor appears to have a role in stabilizing the activated receptor conformation (7,8). In common with other GPCRs, the highly conserved Asp-Arg (DR) sequence at the intracellular boundary of TM3 has an integral role in activation of the GnRH receptor (15).…”

Conserved Helix 7 Tyrosine Acts as a Multistate Conformational Switch in the 5HT2C Receptor

“…Mutagenesis studies (5,19,22), engineered metal-ion activation sites (47), computational modeling (15), and the rhodopsin crystal structure (6) all implicate helix 7 in a network of interactions that modulate receptor activity. In the present study, we investigate the role that the conserved Y7.53 in this domain may play in switching the 5HT2C receptor among multiple states.…”