The Structural Evolution of a P2Y-like G-protein-coupled Receptor

Abstract

Based on the now available crystallographic data of the G-protein-coupled receptor (GPCR) prototype rhodopsin, many studies have been undertaken to build or verify models of other GPCRs. Here, we mined evolution as an additional source of structural information that may guide GPCR model generation as well as mutagenesis studies. The sequence information of 61 cloned orthologs of a P2Y-like receptor (GPR34) enabled us to identify motifs and residues that are important for maintaining the receptor function. The sequence data were compared with available sequences of 77 rhodopsin orthologs. Under a negative selection mode, only 17% of amino acid residues were preserved during 450 million years of GPR34 evolution. On the contrary, in rhodopsin evolution ϳ43% residues were absolutely conserved between fish and mammals. Despite major differences in their structural conservation, a comparison of structural data suggests that the global arrangement of the transmembrane core of GPR34 orthologs is similar to rhodopsin. The evolutionary approach was further applied to functionally analyze the relevance of common scaffold residues and motifs found in most of the rhodopsin-like GPCRs. Our analysis indicates that, in contrast to other GPCRs, maintaining the unique function of rhodopsin requires a more stringent network of relevant intramolecular constrains.Among the different families of transmembrane receptors, G-protein-coupled receptors (GPCRs) 1 form the largest superfamily. Molecular cloning studies and genome data analyses have revealed ϳ1200 -1300 members of the GPCR superfamily in mammalian genomes (1). To predict and understand ligand binding and signal transduction as well as the consequences of structural changes (e.g. disease-causing mutations) within a receptor molecule, detailed information about the native receptor structure in its inactive and active conformations is required. Currently, a high-resolution structure is available only for bovine rhodopsin (2), which now provides the basis to generate models of other GPCRs (3). However, recent studies point out that even with a crystal structure in hand, construction of reliable receptor models still requires time-consuming refinements based on data from mutagenesis, cross-linking, and NMR studies (4).Herein, we mined evolution as an additional source of structural information that may direct GPCR modeling and mutagenesis studies. This idea has been successfully applied in an early stage of GPCR structure/function analysis. The sequences of Ͼ200 different members of the GPCR family were used to predict the approximate arrangements of the seven transmembrane helices (5). To determine more distinct structural determinants that participate in ligand recognition and signal transduction, sequence analysis has to be focused on a single receptor subtype. The structural diversity of a given receptor among different species is the result of a long evolutionary process characterized by a continuous accumulation of mutations. However, the maintenance of vital functions in...

Evolution of GPCR: Change and continuity

“…Indeed, many evolutionary old GPCR genes including the rhodopsins share strong conservation between distantly related species and have low K a /K s ratios throughout their coding regions particularly in the highly conserved 7TM region [86,93,94]. This finding is indicative of continuous negative selection.…”

Altered Immune Response in Mice Deficient for the G Protein-coupled Receptor GPR34

“…Cyclic AMP measurements were performed as described previously (25) using the AlphaScreen technology. Transient co-transfection experiments of COS-7 cells with the GPR34 constructs and the chimeric G protein G␣ qi4 and inositol phosphate accumulation assays (26) were essentially performed as described previously (9). All GPR34 constructs were introduced into the mammalian expression vector pcDps and double-tagged with an N-terminal HA tag and a C-terminal FLAG tag to monitor and quantify total cellular and plasma membrane expression using ELISA (27).…”

“…The functionality of this approach, which links signal transduction of a G i proteincoupled receptor to the phospholipase C/inositol phosphate pathway has been demonstrated for GPR34 (9) and many other GPCR (47). GPR34 activation was measured using an inositol phosphate accumulation assay.…”

“…Phylogenetic studies revealed that GPR34 has been highly conserved over the past 450 million years of vertebrate evolution, and no GPR34-deficient vertebrate has been identified yet (9). To date, there is no report of GPR34 deficiency in humans, and sequencing of more than 100 worldwide samples of human genomic DNA revealed no functionally relevant alleles indicating the physiological importance of the gene (10).…”

Cell Adhesion Receptor GPR133 Couples to Gs Protein

“…Thus, the coupling abilities of several receptors, including "orphan" receptors, have been characterized by overexpression in the absence of an agonist (31)(32)(33). For example, the wild-type adenylate cyclase constitutive activator (ACCA), an orphan GPCR, stimulates the G s protein/adenylyl cyclase system to some extent when expressed in COS-7 cells (31).…”