G-protein-coupled receptors (GPCRs) form the largest class of membrane proteins and are an important target for therapeutic drugs. These receptors are highly dynamic proteins sampling a range of conformational states in order to fulfil their complex signalling roles. In order to fully understand GPCR signalling mechanisms it is necessary to extract the receptor protein out of the plasma membrane. Historically this has universally required detergents which inadvertently strip away the annulus of lipid in close association with the receptor and disrupt lateral pressure exerted by the bilayer. Detergent-solubilized GPCRs are very unstable which presents a serious hurdle to characterization by biophysical methods. A range of strategies have been developed to ameliorate the detrimental effect of removing the receptor from the membrane including amphipols and reconstitution into nanodics stabilized by membrane scaffolding proteins (MSPs) but they all require exposure to detergent. Poly(styrene-co-maleic acid) (SMA) incorporates into membranes and spontaneously forms nanoscale poly(styrene-co-maleic acid) lipid particles (SMALPs), effectively acting like a ‘molecular pastry cutter’ to ‘solubilize’ GPCRs in the complete absence of detergent at any stage and with preservation of the native annular lipid throughout the process. GPCR–SMALPs have similar pharmacological properties to membrane-bound receptor, exhibit enhanced stability compared with detergent-solubilized receptors and being non-proteinaceous in nature, are fully compatible with downstream biophysical analysis of the encapsulated GPCR.

Heteromers of G-protein-coupled receptors (GPCRs) have emerged as potential novel targets for drug development. Accumulating evidence indicates that GPCRs can form homodimers and heteromers, with homodimers being the predominant species and oligomeric receptors being formed as multiples of dimers. Recently, heterotetrameric structures have been proposed for dopamine D 1 receptor (D 1 R)–dopamine D 3 receptor (D 3 R) and adenosine A 2A receptor (A 2A R)–dopamine D 2 receptor (D 2 R) heteromers. The structural model proposed for these complexes is a heteromer constituted by two receptor homodimers. The existence of GPCR homodimers and heteromers provides a structural basis for inter-protomer allosteric mechanisms that might account for a multiplicity of unique pharmacological properties. In this review, we focus on the A 2A R–D 2 R heterotetramer as an example of an oligomeric structure that is key in the modulation of striatal neuronal function. We also review the interfaces involved in this and other recently reported heteromers of GPCRs. Furthermore, we discuss several published studies showing the ex vivo expression of A 2A R–D 2 R heteromers. The ability of A 2A R agonists to decrease the affinity of D 2 R agonists has been reported and, on the basis of this interaction, A 2A R antagonists have been proposed as potential drugs for the treatment of Parkinson's disease. The heterotetrameric structure of the A 2A R–D 2 R complex offers a novel model that can provide new clues about how to adjust the drug dosage to the expected levels of endogenous adenosine.

The understanding of binding interactions between any protein and a small molecule plays a key role in the rationalization of affinity and selectivity and is essential for an efficient structure-based drug discovery (SBDD) process. Clearly, to begin SBDD, a structure is needed, and although there has been fantastic progress in solving G-protein-coupled receptor (GPCR) crystal structures, the process remains quite slow and is not currently feasible for every GPCR or GPCR–ligand complex. This situation significantly limits the ability of X-ray crystallography to impact the drug discovery process for GPCR targets in ‘real-time’ and hence there is still a need for other practical and cost-efficient alternatives. We present here an approach that integrates our previously described hierarchical GPCR modelling protocol (HGMP) and the fragment molecular orbital (FMO) quantum mechanics (QM) method to explore the interactions and selectivity of the human orexin-2 receptor (OX 2 R) and its recently discovered nonpeptidic agonists. HGMP generates a 3D model of GPCR structures and its complexes with small molecules by applying a set of computational methods. FMO allows ab initio approaches to be applied to systems that conventional QM methods would find challenging. The key advantage of FMO is that it can reveal information on the individual contribution and chemical nature of each residue and water molecule to the ligand binding that normally would be difficult to detect without QM. We illustrate how the combination of both techniques provides a practical and efficient approach that can be used to analyse the existing structure–function relationships (SAR) and to drive forward SBDD in a real-world example for which there is no crystal structure of the complex available.

Epacs (exchange proteins directly activated by cAMP) act as guanine-nucleotide-exchange factors for the Ras-like small G-proteins Rap1 and Rap2, and are now recognized as incontrovertible factors leading to complex and diversified cAMP signalling pathways. Given the critical role of cAMP in the regulation of cardiac function, several studies have investigated the functional role of Epacs in the heart, providing evidence that Epacs modulate intracellular Ca 2+ and are involved in several cardiac pathologies such as cardiac hypertrophy and arrhythmia. The present review summarizes recent data on the Epac signalling pathway and its role in cardiac pathophysiology. We also discuss recent advances in the discovery of novel pharmacological modulators of Epacs that were identified by high-throughput screening and their therapeutic potential for the treatment of cardiac disorders.

GPCR (G-protein-coupled receptor) signalling at the plasma membrane is under tight control. In the case of neuropeptides such as SP (substance P), plasma membrane signalling is regulated by cell-surface endopeptidases (e.g. neprilysin) that degrade extracellular neuropeptides, and receptor interaction with β-arrestins, which uncouple receptors from heterotrimeric G-proteins and mediate receptor endocytosis. By recruiting GPCRs, kinases and phosphatases to endocytosed GPCRs, β-arrestins assemble signalosomes that can mediate a second wave of signalling by internalized receptors. Endosomal peptidases, such as ECE-1 (endothelin-converting enzyme-1), can degrade SP in acidified endosomes, which destabilizes signalosomes and allows receptors, freed from β-arrestins, to recycle and resensitize. By disassembling signalosomes, ECE-1 terminates β-arrestin-mediated endosomal signalling. These mechanisms have been studied in model cell systems, and the relative importance of plasma membrane and endosomal signalling to complex pathophysiological processes, such as inflammation, pain and proliferation, is unclear. However, deletion or inhibition of metalloendopeptidases that control neuropeptide signalling at the plasma membrane and in endosomes has marked effects on inflammation. Neprilysin deletion exacerbates inflammation because of diminished degradation of pro-inflammatory SP. Conversely, inhibition of ECE-1 attenuates inflammation by preventing receptor recycling/resensitization, which is required for sustained pro-inflammatory signals from the plasma membrane. β-Arrestin deletion also affects inflammation because of the involvement of β-arrestins in pro-inflammatory signalling and migration of inflammatory cells. Knowledge of GPCR signalling in specific subcellular locations provides insights into pathophysiological processes, and can provide new opportunities for therapy. Selective targeting of β-arrestin-mediated endosomal signalling or of mechanisms of receptor recycling/resensitization may offer more effective and selective treatments than global targeting of cell-surface signalling.

The covalent attachment of palmitic acid to one or more cysteine residues (S-palmitoylation) is a widespread modification of signalling proteins. With the finding that palmitoylation is a dynamic process, it is now widely accepted that repeated cycles of palmitoylation/depalmitoylation could be involved in the regulation of multiple signalling processes. Palmitoylation also represents a common post-translational modification of the GPCRs (G-protein-coupled receptors). Functionally, palmitoylation of GPCRs has been shown to play a central role in the regulation of multiple receptor functions, including determining the efficiency and selectivity of G-protein coupling, receptor phosphorylation and desensitization, endocytosis and transport to the plasma membrane. The present review summarizes our current knowledge of the palmitoylation of serotonin (5-hydroxytryptamine) receptors and its role in the regulation of receptor functions.

The papers resulting from the recent Biochemical Society Focused Meeting ‘G-Protein-Coupled Receptors: from Structural Insights to Functional Mechanisms’ held in Prato in September 2012 are introduced in the present overview. A number of future goals for GPCR (G-protein-coupled receptor) research are considered, including the need to develop biophysical and computational methods to explore the full range of GPCR conformations and their dynamics, the need to develop methods to take this into account for drug discovery and the importance of relating observations on isolated receptors or receptors expressed in model systems to receptor function in vivo .

Recent advances in the structural biology of GPCRs (G-protein-coupled receptors) have provided insights into their structure and function. Comparisons of the visual and ligand-activated receptors highlight the unique elements of rhodopsin that allow it to function as a highly sensitive dim-light photoreceptor in vertebrates, as well as the common elements that it shares with the large class A GPCR family. However, despite progress, a number of questions remain unanswered about how these receptors are activated.

GPCRs (G-protein-coupled receptors) are seven-transmembrane helix proteins that transduce exogenous and endogenous signals to modulate the activity of downstream effectors inside the cell. Despite the relevance of these proteins in human physiology and pharmaceutical research, we only recently started to understand the structural basis of their activation mechanism. In the period 2008–2011, nine active-like structures of GPCRs were solved. Among them, we have determined the structure of light-activated rhodopsin with all the features of the active metarhodopsin-II, which represents so far the most native-like model of an active GPCR. This structure, together with the structures of other inactive, intermediate and active states of rhodopsin constitutes a unique structural framework on which to understand the conserved aspects of the activation mechanism of GPCRs. This mechanism can be summarized as follows: retinal isomerization triggers a series of local structural changes in the binding site that are amplified into three intramolecular activation pathways through TM (transmembrane helix) 5/TM3, TM6 and TM7/TM2. Sequence analysis strongly suggests that these pathways are conserved in other GPCRs. Differential activation of these pathways by ligands could be translated into the stabilization of different active states of the receptor with specific signalling properties.

The lipidic cubic phase method for crystallizing membrane proteins has posted some high-profile successes recently. This is especially true in the area of G-protein-coupled receptors, with six new crystallographic structures emerging in the last 3½ years. Slowly, it is becoming an accepted method with a proven record and convincing generality. However, it is not a method that is used in every membrane structural biology laboratory and that is unfortunate. The reluctance in adopting it is attributable, in part, to the anticipated difficulties associated with handling the sticky viscous cubic mesophase in which crystals grow. Harvesting and collecting diffraction data with the mesophase-grown crystals is also viewed with some trepidation. It is acknowledged that there are challenges associated with the method. However, over the years, we have worked to make the method user-friendly. To this end, tools for handling the mesophase in the pico- to nano-litre volume range have been developed for efficient crystallization screening in manual and robotic modes. Glass crystallization plates have been built that provide unparalleled optical quality and sensitivity to nascent crystals. Lipid and precipitant screens have been implemented for a more rational approach to crystallogenesis, such that the method can now be applied to a wide variety of membrane protein types and sizes. In the present article, these assorted advances are outlined, along with a summary of the membrane proteins that have yielded to the method. The challenges that must be overcome to develop the method further are described.

Phototransduction in vertebrates represents a paradigm of signalling pathways, in particular those mediated by G-protein-coupled receptors. The variety of protein–protein, protein–ion and protein–nucleotide interactions makes up an intricate network which is finely regulated by activating–deactivating molecules and chemical modifications. The holistic systems properties of the network allow for typical adaptation mechanisms, which ultimately result in fine adjustments of sensitivity and electrical response of the photoreceptor cells to the broad range of light stimuli. In the present article, we discuss a novel bottom-up strategy to study the phototransduction cascade in rod cells starting from the underlying biochemistry. The resulting network model can be simulated and the predicted dynamic behaviour directly compared with data from electrophysiological experiments performed on a wide range of illumination conditions. The advantage of applying procedures typical of systems theory to a well-studied signalling pathway is also discussed. Finally, the potential application to the study of the molecular basis of retinal diseases is highlighted through a practical example, namely the simulation of conditions related to Leber congenital amaurosis.

SGLT1 (Na + /glucose co-transporter 1) transports the dietary sugars, D -glucose and D -galactose, from the lumen of the intestine into enterocytes. SGLT1 regulation has important consequences for the provision of glucose to the respiring tissues and is therefore essential for maintaining glucose homoeostasis. SGLT1 expression is directly regulated in response to changes in the sugar content of the diet. To monitor these variations, there is a requirement for a glucose-sensing system located on the luminal membrane of gut cells. This short review focuses on recent findings on intestinal sugar sensing and the downstream mechanisms responsible for enhancement in SGLT1 expression.

Structure-based design has gained credibility as a valuable component of the modern drug discovery process. The technique of SSNMR (solid-state NMR) promises to be a useful counterpart to the conventional experimental techniques of X-ray crystallography and solution-state NMR for providing structural features of drug targets that can guide medicinal chemistry towards drug candidates. This article highlights some recent SSNMR approaches from our group for identifying active compounds, such as enzyme inhibitors, receptor antagonists and peptide agents, that prevent the aggregation of amyloid proteins involved in neurodegenerative diseases. It is anticipated that the use of SSNMR in drug discovery will become more widespread in the wake of advances in hardware and methodological developments.

The secretin receptor is prototypic of Class II GPCRs (G-protein-coupled receptors), based on its structural and functional characteristics and those of its natural agonist ligand. Secretin represents a linear 27-residue peptide with diffuse pharmacophoric domain. The secretin receptor includes the typical signature sequences for this receptor family within its predicted transmembrane segments and the highly conserved six cysteine residues contributing to three intradomain disulfide bonds within its long N-terminus. This domain is critical for secretin binding based on receptor mutagenesis and photoaffinity labelling studies. Full agonist analogues of secretin incorporating a photolabile moiety at various positions throughout the pharmacophore covalently label residues within this region, while only N-terminal probes have labelled the core helical bundle domain. Combining insights coming from receptor structural studies, peptide structure–activity relationship considerations, photoaffinity labelling, and application of fluorescence techniques has resulted in the development of a working model of the secretin–receptor complex. This supports the initial docking of the peptide agonist within a cleft in the receptor N-terminus, providing the opportunity for an endogenous sequence within that domain to interact with the core of the receptor. This interaction is believed to be key in the molecular basis of conformational change associated with activation of this receptor. The site of action of this endogenous agonist could also provide a possible target for small molecule agonists to act.

β-Arrestins 1 and 2 are ubiquitously expressed intracellular adaptor and scaffolding proteins that play important roles in GPCR (G-protein-coupled receptor) desensitization, internalization, intracellular trafficking and G-protein-independent signalling. Recent developments in BRET (bioluminescence resonance energy transfer) technology enable novel insights to be gained from real-time monitoring of GPCR–β-arrestin complexes in live cells for prolonged periods. In concert with confocal microscopy, assays for studying internalization and recycling kinetics such as ELISAs, and techniques for measuring downstream signalling pathways such as those involving MAPKs (mitogen-activated protein kinases), investigators can now use a range of experimental tools to elucidate the ever-expanding roles of β-arrestins in mediating GPCR function.

An actual paradigm for activation and regulation of the GPCR (G-protein-coupled receptors)/seven-transmembrane helix family of receptors essentially emerges from extensive studies of the largest family of receptors, the GPCR-A/rhodopsin family. The mechanisms regulating the GPCR-B family signal transduction are less precisely understood due in part to the lack of the conserved signatures of the GPCR-A family (E/DRY, NPXXY) and in part to the absence of a reliable receptor modelling, although some studies suggest that both families share similar features. Here, we try to highlight the current knowledge of the activation and the regulation of the VIP (vasoactive intestinal peptide) receptors, namely VPAC (VIP/pituitary adenylate cyclase-activating peptide receptor) 1 and 2. This includes search for amino acids involved in the stabilization of the receptor active conformation and in coupling to G-proteins, signalling pathways activated in response to VIP, agonist-dependent receptor down-regulation, phosphorylation and internalization as well as pharmacological consequences of receptor hetero-dimerization.

A full picture of the similarities between Family A and Family B GPCRs (G-protein coupled receptors) has been frustrated by the lack of clear homology between the respective sequences. Here, we review previous computational studies on GPCR dimerization in which the putative dimerization interfaces have been analysed using entropy, the ET (evolutionary trace) method and related methods. The results derived from multiple sequence alignments of Family A subfamilies have been mapped on to the rhodopsin crystal structure using standard alignments. Similarly, the results for the Family B alignments have been mapped on to the rhodopsin crystal structure using the ‘cold-spot’ alignment. For both Family A and Family B GPCRs, the sequence analysis indicates that there are functional sites on essentially all transmembrane helices, consistent with the parallel daisy chain model of GPCR oligomerization in which each GPCR makes interactions with a number of neighbouring GPCRs. The results are not too sensitive to the quality of the alignment. Molecular Dynamics simulations of the activation process within a single transmembrane bundle of the rhodopsin and the β 2 -adrenergic receptor have been reviewed; the key observation, which is consistent with other computational studies, is that there is a translation and bending of helix 6, which contributes to a significant opening out of the intracellular face of the receptor, as shown in the accompanying movies. The simulations required the application of specific experiment-derived harmonic and half-harmonic distance restraints and so the application of such simulations to Family B GPCRs requires considerable care because of the alignment problem. Thus, in order to address the alignment problem, we have exploited the observation that GCR1, a plant GPCR, has homology with Family A, Family B and Family E GPCRs. The resulting alignment for transmembrane helix 3 is presented.

Chemokines are a family of small basic proteins which induce the directed migration of cells, notably leucocytes, by binding to specific GPCRs (G-protein-coupled receptors). Both chemokines and their receptors have been implicated in a host of clinically important diseases, leading to the notion that antagonism of the chemokine–chemokine receptor network may be therapeutically advantageous. Consequently, considerable effort has been put into the development of small-molecule antagonists of chemokine receptors and several such compounds have been described in the literature. One curious by-product of this activity has been the description of several small-molecule agonists of the receptors, which are typically discovered following the optimization of lead antagonists. In this review we discuss these findings and conclude that these small-molecule agonists might be exploited to further our understanding of the molecular mechanisms by which chemokine receptors are activated.

The receptor for GLP-1 [glucagon-like peptide-1-(7–36)-amide] is a member of the ‘Family B’ of GPCRs (G-protein-coupled receptors) comprising an extracellular N-terminal domain containing six conserved cysteine residues (the N-domain) and a core domain (or J-domain) comprising the seven transmembrane helices and interconnecting loop regions. According to the two-domain model for peptide binding, the N-domain is primarily responsible for providing most of the peptide binding energy, whereas the core domain is responsible for binding the N-terminal region of the peptide agonists and transmitting the signal to the intracellular G-protein. Two interesting differences between the binding properties of two GLP-1 receptor agonists, GLP-1 and EX-4 (exendin-4), can be observed. First, while GLP-1 requires its full length to maintain high affinity, the eight N-terminal residues of EX-4 can be removed with little reduction in affinity. Secondly, EX-4 (but not GLP-1) can bind to the fully isolated N-domain of the receptor with an affinity matching that of the full-length receptor. In order to better understand these differences, we have studied the interaction between combinations of full-length or truncated ligands with full-length or truncated receptors.

Agonist efficacy is a measure of how well an agonist can stimulate a response system linked to a receptor. Efficacy can be assessed in functional assays and various parameters ( E max , K A /EC 50 , E max · K A /EC 50 ) determined. The E max · K A /EC 50 parameter provides a good estimate of efficacy across the full range of efficacy. A convenient assay for the efficacy of agonists for some receptors is provided by the [ 35 S]GTP[S] (guanosine 5′-[γ-[ 35 S]thio]triphosphate)-binding assay. In this assay, the normal GTP-binding event in GPCR (G-protein-coupled receptor) activation is replaced by the binding of the non-hydrolysable analogue [ 35 S]GTP[S]. This assay may be used to profile ligands for their efficacy, and an example here is the D 2 dopamine receptor where an efficacy scale has been set up using this assay. The mechanisms underlying the assay have been probed. The time course of [ 35 S]GTP[S] binding follows a pseudo-first-order reaction with [ 35 S]GTP[S] binding reaching equilibrium after approx. 3 h. The [ 35 S]GTP[S]-binding event is the rate-determining step in the assay. Agonists regulate the maximal level of [ 35 S]GTP[S] bound, rather than the rate constant for binding. The [ 35 S]GTP[S]-binding assay therefore determines agonist efficacy on the basis of the amount of [ 35 S]GTP[S] bound rather than the rate of binding.

GPCRs (G-protein-coupled receptors) are a large family of structurally related proteins which mediate their effects by coupling to G-proteins. The V 1a R (V 1a vasopressin receptor) is a member of a family of related GPCRs that are activated by vasopressin {AVP ([Arg 8 ]vasopressin)}, OT (oxytocin) and related peptides. These receptors are members of a subfamily of Family A GPCRs called the neurohypophysial peptide hormone receptor family. GPCRs exhibit a conserved tertiary structure comprising a bundle of seven TM (transmembrane) helices linked by alternating ECLs (extracellular loops) and ICLs (intracellular loops). The cluster of TM helices is functionally important for ligand binding, and, furthermore, activation of GPCRs involves movement of these TM helices. Consequently, it might be assumed that the extracellular face of GPCRs is composed of peptide linkers that merely connect important TM helices. However, using a systematic mutagenesis approach and focusing on the N-terminus and the second ECL of the V 1a R, we have established that these extracellular domains fulfil a range of important roles with respect to GPCR signalling, including agonist binding, ligand selectivity and receptor activation.

The design and development of selective agonists acting at the OT (oxytocin)/AVP (vasopressin) receptors has been and continues to be a difficult task because of the great similarity among the different receptor subtypes as well as the high degree of chemical similarity between the active ligands. In recent decades, at least a thousand synthetic peptides have been synthesized and examined for their ability to bind to and activate the different OT/AVP receptors; an effort that has led to the identification of several receptor subtype-selective agonists in the rat. However, owing to species differences between rat and human AVP/OT receptors, these peptides do not exhibit the same selectivities in human receptor assays. Furthermore, the discovery of receptor promiscuity, which is the ability of a single receptor subtype to couple to several different G-proteins, has led to the definition of a completely new class of compounds, referred to here as coupling-selective ligands, which may activate, within a single receptor subtype, only a specific signalling pathway. Finally, the accumulating evidence that GPCRs (G-protein-coupled receptors) do not function as monomers, but as dimers/oligomers, opens up the design of another class of specific ligands, bivalent ligands, in which two agonist and/or antagonist moieties are joined by a spacer of the appropriate length to allow the simultaneous binding at the two subunits within the dimer. The pharmacological properties and selectivity profiles of these bivalent ligands, which remain to be investigated, could lead to highly novel research tools and potential therapeutic agents.

In April 2007, the Biochemical Society held a meeting to compare and contrast ligand binding and activation of Family A and B GPCRs (G-protein-coupled receptors). Being the largest class, Family A GPCRs usually receive the most attention, although a previous Biochemical Society meeting has focused on Family B GPCRs. The aim of the present meeting was to bring researchers of both families together in order to identify commonalities between the two. The present article introduces the proceedings of the meeting, briefly commenting on the focus of each of the following articles.

GPCRs (G-protein-coupled receptors) such as the M 1 muscarinic receptor have so far proved recalcitrant to direct structure determination. Nevertheless systematic mutagenesis, particularly alanine scanning, has advanced our understanding of their structure–function relationships. GPCRs exhibit multiple conformational states with different affinities for and abilities to activate their cognate G-proteins. Ligand binding alters these conformational equilibria, thus promoting or inhibiting signalling. Alanine-scanning mutagenesis probes the relative contributions of a particular amino acid side chain to the stability of the ground and activated states of the receptor and its complexes. These determine the phenotype of the mutant receptor. Classification of the phenotypes suggests functional roles for particular amino acid side chains, allowing us to group them accordingly. From a rhodopsin-based homology model of the M 1 mAChR, a coherent view emerges of how these clusters of residues function in ligand anchoring, transduction of binding energy, global structural stabilization and selective stabilization of the ground state or the activated state of the receptor. We can identify differences in ligand-binding modes, and suggest inter- and intra-molecular interactions that are weakened or broken, or formed or intensified during acetylcholine-induced activation. In due course, we may be able to extend these insights to activation by unconventional agonists.

This short review provides a broad, and therefore necessarily incomplete and personal, overview of G-protein-coupled receptors, which are often targets for a wide range of important drugs: I will discuss successively their structure, function and interactions with associated proteins. Examples will be drawn from work done over the last 30 years by scientists that worked at different times in my laboratories, mainly in the field of β-adrenoceptors, muscarinic acetylcholine, melatonin and angiotensin receptors.

Cannabinoids have been shown to possess anti-inflammatory and neuroprotective properties, which were proposed to occur mainly via activation of the G-protein-coupled receptor CB 1 (cannabinoid receptor 1). Recently, certain cannabinoids have been reported to be ligands for members of the nuclear receptor transcription factor superfamily known as PPARs (peroxisome-proliferator-activated receptors). This review summarizes the evidence for cannabinoid activation of PPARs and identifies a new intracellular target for cannabinoids as therapeutic agents for neuroprotective treatment.

The interaction of chemokines and GAGs (glycosaminoglycans) on endothelial surfaces is a crucial step for establishing a chemotactic gradient which leads to the functional presentation of chemokines to their GPCRs (G-protein-coupled receptors) and thus to activation of approaching leucocytes. Based on molecular modelling, biophysical investigations, cell-based and in vivo experiments, we have developed a novel concept for therapeutically interfering with chemokine–GAG interactions, namely dominant-negative chemokine mutants with improved GAG binding affinity and knocked-out GPCR activity. These recombinant proteins displace their wild-type chemokine counterparts from the natural proteoglycan co-receptors without being able to activate leucocytes via GPCRs. Our mutant chemokines therefore represent the first protein-based GAG antagonists with high therapeutic potential in inflammatory diseases.

G-protein-coupled receptors (GPCRs) and post-GPCR signalling components are expressed at low overall abundance in plasma membranes, yet they evoke rapid, high-fidelity responses. Considerable evidence suggests that GPCR signalling components are organized together in membrane microdomains, in particular lipid rafts, enriched in cholesterol and sphingolipids, and caveolae, a subset of lipid rafts that also possess the protein caveolin, whose scaffolding domain may serve as an anchor for signalling components. Caveolae were originally identified based on their morphological appearance but their role in compartmentation of GPCR signalling has been primarily studied by biochemical techniques, such as subcellular fractionation and immunoprecipitation. Our recent studies obtained using both microscopic and biochemical methods with adult cardiac myocytes show expression of caveolin not only in surface sarcolemmal domains but also at, or close to, internal regions located at transverse tubules/sarcoplasmic reticulum. Other results show co-localization in lipid rafts/caveolae of AC (adenylyl cyclase), in particular AC6, certain GPCRs, G-proteins and eNOS (endothelial nitric oxide synthase; NOS3), which generates NO, a modulator of AC6. Existence of multiple caveolin-rich microdomains and their expression of multiple modulators of signalling strengthen the evidence that caveolins and lipid rafts/caveolae organize and regulate GPCR signal transduction in eukaryotic cells.

The bioactive phospholipids, LPA (lysophosphatidic acid) and PA (phosphatidic acid), regulate pivotal processes related to the pathogenesis of cancer. Recently, we cloned a novel type of lipid kinase that phosphorylates monoacylglycerols (such as 2-arachidonoylglycerol, an endogenous cannabinoid receptor ligand) and diacylglycerols, to form LPA and PA, respectively. This AGK (acylglycerol kinase) is highly expressed in prostate cancer cell lines and the results reviewed here suggest that AGK might be a critical player in the initiation and progression of prostate cancer. Intriguingly, down-regulation of endogenous AGK inhibited EGF (epidermal growth factor), but not LPA-induced ERK1/2 (extracellular-signal-regulated kinase 1/2) activation and progression through the S-phase of the cell cycle. In this review, we will summarize the evidence demonstrating that AGK amplifies EGF growth signalling pathways that play an important role in the pathophysiology of prostate cancer. Because LPA has long been implicated as an autocrine and paracrine growth stimulatory factor for prostate cancer cells, the identification of this novel lipid kinase that regulates its production could provide new and useful targets for preventive or therapeutic measures.

The GABA B (γ-aminobutyric acid-B) receptor is composed of two subunits, GABA B1 and GABA B2 . Both subunits share structural homology with other class-III G-protein-coupled receptors. They contain two main domains, a heptahelical domain typical of all G-protein-coupled receptors and a large ECD (extracellular domain). It has not been demonstrated whether the association of these two subunits is always required for function. However, GABA B2 plays a major role in coupling with G-proteins, and GABA B1 has been shown to bind GABA. To date, only ligands interacting with GABA B1 -ECD have been identified. In the present study, we explored the mechanism of action of CGP7930, a compound described as a positive allosteric regulator of the GABA B receptor. We have shown that it can weakly activate the wild-type GABA B receptor, but also the GABA B2 expressed alone, thus being the first described agonist of GABA B2 . CGP7930 retains its weak agonist activity on a GABA B2 subunit deleted of its ECD. Thus the heptahelical domain of GABA B2 behaves similar to a rhodopsin-like receptor. These results open new strategies for studying the mechanism of activation of GABA B receptor and examine any possible role of GABA B2 .

GPCRs (G-protein-coupled receptors) are a large family of structurally related proteins, which mediate their effects by coupling with G-proteins. Despite responding to a range of very diverse stimuli, these receptors exhibit a conserved tertiary structure comprising a bundle of seven TM (transmembrane) helices linked by alternating ECLs (extracellular loops) and ICLs (intracellular loops). The hydrophobic environment formed by the cluster of TM helices is functionally important. For example, the 11- cis retinal chromophore of rhodopsin forms a protonated Schiff base linkage to a lysine in TM7, deep within the helical bundle, and small ligands, such as amine neurotransmitters and non-peptide analogues of peptide hormones, also bind within the corresponding region of their cognate receptors. In addition, activation of GPCRs involves relative movement of TM helices to present G-protein interaction sites across the intracellular face of the receptor. Consequently, it might be assumed that the ECLs of the GPCR are inert peptide linkers that merely connect important TM helices. Focusing on ECL3 (third ECL), it is becoming increasingly apparent that this extracellular domain can fulfil a range of important roles with respect to GPCR signalling, including agonist binding, ligand selectivity and receptor activation.

The kinetics of G-protein-coupled receptor activation and deactivation has, so far, been measured only indirectly, most frequently by assessing the production of various second messengers. We have developed methods based on fluorescence resonance energy transfer to quantify the kinetics of receptor activation by agonist (measured as conformational change in the receptor), the kinetics of G-protein activation (measured as G-protein subunit rearrangement) and the kinetics of receptor inactivation by arrestins (measured as receptor–arrestin interaction). Using these methods, we show that receptor activation by agonists and signalling to G-proteins occur on the subsecond time scale, whereas receptor desensitization is limited by receptor phosphorylation and proceeds more slowly.

The ternary complex model suggests that G-protein-coupled receptors resonate between inactive (R) and active (R*) forms. Physiologically, R sites ordinarily predominate with a few R* sites giving rise to basal activity. Agonists recognize, stabilize and increase the R* population, thus altering intracellular activity. There is evidence to suggest the possibility of a spectrum of conformations between R and R*. Our aim is to study the consequences of putative GR (glucagon receptor)-activating mutations using glucagon and partial agonist des-His 1 -[Glu 9 ]glucagon amide (glucagon-NH 2 ). Alanine substitution in TM (transmembrane) helix 2 of Arg 173 or of His 177 detrimentally affected glucagon and glucagon-NH 2 response maxima. TM2 receptor mutant, Phe 181 -Ala, displayed reduced maximum cAMP accumulation in response to glucagon-NH 2 . Thr 353 -Cys (TM6) and Glu 406 -Ala (TM7) receptors demonstrated constitutive activity and enhanced EC 50 values for glucagon-NH 2 ; Arg 346 -Ala (TM6) and Asn 404 -Ala (TM7) receptors were activated by sub-fmol glucagon concentrations, yet were not constitutively active and demonstrated wild-type receptor-like EC 50 values for glucagon-NH 2 . Unlike Arg 346 -Ala receptors, Thr 353 -Cys, Asn 404 -Ala and Glu 406 -Ala receptors demonstrated improved EC 50 values for glucagon, whereas their maximal responses to and their affinity for glucagon were comparable with the wild-type receptor. In contrast, despite slightly reduced glucagon-NH 2 affinity, Arg 346 -Ala, Thr 353 -Cys, Asn 404 -Ala and Glu 406 -Ala receptors displayed glucagon-NH 2 response maxima that exceeded those seen for wild-type receptors. Interestingly, we observed biphasic glucagon-mediated signalling responses. Our results are consistent with the concept of different agonists promoting the formation of distinct active states from partially active R* low to fully active R* high forms.

Cardiac function is controlled by GPCRs (G-protein-coupled receptors) which exert their function by triggering numerous signalling pathways, including the activation of PI3K (phosphoinositide 3-kinase). The GPCR-activated PI3Kγ is weakly expressed in the heart, but the deletion of its expression in mice causes remarkable phenotypes. Indeed, the lack of PI3Kγ does not modify heart rate and blood pressure, but does increase contractility, particularly in response to stimuli that enhance cardiac contractile force, such as catecholamines. Consistently, treatment of mutant cardiomyocytes with β-adrenergic agonists causes an abnormal increase in the elevation of cAMP production. On the other hand, PI3Kγ appears to play a role in mediating the contractile depression exerted by other GPCR agonists, such as PAF (platelet-activating factor), that are released in pathological conditions, such as after an ischaemic insult. The receptor for PAF coupled to G i activates PI3Kγ, which, in turn, is essential to promote Akt phosphorylation, NOSIII (nitric oxide synthase isoform III) activation and the production of nitric oxide, a well characterized cardiodepressing agent. As a whole, PI3Kγ appears to negatively control cardiac contractility through different signalling mechanisms, thus becoming a possible drug target for the treatment of critical human cardiac pathologies, such as infarction or heart failure.

The EGFR (epidermal growth factor receptor) plays a key role in the regulation of essential normal cellular processes and in the pathophysiology of hyperproliferative diseases such as cancer. Recent investigations have demonstrated that GPCRs (G-protein-coupled receptors) are able to utilize the EGFR as a downstream signalling partner in the generation of mitogenic signals. This cross-talk mechanism combines the broad diversity of GPCRs with the signalling capacities of the EGFR and has emerged as a general concept in a multitude of cell types. The molecular mechanisms of EGFR signal transactivation involve processing of transmembrane growth factor precursors by metalloproteases which have been recently identified as members of the ADAM ( a d isintegrin a nd m etalloprotease) family of zinc-dependent proteases. Subsequently, the EGFR transmits signals to prominent downstream pathways, such as mitogen-activated protein kinases, the phosphoinositide 3-kinase/Akt pathway and modulation of ion channels. Analysis of GPCR-induced EGFR activation in more than 60 human carcinoma cell lines derived from different tissues has demonstrated the broad relevance of this signalling mechanism in cancer. Moreover, EGFR signal transactivation was linked to diverse biological processes in human cancer cells, such as cell proliferation, migration and anti-apoptosis. Together with investigations revealing the importance of this GPCR–EGFR cross-talk mechanism in cardiac hypertrophy, Helicobacter pylori -induced pathophysiological processes and cystic fibrosis, these findings support an important role for GPCR ligand-dependent EGFR signal transactivation in diverse pathophysiological disorders.

S1P (sphingosine 1-phosphate) is the ligand for a family of specific G-protein-coupled receptors that regulate a wide variety of important cellular functions, including vascular maturation, angiogenesis, cell growth, survival, cytoskeletal rearrangements and cell motility. However, S1P also may have intracellular functions. In this review, we discuss two examples that clearly indicate that intracellularly generated and exogenous S1P can regulate biological processes by divergent pathways.