Evolutionary Trace of G Protein-coupled Receptors Reveals Clusters of Residues That Determine Global and Class-specific Functions

Abstract

G protein-coupled receptor (GPCR) activation mediated by ligand-induced structural reorganization of its helices is poorly understood. To determine the universal elements of this conformational switch, we used evolutionary tracing (ET) to identify residue positions commonly important in diverse GPCRs. When mapped onto the rhodopsin structure, these trace residues cluster into a network of contacts from the retinal binding site to the G protein-coupling loops. Their roles in a generic transduction mechanism were verified by 211 of 239 published mutations that caused functional defects. When grouped according to the nature of the defects, these residues subdivided into three striking sub-clusters: a trigger region, where mutations mostly affect ligand binding, a coupling region near the cytoplasmic interface to the G protein, where mutations affect G protein activation, and a linking core in between where mutations cause constitutive activity and other defects. Differential ET analysis of the opsin family revealed an additional set of opsin-specific residues, several of which form part of the retinal binding pocket, and are known to cause functional defects upon mutation. To test the predictive power of ET, we introduced novel mutations in bovine rhodopsin at a globally important position, Leu-79, and at an opsin-specific position, Trp-175. Both were functionally critical, causing constitutive G protein activation of the mutants and rapid loss of regeneration after photobleaching. These results define in GPCRs a canonical signal transduction mechanism where ligand binding induces conformational changes propagated through adjacent trigger, linking core, and coupling regions.The profusion and diversity of G protein-coupled receptors (GPCRs) 1 give them a central role in health and disease. In humans, over 1000 genes encode these receptors (1), each of which responds to a single or few ligands by activating G proteins, which then modulate enzymes and channels to initiate highly amplified signaling cascades. Such cascades control sight, taste, smell, slow neurotransmission and the responses to most water-soluble hormones and chemokines. In fact, GPCRs are so ubiquitous that, although they are the targets of nearly 50% of current drugs (2), this is still a small fraction of their pharmacological potential (3).Some of the major questions relevant to GPCR pharmacology include the following: What residues are critical for ligand binding and G protein activation? What do different receptor families have in common with regard to their activation mechanism? From a structural perspective, it is known that all GPCRs form a seven transmembrane (TM) ␣-helical bundle, connected by three intracellular and three extracellular loops, with an extracellular N terminus and an intracellular C terminus (4, 5). However, low overall sequence identity of 25% even within class A GPCRs (6 -8) suggests that significant deviations can occur in ligand binding pockets and in interhelical contacts that stabilize or mediate the transition between a...

A two‐entropies analysis to identify functional positions in the transmembrane region of class A G protein‐coupled receptors

“…This is also the unaccounted assumption in both evolutionary trace and correlated mutation analysis during the process of identifying binding sites of GPCRs. 14,17 The second assumption is that endogenous binding sites are located in a region embedded between TM helices. This has been shown experimentally for a large number of receptors, including those for biogenic amines, 49 nucleotides, 50 melatonin, 51 and prostacyclin.…”

“…Only the opsin subfamily was subjected to differential trace analysis, and finally 17 opsin "specific" conserved residues were identified. 17 However, the identified 39 "globally" conserved residues based on only four subfamilies are not conserved in all subfamilies of class A GPCRs. For example, position 3.33 was identified as one of the 39 "globally" conserved residues.…”

“…[53][54][55][56][57][58][59] Recently, this method was used to analyze class A GPCR sequences to identify globally conserved residues and opsin subfamily-specific residues. 17 In that study, only four subfamilies of class A GPCRs-visual opsin, bioamine, olfactory, and chemokine-were included to trace 39 "globally" conserved residues. Only the opsin subfamily was subjected to differential trace analysis, and finally 17 opsin "specific" conserved residues were identified.…”

“…10,12,13,15 The fingerprints identified at the family level show a certain correlation to the endogenous ligand binding, whereas the evolutionary trace method has recently been used to reveal global and subfamilyspecific conserved residues of class A GPCRs. 17 It was reported that globally conserved residues relate to a canonical conformational switch, while some class-specific conserved residues form part of the ligand binding pocket. 17 Correlated mutation analysis and entropy-variability plots were also used to detect networks of functional residues in GPCRs.…”

“…17 It was reported that globally conserved residues relate to a canonical conformational switch, while some class-specific conserved residues form part of the ligand binding pocket. 17 Correlated mutation analysis and entropy-variability plots were also used to detect networks of functional residues in GPCRs. 11,14 The combination of these latter two methods allowed the identification of three groups of positions: residues responsible for G protein coupling, residues in the binding site, and residues in between these two groups.…”