Ras GTPases: integrins' friends or foes?

Integrins are cell-surface receptors that mediate and coordinate cellular responses to the extracellular matrix (ECM). Cellular signalling pathways can regulate cell adhesion by altering the affinity and avidity of integrins for ECM. The Ras family of small G proteins, which includes H-ras, R-ras and Rap, are important elements in cellular signalling pathways that control integrin function.     

A role for G proteins in plant hormone signalling?

The G protein signalling pathway is one of the most highly conserved mechanisms that enables cells to sense and respond to changes in their environment. Essential components of this are cell surface G protein-coupled receptors (GPCRs) that perceive extracellular ligands, and heterotrimeric G proteins (G proteins) that transduce information from activated GPCRs to down-stream effectors such as enzymes or ion channels. It is now clear from a range of biochemical and molecular studies that some potential G protein signalling components exist in plants. The best examples of these are the seven transmembrane receptor homologue GCR1 and the G_α (GPA1) and G_β (Gβ1) subunit homologues of heterotrimeric G proteins. G protein agonists and antagonists are known to influence a variety of signalling events in plants and have been used to implicate G proteins in a range of signalling pathways that include the plant hormones gibberellin and auxin. Furthermore, antisense suppression of GCR1 expression in Arabidopsis leads to a phenotype that supports a role for this receptor in cytokinin signalling. This review considers the current evidence for and against functional G protein signalling pathways in higher plants and questions whether or not these might be involved in the action of certain plant hormones.     

Human herpesvirus-8-encoded signalling ligands and receptors

Analysis of the genome of human herpesvirus 8 (HHV-8) led to the discovery of several novel genes, unique among the characterized gammaherpesviruses. These include cytokines (interleukin-6 and chemokine homologues), two putative signal-transducing transmembrane proteins encoded by genes K1 and K15 at the genome termini, and an OX-2 (CD200) receptor homologue that had not previously been identified in a gammaherpesvirus. HHV-8 also specifies a diverged version of the gammaherpesvirus-conserved G protein-coupled chemokine receptor (vGCR) and a latently expressed protein unique to HHV-8 specified by open reading frame (ORF) K12. These cytokine and receptor homologues mediate signal transduction or modulate the activities of other endogenous cytokines and receptors to enhance viral productive replication, regulate latent-lytic switching, evade host attack, or mediate cell survival. The viral signalling ligands and receptors are also potential contributors to virus-associated diseases, Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease, and so represent potentially important targets for therapeutic and antiviral drugs. Understanding these proteins' modes of action and functions in viral biology and disease is therefore of considerable importance, and the subject of this review.     

Biochemical and genetic analysis of dominant-negative mutations affecting a yeast G-protein gamma subunit.

Heterotrimeric guanine nucleotide-binding proteins (G proteins) consisting of alpha, beta, and gamma subunits mediate signalling between cell surface receptors and intracellular effectors in eukaryotic cells. To define signalling functions of G gamma subunits (STE18 gene product) involved in pheromone response and mating in the yeast Saccharomyces cerevisiae, we isolated and characterized dominant-negative STE18 alleles. We obtained dominant-negative mutations that disrupt C-terminal sequences required for prenylation of G gamma precursors (CAAX box) and that affect residues in the N-terminal half of Ste18p. Overexpression of mutant G gamma subunits in wild-type cells blocked signal transduction; this effect was suppressed upon overexpression of G beta subunits. Mutant G gamma subunits may therefore sequester G beta subunits into nonproductive G beta gamma dimers. Because mutant G gamma subunits blocked the constitutive signal resulting from disruption of the G alpha subunit gene (GPA1), they are defective in functions required for downstream signalling. Ste18p bearing a C107Y substitution in the CAAX box displayed reduced electrophoretic mobility, consistent with a prenylation defect. G gamma subunits carrying N-terminal substitutions had normal electrophoretic mobilities, suggesting that these proteins were prenylated. G gamma subunits bearing substitutions in their N-terminal region or C-terminal CAAX box (C107Y) supported receptor-G protein coupling in vitro, whereas C-terminal truncations caused partial defects in receptor coupling.     

Bradykinin signalling to MAP kinase: cell-specific connections versus principle mitogenic pathways

Mitogenic signalling pathways from G protein-coupled receptors (GPCRs) to the mitogen-activated protein kinase (MAPK) cascade may involve alpha- or betagamma-subunits of heterotrimeric G proteins, receptor or non-receptor tyrosine kinases, adaptor molecules, phosphoinositide 3-kinases, protein kinase C, and probably other proteins. The majority of models describing the connection of different signalling proteins within a mitogenic pathway are based on experimental data obtained by co- and overexpression of epitope-tagged MAPK together with the respective GPCR and other signalling proteins of interest in transfectable cell lines. Here the link of the bradykinin B2 receptor (B2R) to MAPK in the COS-7 cell expression system is compared with mitogenic signalling pathways of bradykinin in various tumour cell lines. It becomes evident that in natural or tumour cells expressing individual amounts and different isoforms of signalling proteins completely other relations between B2R and MAPK may exist than in COS-7 cells, suggesting a high degree of cellular specificity in mitogenic signalling.     

Structure and functions of arrestins.

Transmembrane signal transductions in a variety of cell types that mediate signals as diverse as those carried by neurotransmitters, hormones, and sensory signals share basic biochemical mechanisms that include: (1) an extracellular perturbation (neurotransmitter, hormone, odor, light); (2) specific receptors; (3) coupling proteins, such as G proteins; and (4) effector enzymes or ion channels. Parallel to these amplification reactions, receptors are precisely inactivated by mechanisms that involve protein kinases and regulatory proteins called arrestins. The structure and functions of arrestins are the focus of this review.     

Cell signalling diversity of the Gqalpha family of heterotrimeric G proteins

Many receptors for neurotransmitters and hormones rely upon members of the Gqalpha family of heterotrimeric G proteins to exert their actions on target cells. Galpha subunits of the Gq class of G proteins (Gqalpha, G11alpha, G14alpha and G15/16alpha) directly link receptors to activation of PLC-beta isoforms which, in turn, stimulate inositol lipid (i.e. calcium/PKC) signalling. Although Gqalpha family members share a capacity to activate PLC-beta, they also differ markedly in their biochemical properties and tissue distribution which predicts functional diversity. Nevertheless, established models suggest that Gqalpha family members are functionally redundant and that their cellular responses are a result of PLC-beta activation and downstream calcium/PKC signalling. Growing evidence, however, indicates that Gqalpha, G11alpha, G14alpha and G15/16alpha are functionally diverse and that many of their cellular actions are independent of inositol lipid signalling. Recent findings show that Gqalpha family members differ with regard to their linked receptors and downstream binding partners. Reported binding partners distinct from PLC-beta include novel candidate effector proteins, various regulatory proteins, and a growing list of scaffolding/adaptor proteins. Downstream of these signalling proteins, Gqalpha family members exhibit unexpected differences in the signalling pathways and the gene expression profiles they regulate. Finally, genetic studies using whole animal models demonstrate the importance of certain Gqalpha family members in cardiac, lung, brain and platelet functions among other physiological processes. Taken together, these findings demonstrate that Gqalpha, G11alpha, G14alpha and G15/16alpha regulate both overlapping and distinct signalling pathways, indicating that they are more functionally diverse than previously thought.     

Molecular and functional characterization of proteins interacting with the C-terminal domains of 5-HT2 receptors: emergence of 5-HT2 "receptosomes"

Many cellular functions are carried out by multiprotein complexes. The last five years of research have revealed that many G-protein coupled receptor (GPCR) functions that are not mediated by G proteins involve protein networks, which interact with their intracellular domains. This review focuses on one family of GPCRs activated by serotonin, the 5-HT(2) receptor family, which comprises three closely related subtypes, the 5-HT(2A), the 5-HT(2B) and the 5-HT(2c) receptors. These receptors still raise particular interest, because a large number of psychoactive drugs including hallucinogens, anti-psychotics, anxiolytics and anti-depressants, mediate their action, at least in part, through activation of 5-HT(2) receptors. Recent studies based on two-hybrid screens, proteomic, biochemical and cell biology approaches, have shown that the C-terminal domains of 5-HT(2) receptors interact with intracellular proteins. To date, the protein network associated with the C-terminus of the 5-HT(2C) receptor has been the most extensively characterized, using a proteomic approach combining affinity chromatography, mass spectrometry and immunoblotting. It includes scaffolding proteins containing one or several PDZ domains, signalling proteins and proteins of the cytoskeleton. Data indicating that the protein complexes interacting with 5-HT(2) receptor C-termini tightly control receptor trafficking and receptor-mediated signalling will also be reviewed.     

Non-canonical functions of RGS proteins

Regulators of G protein signalling (RGS) proteins are united into a family by the presence of the RGS domain which serves as a GTPase-activating protein (GAP) for various Galpha subunits of heterotrimeric G proteins. Through this mechanism, RGS proteins regulate signalling of numerous G protein-coupled receptors. In addition to the RGS domains, RGS proteins contain diverse regions of various lengths that regulate intracellular localization, GAP activity or receptor selectivity of RGS proteins, often through interaction with other partners. However, it is becoming increasingly appreciated that through these non-RGS regions, RGS proteins can serve non-canonical functions distinct from inactivation of Galpha subunits. This review summarizes the data implicating RGS proteins in the (i) regulation of G protein signalling by non-canonical mechanisms, (ii) regulation of non-G protein signalling, (iii) signal transduction from receptors not coupled to G proteins, (iv) activation of mitogen-activated protein kinases, and (v) non-canonical functions in the nucleus.     

The calcium-sensing receptor and its interacting proteins

Seven membrane-spanning, or G protein-coupled receptors were originally thought to act through het-erotrimeric G proteins that in turn activate intracellular enzymes or ion channels, creating relatively simple, linear signalling pathways. Although this basic model remains true in that this family does act via a relatively small number of G proteins, these signalling systems are considerably more complex because the receptors interact with or are located near additional proteins that are often unique to a receptor or subset of receptors. These additional proteins give receptors their unique signalling personalities. The extracellular Ca-sensing receptor (CaR) signals via Galpha(i), Galpha(q) and Galpha(12/13), but its effects in vivo demonstrate that the signalling pathways controlled by these subunits are not sufficient to explain all its biologic effects. Additional structural or signalling proteins that interact with the CaR may explain its behaviour more fully. Although the CaR is less well studied in this respect than other receptors, several CaR-interacting proteins such as filamin, a potential scaffolding protein, receptor activity modifying proteins (RAMPs) and potassium channels may contribute to the unique characteristics of the CaR. The CaR also appears to interact with additional proteins common to other G protein-coupled receptors such as arrestins, G protein receptor kinases, protein kinase C, caveolin and proteins in the ubiquitination pathway. These proteins probably represent a few initial members of CaR-based signalling complex. These and other proteins may not all be associated with the CaR in all tissues, but they form the basis for understanding the complete nature of CaR signalling.     

Gαq signalling: The new and the old

In the last few years the interactome of Gαq has expanded considerably, contributing to improve our understanding of the cellular and physiological events controlled by this G alpha subunit. The availability of high-resolution crystal structures has led the identification of an effector-binding region within the surface of Gαq that is able to recognise a variety of effector proteins. Consequently, it has been possible to ascribe different Gαq functions to specific cellular players and to identify important processes that are triggered independently of the canonical activation of phospholipase Cβ (PLCβ), the first identified Gαq effector. Novel effectors include p63RhoGEF, that provides a link between G protein-coupled receptors and RhoA activation, phosphatidylinositol 3-kinase (PI3K), implicated in the regulation of the Akt pathway, or the cold-activated TRPM8 channel, which is directly inhibited upon Gαq binding. Recently, the activation of ERK5 MAPK by Gq-coupled receptors has also been described as a novel PLCβ-independent signalling axis that relies upon the interaction between this G protein and two novel effectors (PKCζ and MEK5). Additionally, the association of Gαq with different regulatory proteins can modulate its effector coupling ability and, therefore, its signalling potential. Regulators include accessory proteins that facilitate effector activation or, alternatively, inhibitory proteins that downregulate effector binding or promote signal termination. Moreover, Gαq is known to interact with several components of the cytoskeleton as well as with important organisers of membrane microdomains, which suggests that efficient signalling complexes might be confined to specific subcellular environments. Overall, the complex interaction network of Gαq underlies an ever-expanding functional diversity that puts forward this G alpha subunit as a major player in the control of physiological functions and in the development of different pathological situations.     

Cannabinoid signalling

After their discovery, the two known cannabinoid receptors, CB(1) and CB(2), have been the focus of research into the cellular signalling mechanisms of cannabinoids. The initial assessment, mainly derived from expression studies, was that cannabinoids, via G(i/o) proteins, negatively modulate cyclic AMP levels, and activate inward rectifying K(+) channels. Recent findings have complicated this assessment on different levels: (1) cannabinoids include a wide range of compounds with varying profiles of affinity and efficacy at the known CB receptors, and these profiles do not necessarily match their biological activity; (2) CB receptors appear to be intrinsically active and possibly coupled to more than one type of G protein; (3) CB receptor signalling mechanisms are diverse and dependent on the system studied; (4) cannabinoids have other targets than CB receptors. The aim of this mini review is to discuss the current literature regarding CB receptor signalling pathways. These include regulation of adenylyl cyclase, MAP kinase, intracellular Ca(2+), and ion channels. In addition, actions of cannabinoids that are not mediated by CB(1) or CB(2) receptors are discussed.     

Recognition in cell adhesion. A comparative study of the conformations of RGD-containing peptides by Monte Carlo and NMR methods

Many of the proteins that mediate cell adhesion processes processes-fibronectin, fibrinogen, vitronectin, von Willebrand factor, osteopontin, laminin and various collagens--contain the amino acid sequence Arg-Gly-Asp. Short peptides that include this sequence have been shown to inhibit the interactions of cell adhesion proteins with their receptors and to have dramatic effects on developmental processes involving cellular recognition. In order to determine which conformations are accessible to Arg-Gly-Asp containing peptides, we analyzed tri-, tetra- and pentapeptides using molecular mechanics and Monte Carlo methods, and studied the solution conformations of the pentapeptide Gly-Arg-Gly-Asp-Ser using nuclear magnetic resonance techniques. The Monte Carlo method was used to: (a) identify the low energy conformations of the peptides and (b) evaluate their thermodynamic properties. In the case of the pentapeptide Gly-Arg-Gly-Asp-Gly, the four stable conformations include three with reverse turns and one open structure. The conformations found in this analysis are compatible with the nuclear magnetic resonance (nuclear Overhauser effect) data.     

Notch signalling in B cells

Notch signalling is likely to regulate multiple aspects of lymphoid development and function. During T cell development, Notch signalling is required for commitment of the earliest progenitor, and may also function during other developmental stages. T cell commitment from a common lymphoid progenitor occurs at the expense of B cell development, suggesting that Notch signalling inhibits the earliest stage of B lymphopoiesis. In contrast, recent evidence suggests that Notch promotes the development of marginal zone lymphocytes. Not only is Notch required for later stages of B cell development, but several viral proteins appear to utilize Notch signalling in B cells to mediate their functions. In this review, we will focus on potential roles of Notch signalling in B lymphopoiesis and also consider how viral proteins may utilize Notch signalling in B cells.     

A constitutively active GPCR retains its G protein specificity and the ability to form dimers

G protein-coupled receptors (GPCRs) are cell surface proteins which help to regulate the physiology of all the major organ systems within higher eukaryotes. They are stimulated by multiple ligands and activate a range of effector molecules to bring about changes in cell behaviour. The use of constitutively active mutants (CAMs) of GPCRs has enabled a better understanding of receptor activation as CAMs exhibit ligand-independent signalling negating the use of ligands. Here we introduce the fission yeast Schizosaccharomyces pombe as a host for producing CAMs, by describing the isolation and characterization of constitutive mutants of the P-factor receptor (Mam2). One mutant Mam2[P261L] contained a single-amino-acid substitution (Pro261 to Leu) within a region of high homology in GPCRs. Substitution of this proline leads to an 18-fold increase in ligand-independent signalling. We utilized Mam2[P261L] to investigate CAM activity by demonstrating that Mam2[P261L] is efficiently trafficked to the cell surface where it can form fully functional oligomeric complexes with the native receptor. Mam2[P261L] also retains the G protein specificity (RG-profile) of the native receptor and only induces constitutive signalling in the same G proteins. Finally, evidence is provided to indicate that CAM activity results from a reduction in the kinetics of G protein binding. This is the first time that S. pombe has been utilized for isolating and characterizing CAMs and the techniques employed will complement the current systems available for studying these important receptors.     

Two tumour necrosis factor receptors: structure and function

Tumour necrosis factor (TNF) exerts two main effects: a beneficial one as an anti-infection, anti-tumour cytokine, and a detrimental one in the systemic inflammatory response syndrome (SIRS). Two receptors (TNF-R) mediate these effects, but their precise role in different cell types is far from solved. TNF induces receptor oligomerization, an event that is believed to connect the receptors to downstream signalling pathways. Recent research suggests that several TNF-R-associated proteins, including kinases, may initiate cytoplasmic signal transduction.     

Wnt3a stimulation elicits G-protein-coupled receptor properties of mammalian Frizzled proteins

Receptors of the Fz (Frizzled) family initiate Wnt ligand-dependent signalling controlling multiple steps in organism development and carcinogenesis. Fz proteins possess seven transmembrane domains, and their signalling depends on heterotrimeric G-proteins in various organisms; however, Fz proteins constitute a distinct group within the GPCR (G-protein-coupled receptor) superfamily, and Fz signalling can be G-protein-independent in some experimental setups, leading to concerns about the GPCR nature of these proteins. In the present study, we demonstrate that mammalian Fz proteins act as GPCRs on heterotrimeric G(o/i) proteins. Addition of the Wnt3a ligand to rat brain membranes or cultured cells elicits Fz-dependent guanine-nucleotide exchange on G(o/i) proteins. These responses were sensitive to a Wnt antagonist and to pertussis toxin, which decouples the G(o/i) proteins from their receptors through covalent modification. The results of the present study provide the long-awaited biochemical proof of the GPCR nature of Fz receptors.     

Receptor crosstalk. Implications for cardiovascular function, disease and therapy.

There are at least three well-defined signalling cascades engaged directly in the physiological regulation of cardiac circulatory function: the beta1-adrenoceptors that control the cardiac contractile apparatus, the renin-angiotensin-aldosterone system involved in regulating blood pressure and the natriuretic peptides contributing at least to the factors determining circulating volume. Apart from these pathways, other cardiac receptor systems, particularly the alpha1-adrenoceptors, adenosine, endothelin and opioid receptors, whose physiological role may not be immediately evident, are also important with respect to regulating cardiovascular function especially in disease. These and the majority of other cardiovascular receptors identified to date belong to the guanine nucleotide binding (G) protein-coupled receptor families that mediate signalling by coupling primarily to three G proteins, the stimulatory (Gs), inhibitory (Gi) and Gq/11 proteins to stimulate the adenylate cyclases and phospholipases, activating a small but diverse subset of effectors and ion channels. These receptor pathways are engaged in crosstalk utilizing second messengers and protein kinases as checkpoints and hubs for diverting, converging, sieving and directing the G protein-mediated messages resulting in different signalling products. Besides, the heart itself is endowed with the means to harmonize these signalling mechanisms and to fend off potentially fatal consequences of functional loss of the essential signalling pathways via compensatory reserve pathways, or by inducing some adaptive mechanisms to be turned on, if and when required. This receptor crosstalk constitutes the underlying basis for sustaining a coherently functional circulatory entity comprising mechanisms controlling the contractile apparatus, blood pressure and circulating volume, both in normal physiology and in disease.