The structural evolution of a P2Y-like G-protein-coupled receptor

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 approximately 43% 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.     

Simulations of a G protein-coupled receptor homology model predict dynamic features and a ligand binding site

A computational approach to predict structures of rhodopsin-like G protein-coupled receptors (GPCRs) is presented and evaluated by comparison to the X-ray structural models. By combining sequence alignment, the rhodopsin crystal structure, and point mutation data on the beta2 adrenoreceptor (b2ar), we predict a (-)-epinephrine-bound computational model of the beta2 adrenoreceptor. The model is evaluated by molecular dynamics simulations and by comparison with the recent X-ray structures of b2ar. The overall correspondence between the predicted and the X-ray structural model is high. Especially the prediction of the ligand binding site is accurate. This shows that the proposed dynamic homology modelling approach can be used to create reasonable models for the understanding of structure and dynamics of other rhodopsin-like GPCRs.     

Sequence,Structure and Ligand Binding Evolution of Rhodopsin-Like G Protein-Coupled Receptors: A Crystal Structure-Based Phylogenetic Analysis

G protein-coupled receptors (GPCRs) form the largest family of membrane receptors in the human genome. Advances in membrane protein crystallization so far resulted in the determination of 24 receptors available as high-resolution atomic structures. We performed the first phylogenetic analysis of GPCRs based on the available set of GPCR structures. We present a new phylogenetic tree of known human rhodopsin-like GPCR sequences based on this structure set. We can distinguish the three separate classes of small-ligand binding GPCRs, peptide binding GPCRs, and olfactory receptors. Analyzing different structural subdomains, we found that small molecule binding receptors most likely have evolved from peptide receptor precursors, with a rhodopsin/S1PR1 ancestor, most likely an ancestral opsin, forming the link between both classes. A light-activated receptor therefore seems to be the origin of the small molecule hormone receptors of the central nervous system. We find hints for a common evolutionary path of both ligand binding site and central sodium/water binding site. Surprisingly, opioid receptors exhibit both a binding cavity and a central sodium/water binding site similar to the one of biogenic amine receptors instead of peptide receptors, making them seemingly prone to bind small molecule ligands, e.g. opiates. Our results give new insights into the relationship and the pharmacological properties of rhodopsin-like GPCRs.     

A beta-arrestin binding determinant common to the second intracellular loops of rhodopsin family G protein-coupled receptors

beta-Arrestins have been shown to inhibit competitively G protein-dependent signaling and to mediate endocytosis for many of the hundreds of nonvisual rhodopsin family G protein-coupled receptors (GPCR). An open question of fundamental importance concerning the regulation of signal transduction of several hundred rhodopsin-like GPCRs is how these receptors of limited sequence homology, when considered in toto, can all recruit and activate the two highly conserved beta-arrestin proteins as part of their signaling/desensitization process. Although the serine and threonine residues that form GPCR kinase phosphorylation sites are common beta-arrestin-associated receptor determinants regulating receptor desensitization and internalization, the agonist-activated conformation of a GPCR probably reveals the most fundamental determinant mediating the GPCR and arrestin interaction. Here we identified a beta-arrestin binding determinant common to the rhodopsin family GPCRs formed from the proximal 10 residues of the second intracellular loop. We demonstrated by both gain and loss of function studies for the serotonin 2C, beta2-adrenergic, alpha2a)adrenergic, and neuropeptide Y type 2 receptors that the highly conserved amino acids, proline and alanine, naturally occurring in rhodopsin family receptors six residues distal to the highly conserved second loop DRY motif regulate beta-arrestin binding and beta-arrestin-mediated internalization. In particular, as demonstrated for the beta2 AR, this occurs independently of changes in GPCR kinase phosphorylation. These results suggest that a GPCR conformation directed by the second intracellular loop, likely using the loop itself as a binding patch, may function as a switch for transitioning beta-arrestin from its inactive form to its active receptor-binding state.     

Structure and pH sensitivity of the transmembrane segment 3 of rhodopsin

Activation of G protein-coupled receptors (GPCRs) originates in ligand-induced protein conformational changes that are transmitted to the cytosolic receptor surface. In the photoreceptor rhodopsin, and possibly other rhodopsin-like GPCRs, protonation of a carboxylic acid in the conserved E(D)RY motif at the cytosolic end of transmembrane helix 3 (TM3) is coupled to receptor activation. Here, we have investigated the structure of synthetic peptides derived from rhodopsin TM3. Polarized FTIR spectroscopy reveals a helical structure of a 31-mer TM3 peptide reconstituted into PC vesicles with a large tilt of 40-50 degrees of the helical axis relative to the membrane normal. Helical structure is also observed for the TM3 peptide in detergent micelles and depends on pH, especially in the C-terminal sequence. In addition, the fluorescence emission of the single tyrosine of the D(E)RY motif in the TM3 peptide exhibits a pronounced pH sensitivity that is abolished when Glu is replaced by Gln, demonstrating that protonation of the conserved Glu side chain affects the structure in the environment of the D(E)RY motif of TM3. The pH regulation of the C-terminal TM3 structure may be an intrinsic feature of the E(D)RY motif in other class I receptors, allowing the coupling of protonation and conformation of membrane-exposed residues in full-length GPCRs.     

The serendipitous origin of chordate secretin peptide family members

Background  

The secretin family is a pleotropic group of brain-gut peptides with affinity for class 2 G-protein coupled receptors (secretin family GPCRs) proposed to have emerged early in the metazoan radiation via gene or genome duplications. In human, 10 members exist and sequence and functional homologues and ligand-receptor pairs have been characterised in representatives of most vertebrate classes. Secretin-like family GPCR homologues have also been isolated in non-vertebrate genomes however their corresponding ligands have not been convincingly identified and their evolution remains enigmatic.     

3D modeling of the activated states of constitutively active mutants of rhodopsin

The activated (R*) states in constitutively active mutants (CAMs) of G-protein-coupled receptors (GPCRs) are presumably characterized by lower energies than the resting (R) states. If specific configurations of TM helices differing by rotations along the long transmembrane axes possess energies lower than that in the R state for pronounced CAMs, but not for non-CAMs, these particular configurations of TM helices are candidate 3D models for the R* state. The hypothesis was studied in the case of rhodopsin, the only GPCR for which experimentally determined 3D models of the R and R* states are currently available. Indeed, relative energies of the R* state were significantly lower than that of the R state for the rhodopsin mutants G90D/M257Y and E113Q/M257Y (strong CAMs), but not for G90D, E113Q, and M257Y (not CAMs). Next, the developed build-up procedure successfully identified few similar configurations of the TM helical bundle of G90D/M257Y and E113Q/M257Y as possible candidates for the 3D model of the R* state of rhodopsin, all of them being in good agreement with the model suggested by experiment. Since constitutively active mutants are known for many of GPCRs belonging to the large rhodopsin-like family, this approach provides a way for predicting possible 3D structures corresponding to the activated states of the TM regions of many GPCRs for which CAMs have been identified.     

GPCRs: an update on structural approaches to drug discovery

G-protein-coupled receptors (GPCRs) are a major opportunity for drug discovery in the post-genomic era. There are thought to be more than 500 therapeutically relevant GPCRs out of a total of over 700 identified to date, although only one, rhodopsin, has been the subject of a full 3D X-ray crystallography study. Two structurally related proteins, bacteriorhodopsin and sensory rhodopsin, which are not GPCRs but are part of the seven-helix membrane receptor family, have also been the subject of X-ray crystallographic studies and have been used in GPCR modeling studies. The significant differences between these rhodopsin structures, the relatively low sequence homology between individual GPCRs, and some difficulties in rationalizing point-mutation data suggests that homology-based molecular modeling alone will not provide the accurate structural information on individual receptors required for ligand design and in silico screening. In the absence of such structural information, several approaches can be used to assist in the discovery of ligands.     

Membrane topology of a metabotropic glutamate receptor

The metabotropic glutamate receptors (mGluRs) have been predicted to have a classical seven transmembrane domain structure similar to that seen for members of the G-protein-coupled receptor (GPCR) superfamily. However, the mGluRs (and other members of the family C GPCRs) show no sequence homology to the rhodopsin-like GPCRs, for which this seven transmembrane domain structure has been experimentally confirmed. Furthermore, several transmembrane domain prediction algorithms suggest that the mGluRs have a topology that is distinct from these receptors. In the present study, we set out to test whether mGluR5 has seven true transmembrane domains. Using a variety of approaches in both prokaryotic and eukaryotic systems, our data provide strong support for the proposed seven transmembrane domain model of mGluR5. We propose that this membrane topology can be extended to all members of the family C GPCRs.     

The second intracellular loop of metabotropic glutamate receptors recognizes C termini of G-protein alpha-subunits

Heptahelical receptor coupling selectivity to G-proteins is controlled by a large contact area that involves several portions of the receptor and each subunit of the G-protein. In the G-protein alpha subunit, the C-terminal 5 residues, the N terminus, and the alpha N-beta 1 and alpha 4-alpha 5 loops play important roles. On the receptor side, both the second and third (i2 and i3) intracellular loops as well as the C-terminal tail probably contact these different regions of the G-protein. It is now accepted that the C terminus of the alpha subunit binds in a cavity formed by the i2 and i3 loops. Among the various G-protein-coupled receptors (GPCRs), class III receptors that include metabotropic glutamate (mGlu) receptors greatly differ from the rhodopsin-like GPCRs, but the contact zone between these receptors and the G-protein is less understood. The C terminus of the alpha subunit has been shown to play a pivotal role in the selective recognition of class III GPCRs. Indeed, the mGlu2 and mGlu4 and -8 receptors can discriminate between alpha subunits that differ at the level of their C-terminal end only (such as Gqo and Gqz). Here, we examine the role of the i2 loop of mGluRs in the selective recognition of this region of the alpha subunit. To that aim, we analyzed the coupling properties of mGlu2 and mGlu4 or -8 receptors and chimeras containing the i2 loop of the converse receptor to G-protein alpha subunits that only differ by their C termini (Gqo,Gqz, and their point mutants). Our data demonstrate that the central portion of the i2 loop is responsible for the selective recognition of the C-terminal end of the alpha subunit, especially the residue on position -4. These data are consistent with the proposal that the C-terminal end of the G-protein alpha subunit interacts with residues in a cavity formed by the i2 and i3 loops in class III GPCRs, as reported for class I GPCRs.     

Homo- and hetero-oligomerization of G protein-coupled receptors

G protein-coupled receptors (GPCRs) form homo-oligomeric and hetero-oligomeric complexes. This understanding has prompted a re-evaluation of many aspects of GPCR biology, however the concept of receptor complexes has not been fully integrated into the current thinking about GPCR structure and function. Nevertheless, receptor oligomerization is a pivotal aspect of the structure and function of GPCRs that has been shown to have implications for receptor trafficking, signaling, and pharmacology and more intricate models for understanding the physiological roles of these receptors are emerging. Here, we summarize some of the advances made in understanding the structural basis and the functional roles of homo- and hetero- oligomerization in this important group of receptors. Although this discussion focuses primarily on the dopamine receptors, particularly the D2 dopamine receptor, and the opioid and serotonin receptors, we discuss the principles governing the oligomerization of all rhodopsin-like GPCRs and potentially of the entire superfamily of these receptors.     

Seven evolutionarily conserved human rhodopsin G protein-coupled receptors lacking close relatives

We report seven new members of the superfamily of human G protein-coupled receptors (GPCRs) found by searches in the human genome databases, termed GPR100, GPR119, GPR120, GPR135, GPR136, GPR141, and GPR142. We also report 16 orthologues of these receptors in mouse, rat, fugu (pufferfish) and zebrafish. Phylogenetic analysis shows that these are additional members of the family of rhodopsin-type GPCRs. GPR100 shows similarity with the orphan receptor SALPR. Remarkably, the other receptors do not have any close relative among other known human rhodopsin-like GPCRs. Most of these orphan receptors are highly conserved through several vertebrate species and are present in single copies. Analysis of expressed sequence tag (EST) sequences indicated individual expression patterns, such as for GPR135, which was found in a wide variety of tissues including eye, brain, cervix, stomach and testis. Several ESTs for GPR141 were found in marrow and cancer cells, while the other receptors seem to have more restricted expression patterns.     

Monomeric G-protein-coupled receptor as a functional unit

Rhodopsin, the first purified G-protein-coupled receptor (GPCR), was characterized as a functional monomer 30 year ago, but dimerization of GPCRs recently became the new paradigm of signal transduction. It has even been claimed, on the basis of recent biophysical and biochemical studies, that this new concept could be extended to higher-order oligomerization. Here this view is challenged. The new studies of rhodopsin and other simple (class 1a) GPCRs solubilized in detergent are re-assessed and are compared to the earlier classical studies of rhodopsin and other membrane proteins solubilized in detergent. The new studies are found to strengthen rather than invalidate the conclusions of the early ones and to support a monomeric model for rhodopsin and other class 1a GPCRs. A molecular model is proposed for the functional coupling of a rhodopsin monomeric unit with a G-protein heterotrimer. This model should be valid even for GPCRs that exist as structural dimers.     

Linking agonist binding to histamine H1 receptor activation

G protein-coupled receptors (GPCRs) constitute a large and functionally diverse family of transmembrane proteins. They are fundamental in the transfer of extracellular stimuli to intracellular signaling pathways and are among the most targeted proteins in drug discovery. The detailed molecular mechanism for agonist-induced activation of rhodopsin-like GPCRs has not yet been described. Using a combination of site-directed mutagenesis and molecular modeling, we characterized important steps in the activation of the human histamine H1 receptor. Both Ser3.36 and Asn7.45 are important links between histamine binding and previously proposed conformational changes in helices 6 and 7. Ser3.36 acts as a rotamer toggle switch that, upon agonist binding, initiates the activation of the receptor through Asn7.45. The proposed transduction involves specific residues that are conserved among rhodopsin-like GPCRs.     

Identifying G-protein coupled receptors using weighted Levenshtein distance and the nearest neighbor method

G-protein coupled receptors （GPCRs） are a class of seven-helix transmembrane proteins that have been used in bioinformatics as the targets to facilitate drug discovery for human diseases. Although thousands of GPCR sequences have been collected, the ligand specificity of many GPCRs is still unknown and only one crystal structure of the rhodopsin-like family has been solved. Therefore, identifying GPCR types only from sequence data has become an important research issue. In this study, a novel technique for identifying GPCR types based on the weighted Levenshtein distance between two receptor sequences and the nearest neighbor method （NNM） is introduced, which can deal with receptor sequences with different lengths directly. In our experiments for classifying four classes （acetylcholine, adrenoceptor, dopamine, and serotonin） of the rhodopsin-like family of GPCRs, the error rates from the leave-one-out procedure and the leave-half-out procedure were 0.62% and 1.24%, respectively. These results are prior to those of the covariant discriminant algorithm, the support vector machine method, and the NNM with Euclidean distance.     

Regulation of the rhodopsin protein phosphatase, RDGC, through interaction with calmodulin.

Hundreds of G protein-coupled receptors (GPCRs) and at least six GPCR kinases have been identified, but the only GPCR phosphatase that has been definitively demonstrated is the rhodopsin phosphatase encoded by the rdgC locus of Drosophila. Mutations in rdgC result in defects in termination of the light response and cause severe retinal degeneration. In the current work, we demonstrate that RDGC binds to calmodulin, and a mutation in an IQ motif that eliminates the calmodulin/RDGC interaction prevents dephosphorylation of rhodopsin in vivo and disrupts termination of the photoresponse. Our data indicate that RDGC is a novel calmodulin-dependent protein phosphatase and raise the possibility that regulation of other GPCRs through dephosphorylation may be controlled by calmodulin-dependent protein phosphatases related to RDGC.     

Activation of rhodopsin: new insights from structural and biochemical studies

G-protein-coupled receptors (GPCRs) are involved in a vast variety of cellular signal transduction processes from visual, taste and odor perceptions to sensing the levels of many hormones and neurotransmitters. As a result of agonist-induced conformation changes, GPCRs become activated and catalyze nucleotide exchange within the G proteins, thus detecting and amplifying the signal. GPCRs share a common heptahelical transmembrane structure as well as many conserved key residues and regions. Rhodopsins are prototypical GPCRs that detect photons in retinal photoreceptor cells and trigger a phototransduction cascade that culminates in neuronal signaling. Biophysical and biochemical studies of rhodopsin activation, and the recent crystal structure determination of bovine rhodopsin, have provided new information that enables a more complete mechanism of vertebrate rhodopsin activation to be proposed. In many aspects, rhodopsin might provide a structural and functional template for other members of the GPCR family.     

The transmembrane 7-alpha-bundle of rhodopsin: distance geometry calculations with hydrogen bonding constraints.

A 3D model of the transmembrane 7-alpha-bundle of rhodopsin-like G-protein-coupled receptors (GPCRs) was calculated using an iterative distance geometry refinement with an evolving system of hydrogen bonds, formed by intramembrane polar side chains in various proteins of the family and collectively applied as distance constraints. The alpha-bundle structure thus obtained provides H bonding of nearly all buried polar side chains simultaneously in the 410 GPCRs considered. Forty evolutionarily conserved GPCR residues form a single continuous domain, with an aliphatic "core" surrounded by six clusters of polar and aromatic side chains. The 7-alpha-bundle of a specific GPCR can be calculated using its own set of H bonds as distance constraints and the common "average" model to restrain positions of the helices. The bovine rhodopsin model thus determined is closely packed, but has a few small polar cavities, presumably filled by water, and has a binding pocket that is complementary to 11-cis (6-s-cis, 12-s-trans, C = N anti)-retinal or to all-trans-retinal, depending on conformations of the Lys296 and Trp265 side chains. A suggested mechanism of rhodopsin photoactivation, triggered by the cis-trans isomerization of retinal, involves rotations of Glu134, Tyr223, Trp265, Lys296, and Tyr306 side chains and rearrangement of their H bonds. The model is in agreement with published electron cryomicroscopy, mutagenesis, chemical modification, cross-linking, Fourier transform infrared spectroscopy, Raman spectroscopy, electron paramagnetic resonance spectroscopy, NMR, and optical spectroscopy data. The rhodopsin model and the published structure of bacteriorhodopsin have very similar retinal-binding pockets.     

Visual rhodopsin sees the light: structure and mechanism of G protein signaling

The availability of crystal structures for the dark, inactive, and several light-activated photointermediate states of vertebrate visual rhodopsin has provided important mechanistic and energetic insights into the transformations underlying agonist-dependent activation of this and other G protein-coupled receptors (GPCRs). The high natural abundance of rhodopsin in the vertebrate retina, together with its specific localization to the disk membranes of the rod cell, has also enabled direct imaging of rhodopsin in its native environment. These advances have provided compelling evidence that rhodopsin, like many other GPCRs, forms highly organized oligomeric structures that, in all likelihood, are important for receptor biosynthesis, optimal activation, and signaling.     

Identification of cholesterol recognition amino acid consensus (CRAC) motif in G-protein coupled receptors

G-protein coupled receptors (GPCRs) are the largest class of molecules involved in signal transduction across membranes, and represent major targets in the development of novel drug candidates in all clinical areas. Membrane cholesterol has been reported to have an important role in the function of a number of GPCRs. Several structural features of proteins, believed to result in preferential association with cholesterol, have been recognized. Cholesterol recognition/interaction amino acid consensus (CRAC) sequence represents such a motif. Many proteins that interact with cholesterol have been shown to contain the CRAC motif in their sequence. We report here the presence of CRAC motifs in three representative GPCRs, namely, rhodopsin, the β(2)-adrenergic receptor, and the serotonin(1A) receptor. Interestingly, the function of these GPCRs has been previously shown to be dependent on membrane cholesterol. The presence of CRAC motifs in GPCRs indicates that interaction of cholesterol with GPCRs could be specific in nature. Further analysis shows that CRAC motifs are inherent characteristic features of the serotonin(1A) receptor and are conserved over natural evolution. These results constitute the first report of the presence of CRAC motifs in GPCRs and provide novel insight in the molecular nature of GPCR-cholesterol interaction.