Allosteric functioning of dimeric class C G-protein-coupled receptors

Whereas most membrane receptors are oligomeric entities, G-protein-coupled receptors have long been thought to function as monomers. Within the last 15 years, accumulating data have indicated that G-protein-coupled receptors can form dimers or even higher ordered oligomers, but the general functional significance of this phenomena is not yet clear. Among the large G-protein-coupled receptor family, class C receptors represent a well-recognized example of constitutive dimers, both subunits being linked, in most cases, by a disulfide bridge. In this review article, we show that class C G-protein-coupled receptors are multidomain proteins and highlight the importance of their dimerization for activation. We illustrate several consequences of this in terms of specific functional properties and drug development.     

Multidimensional scaling reveals the main evolutionary pathways of class A G-protein-coupled receptors

Class A G-protein-coupled receptors (GPCRs) constitute the largest family of transmembrane receptors in the human genome. Understanding the mechanisms which drove the evolution of such a large family would help understand the specificity of each GPCR sub-family with applications to drug design. To gain evolutionary information on class A GPCRs, we explored their sequence space by metric multidimensional scaling analysis (MDS). Three-dimensional mapping of human sequences shows a non-uniform distribution of GPCRs, organized in clusters that lay along four privileged directions. To interpret these directions, we projected supplementary sequences from different species onto the human space used as a reference. With this technique, we can easily monitor the evolutionary drift of several GPCR sub-families from cnidarians to humans. Results support a model of radiative evolution of class A GPCRs from a central node formed by peptide receptors. The privileged directions obtained from the MDS analysis are interpretable in terms of three main evolutionary pathways related to specific sequence determinants. The first pathway was initiated by a deletion in transmembrane helix 2 (TM2) and led to three sub-families by divergent evolution. The second pathway corresponds to the differentiation of the amine receptors. The third pathway corresponds to parallel evolution of several sub-families in relation with a covarion process involving proline residues in TM2 and TM5. As exemplified with GPCRs, the MDS projection technique is an important tool to compare orthologous sequence sets and to help decipher the mutational events that drove the evolution of protein families.     

Proton-sensing and lysolipid-sensitive G-protein-coupled receptors: a novel type of multi-functional receptors

OGR1, GPR4, G2A, and TDAG8 share 40% to 50% homology with each other and seem to form a family of GPCRs. They have been described as receptors for lipid molecules such as sphingosylphosphorylcholine, lysophosphatidylcholine, and psychosine. Recent studies, however, have revealed that these receptors also sense extracellular protons or pH through histidine residues of receptors and stimulate a variety of intracellular signaling pathways through several species of hetero-trimeric G-proteins, including G_s, G_i, G_q, and G_12/13. Thus, this family of GPCR seems to recognize both lipid molecules and protons as ligands. Although our knowledge of proton-sensing and lysolipid-sensitive GPCRs is preliminary, the receptor levels and ligand levels especially protons are both sensitively modulated in response to a variety of microenvironmental changes. These results suggest a multiple role of proton-sensing GPCRs in a variety of physiological and pathophysiological states.     

Current developments in G-protein-coupled receptors.

The rate at which receptors have been cloned has recently increased dramatically--existing families have been extended and new families created. The rapid cloning by homology of 'orphan receptors' has also stimulated the development of a new reverse pharmacology.     

Discovery and validation of novel peptide agonists for G-protein-coupled receptors

G-protein-coupled receptors (GPCRs) represent an important group of targets for pharmaceutical therapeutics. The completion of the human genome revealed a large number of putative GPCRs. However, the identification of their natural ligands, and especially peptides, suffers from low discovery rates, thus impeding development of therapeutics based on these potential drug targets. We describe the discovery of novel GPCR ligands encrypted in the human proteome. Hundreds of potential peptide ligands were predicted by machine learning algorithms. In vitro screening of selected 33 peptides on a set of 152 GPCRs, including a group of designated orphan receptors, was conducted by intracellular calcium measurements and cAMP assays. The screening revealed eight novel peptides as potential agonists that specifically activated six different receptors in a dose-dependent manner. Most of the peptides showed distinct stimulatory patterns targeted at designated and orphan GPCRs. Further analysis demonstrated a significant in vivo effect for one of the peptides in a mouse inflammation model.     

Length analyses of mammalian G-protein-coupled receptors.

G-protein-coupled receptors (GPCRs) play a crucial role in mediating effects of extracellular messengers in a wide variety of biological systems, comprising the largest gene superfamily at least in mammals. Mammalian GPCRs are broadly classified into three families based on pharmacological properties and sequence similarities. These sequence similarities are largely confined to the seven transmembrane domains, and much less in the extracellular and intracellular loops and terminals (LTs). Together with the fact that the LTs vary considerably in length and sequence, the LT length of GPCRs has not been studied systematically. Here we have applied a statistical analysis to the length of the LTs of a wide variety of mammalian GPCRs in order to examine the existence of any trends in molecular architecture among a known mammalian GPCR population. Tree diagrams constructed by cluster analyses, using eight length factors in a given GPCR, revealed possible length relations among GPCRs and defined at least three groups. Most samples in Group J (joined) and Group M (minor) had an exceptionally long N-terminal and I3 loop, respectively; and other samples were considered as Group O (other/original). This length-based classification largely coincided with the conventional sequence- and pharmacology-based classification, suggesting that the LT length contains some biological information when analysed at the population level. Principle component analyses suggested the existence of inherent length differences between loops and terminals as well as between extracellular and intracellular LTs. Wilcoxon rank transformation tests unveiled statistically significant differences between Group O and Group J, not only in the N-terminal and I3 loop, but also in the E3 loop. Correlation analyses identified an E1-I2 length-correlation in Group O and Group J and an N-E3 length-correlation in Group J. Taken together, these results suggest a possible functional importance of LT length in the GPCR superfamily.     

Modelling G-protein-coupled receptors for drug design.

The G-protein coupled receptors form a large and diverse multi-gene superfamily with many important physiological functions. As such, they have become important targets in pharmaceutical research. Molecular modelling and site-directed mutagenesis have played an important role in our increasing understanding of the structural basis of drug action at these receptors. Aspects of this understanding, how these techniques can be used within a drug-design programme, and remaining challenges for the future are reviewed.     

G-protein-coupled receptors and melanoma

G-protein-coupled receptors (GPCR) are the largest family of receptors with over 500 members. Evaluation of GPCR gene expression in primary human tumors identified over-expression of GPCR in several tumor types. Analysis of cancer samples in different disease stages also suggests that some GPCR may be involved in early tumor progression and others may play a critical role in tumor invasion and metastasis. Currently, >50% of drug targets to various human diseases are based on GPCR. In this review, the relationships between several GPCR and melanoma development and/or progression will be discussed. Finally, the possibility of using one or more of these GPCR as therapeutic targets in melanoma will be summarized.     

A novel class of human type I interferons

The screening of a cDNA library prepared from mRNA of Sendai virus induced Namalwa (human Burkitt's lymphoma) cells, using a human IFN-alpha 2 DNA probe under conditions of low stringency, identified two weakly hybridizing clones containing sequences related to, but discernably different from those of the IFN-alpha class. Sequence and hybridization analysis of these cDNAs as well as expression in E. coli provided evidence that they encode proteins which have the characteristics of IFN type I but which are sufficiently diverged in sequence from both IFN-alpha s and IFN-beta to suggest that they are representatives of a new and distinct class of interferons named interferon-omega. Hybridization of these sequences to genomic DNA reveals that this class contains at least four members.     

Bioinformatical analysis of G-protein-coupled receptors

G-protein-coupled receptors play a key role in cellular signaling networks that regulate various physiological processes, such as vision, smell, taste, neurotransmission, secretion, inflammatory, immune responses, cellular metabolism, and cellular growth. These proteins are very important for understanding human physiology and disease. Many efforts in pharmaceutical research have been aimed at understanding their structure and function. Unfortunately, because they are difficult to crystallize and most of them will not dissolve in normal solvents, so far very few G-protein-coupled receptor structures have been determined. In contrast, more than 1000 G-protein-coupled receptor sequences are known, and many more are expected to become known soon. In view of the extremely unbalanced state, it would be very useful to develop a fast sequence-based method to identify their different types. This would no doubt have practical value for both basic research and drug discovery because the function or binding specificity of a G-protein coupled receptor is determined by the particular type it belongs to. To realize this, a statistical analysis has been performed for 566 G-protein-coupled receptors classified into seven different types. The results indicate that the types of G-protein-coupled receptors are predictable to a considerable accurate extent if a good training data set can be established for such a goal.     

A novel ubiquitously expressed alpha-latrotoxin receptor is a member of the CIRL family of G-protein-coupled receptors

Poisoning with alpha-latrotoxin, a neurotoxic protein from black widow spider venom, results in a robust increase of spontaneous synaptic transmission and subsequent degeneration of affected nerve terminals. The neurotoxic action of alpha-latrotoxin involves extracellular binding to its high affinity receptors as a first step. One of these proteins, CIRL, is a neuronal G-protein-coupled receptor implicated in the regulation of secretion. We now demonstrate that CIRL has two close homologs with a similar domain structure and high degree of overall identity. These novel receptors, which we propose to name CIRL-2 and CIRL-3, together with CIRL (CIRL-1) belong to a recently identified subfamily of large orphan receptors with structural features typical of both G-protein-coupled receptors and cell adhesion proteins. Northern blotting experiments indicate that CIRL-2 is expressed ubiquitously with highest concentrations found in placenta, kidney, spleen, ovary, heart, and lung, whereas CIRL-3 is expressed predominantly in brain similarly to CIRL-1. It appears that CIRL-2 can also bind alpha-latrotoxin, although its affinity to the toxin is about 14 times less than that of CIRL-1. When overexpressed in chromaffin cells, CIRL-2 increases their sensitivity to alpha-latrotoxin stimulation but also inhibits Ca2+-regulated secretion. Thus, CIRL-2 is a functionally competent receptor of alpha-latrotoxin. Our findings suggest that although the nervous system is the primary target of low doses of alpha-latrotoxin, cells of other tissues are also susceptible to the toxic effects of alpha-latrotoxin because of the presence of CIRL-2, a low affinity receptor of the toxin.     

Oligomerization of G-protein-coupled transmitter receptors

Examples of G-protein-coupled receptors that can be biochemically detected in homo- or heteromeric complexes are emerging at an accelerated rate. Biophysical approaches have confirmed the existence of several such complexes in living cells and there is strong evidence to support the idea that dimerization is important in different aspects of receptor biogenesis and function. While the existence of G-protein-coupled-receptor homodimers raises fundamental questions about the molecular mechanisms involved in transmitter recognition and signal transduction, the formation of heterodimers raises fascinating combinatorial possibilities that could underlie an unexpected level of pharmacological diversity, and contribute to cross-talk regulation between transmission systems. Because G-protein-coupled receptors are major pharmacological targets, the existence of dimers could have important implications for the development and screening of new drugs. Here, we review the evidence supporting the existence of G-protein-coupled-receptor dimerization and discuss its functional importance.     

Metabotropic G-protein-coupled glutamate receptors as therapeutic targets.

Metabotropic glutamate receptors have received considerable attention over the past decade in view of their relevance in multiple aspects of glutamatergic transmission. Recent advances in the molecular biology, pharmacology and medicinal chemistry of this family of G-protein-coupled receptors have led to therapeutic opportunities for subtype-selective modulators in brain disorders and diseases such as ischemia and schizophrenia.     

A green-fluorescent-protein-based assay for the characterization of G-protein-coupled receptors

A simple and sensitive system to monitor activation and pharmacology of G-protein-coupled receptors in mammalian cells is described. It is based on cAMP-responsible-element-regulated expression of green fluorescent protein (GFP). Cotransfection with appropriate G-protein-coupled receptors and subsequent activation with agonists induces expression of GFP in a dose-dependent manner. This system is suited for the analysis of most G-protein-coupled receptors, including those that are coupled to Gs, Gi, and Gq. It can replace reporter systems for G-protein-coupled receptors currently in use. Time-consuming and labor-intensive analysis is avoided because it is a noninvasive system, which allows multiple reprobing without disturbing the cells. In addition, adaptation to high-throughput approaches is possible. Together with human embryonic kidney cells, it is a zero-background expression system, making it ideally suited to the pharmacological characterization of cloned receptors, to expression cloning experiments, and to the identification of the natural ligand of orphan G-protein-coupled receptors.     

Dimerization in aminergic G-protein-coupled receptors: application of a hidden-site class model of evolution

G-Protein-coupled receptors (GPCRs) are an important superfamily of transmembrane proteins involved in cellular communication. Recently, it has been shown that dimerization is a widely occurring phenomenon in the GPCR superfamily, with likely important physiological roles. Here we use a novel hidden-site class model of evolution as a sequence analysis tool to predict possible dimerization interfaces in GPCRs. This model aims to simulate the evolution of proteins at the amino acid level, allowing the analysis of their sequences in an explicitly evolutionary context. Applying this model to aminergic GPCR sequences, we first validate the general reasoning behind the model. We then use the model to perform a family specific analysis of GPCRs. Accounting for the family structure of these proteins, this approach detects different evolutionarily conserved and accessible patches on transmembrane (TM) helices 4-6 in different families. On the basis of these findings, we propose an experimentally testable dimerization mechanism, involving interactions among different combinations of these helices in different families of aminergic GPCRs.     

G-protein-coupled receptors in intestinal chemosensation

Food intake is detected by the chemical senses of taste and smell and subsequently by chemosensory cells?in the gastrointestinal tract that link the composition of ingested foods to feedback circuits controlling gut motility/secretion, appetite, and peripheral nutrient disposal. G-protein-coupled receptors responsive to?a range of nutrients and other food components have been identified, and many are localized to intestinal chemosensory cells, eliciting hormonal and neuronal signaling to the brain and periphery. This review examines the role of G-protein-coupled receptors as signaling molecules in the gut, with a particular focus on pathways relevant to appetite and glucose homeostasis.