Src tyrosine kinase is a novel direct effector of G proteins

Heterotrimeric G proteins transduce signals from cell surface receptors to modulate the activity of cellular effectors. Src, the product of the first characterized proto-oncogene and the first identified protein tyrosine kinase, plays a critical role in the signal transduction of G protein-coupled receptors. However, the mechanism of biochemical regulation of Src by G proteins is not known. Here we demonstrate that Galphas and Galphai, but neither Galphaq, Galpha12 nor Gbetay, directly stimulate the kinase activity of downregulated c-Src. Galphas and Galphai similarly modulate Hck, another member of Src-family tyrosine kinases. Galphas and Galphai bind to the catalytic domain and change the conformation of Src, leading to increased accessibility of the active site to substrates. These data demonstrate that the Src family tyrosine kinases are direct effectors of G proteins.     

Control of voltage-dependent Ca2+ channels by G protein-coupled receptors

G proteins act as transducers between membrane receptors activated by extracellular signals and enzymatic effectors controlling the concentration of cytosolic signal molecules such as cAMP, cGMP, inositol phosphates and Ca2+. In some instances, the receptor/G protein-induced changes in the concentration of cytosolic signal molecules correlate with activity changes of voltage-dependent Ca2+ channels. Ca2+ channel modulation, in these cases, requires the participation of protein kinases whose activity is stimulated by cytosolic signal molecules. The respective protein kinases phosphorylate Ca2+ channel-forming proteins or unknown regulatory components. More recent findings suggest another membrane-confined mechanism that does not involve cytosolic signal molecules but rather a more direct control of voltage-dependent Ca2+ channels by G proteins. Modulation of Ca2+ channel activity that follows this apparently membrane-confined mechanism has been described to occur in neuronal, cardiac, and endocrine cells. The G protein involved in the hormonal stimulation of Ca2+ channels in endocrine cells may belong to the family of Gi-type G proteins, which are functionally uncoupled from activating receptors by pertussis toxin. The G protein Gs, which is activated by cholera toxin, may stimulate cardiac Ca2+ channels without the involvement of a cAMP-dependent intermediate step. Hormonal inhibition of Ca2+ channels in neuronal and endocrine cells is mediated by a pertussis toxin-sensitive G protein, possibly Go. Whether G proteins act by binding directly to Ca2+ channels or through interaction with as yet undetermined regulatory components of the plasma membrane remains to be clarified.     

The G protein G alpha(13) is required for growth factor-induced cell migration

Heterotrimeric G proteins are critical cellular signal transducers. They are known to directly relay signals from seven-transmembrane G protein-coupled receptors (GPCRs) to downstream effectors. On the other hand, receptor tyrosine kinases (RTKs), a different family of membrane receptors, signal through docking sites in their carboxy-terminal tails created by autophosphorylated tyrosine residues. Here we show that a heterotrimeric G protein, G alpha(13), is essential for RTK-induced migration of mouse fibroblast and endothelial cells. G alpha(13) activity in cell migration is retained in a C-terminal mutant that is defective in GPCR coupling, suggesting that the migration function is independent of GPCR signaling. Thus, G alpha(13) appears to be a critical signal transducer for RTKs as well as GPCRs. This broader role of G alpha(13) in cell migration initiated by two types of receptors could provide a molecular basis for the vascular system defects exhibited by G alpha(13) knockout mice.     

G protein-coupled receptor cross-talk: pivotal roles of protein phosphorylation and protein-protein interactions

G protein-coupled receptors are dynamically regulated. Such regulation is frequently associated with covalent posttranslational modifications, such as phosphorylation, and with regulatory elements. G protein-coupled receptor kinases and casein kinase 1alpha play key roles in agonist-dependent receptor phosphorylations. Cross-talk between different receptors frequently involves second messenger-activated proteins, such as protein kinase C and protein kinase A. There is some evidence indicating that such kinases may not only turn off receptors but also switch their coupling to different G proteins. Receptor tyrosine kinases may phosphorylate and regulate G protein-coupled receptors and recent evidence indicates that other kinases, such as Akt/protein kinase B and phosphoinositide 3-kinase, may participate in such regulations as integrators of signalling.Recent approaches have shed new light on G protein-coupled receptor interactions that provide novel mechanisms of action and regulation. G protein-coupled receptor activities go beyond G proteins and receptors can be partners of exquisitely assembled signalling complexes through molecular bridges composed of multidomain proteins. The possibilities of interaction increase enormously through the diversity of structural and functional domains present in complex proteins, many of them just known as predicted sequences.     

Functional analysis of cloned opioid receptors in transfected cell lines

Opioids modulate numerous central and peripheral processes including pain perception, neuroendocrine secretion and the immune response. The opioid signal is transduced from receptors through G proteins to various different effectors. Heterogeneity exists at all levels of the transduction process. There are numerous endogenous ligands with differing selectivities for at least three distinct opioid receptors (μ, δ, κ). G proteins activated by opioid receptors are generally of the pertussis toxin-sensitive Gi/Go class, but there are also opioid actions that are thought to involve Gq and cholera toxin-sensitive G proteins. To further complicate the issue, the actions of opioid receptors may be mediated by G-protein α subunits and/or βγ subunits. Subsequent to G protein activation several effectors are known to orchestrate the opioid signal. For example activation of opioid receptors increases phosphatidyl inositol turnover, activates K⁺ channels and reduces adenylyl cyclase and Ca²⁺ channel activities. Each of these effectors shows considerable heterogeneity. In this review we examine the opioid signal transduction mechanism. Several important questions arise: Why do opioid ligands with similar binding affinities have different potencies in functional assays? To which Ca²⁺ channel subtypes do opioid receptors couple? Do opioid receptors couple to Ca²⁺ channels through direct G protein interactions? Does the opioid-induced inhibition of vesicular release occur through modulation of multiple effectors? We are attempting to answer these questions by expressing cloned opioid receptors in GH₃ cells. Using this well characterized system we can study the entire opioid signal transduction process from ligand-receptor interaction to G protein-effector coupling and subsequent inhibition of vesicular release.     

G proteins: critical control points for transmembrane signals.

Heterotrimeric GTP-binding proteins (G proteins) that are made up of alpha and beta gamma subunits couple many kinds of cell-surface receptors to intracellular effector enzymes or ion channels. Every cell contains several types of receptors, G proteins, and effectors. The specificity with which G protein subunits interact with receptors and effectors defines the range of responses a cell is able to make to an external signal. Thus, the G proteins act as a critical control point that determines whether a signal spreads through several pathways or is focused to a single pathway. In this review, I will summarize some features of the structure and function of mammalian G protein subunits, discuss the role of both alpha and beta gamma subunits in regulation of effectors, the role of the beta gamma subunit in macromolecular assembly, and the mechanisms that might make some responses extremely specific and others rather diffuse.     

Arabidopsis G‐protein interactome reveals connections to cell wall carbohydrates and morphogenesis

The heterotrimeric G‐protein complex is minimally composed of Gα, Gβ, and Gγ subunits. In the classic scenario, the G‐protein complex is the nexus in signaling from the plasma membrane, where the heterotrimeric G‐protein associates with heptahelical G‐protein‐coupled receptors (GPCRs), to cytoplasmic target proteins called effectors. Although a number of effectors are known in metazoans and fungi, none of these are predicted to exist in their canonical forms in plants. To identify ab initio plant G‐protein effectors and scaffold proteins, we screened a set of proteins from the G‐protein complex using two‐hybrid complementation in yeast. After deep and exhaustive interrogation, we detected 544 interactions between 434 proteins, of which 68 highly interconnected proteins form the core G‐protein interactome. Within this core, over half of the interactions comprising two‐thirds of the nodes were retested and validated as genuine in planta. Co‐expression analysis in combination with phenotyping of loss‐of‐function mutations in a set of core interactome genes revealed a novel role for G‐proteins in regulating cell wall modification.     

Arrestin-mediated signaling: Is there a controversy?

The activation of the mitogen-activated protein(MAP) kinases extracellular signal-regulated kinase(ERK)1/2 was traditionally used as a readout of signaling of G protein-coupled receptors(GPCRs) via arrestins, as opposed to conventional GPCR signaling via G proteins. Several recent studies using HEK293 cells where all G proteins were genetically ablated or inactivated, or both non-visual arrestins were knocked out, demonstrated that ERK1/2 phosphorylation requires G protein activity, but does not necessarily require the presence of non-visual arrestins. This appears to contradict the prevailing paradigm. Here we discuss these results along with the recent data on gene edited cells and arrestinmediated signaling. We suggest that there is no real controversy. G proteins might be involved in the activation of the upstream-most MAP3Ks, although in vivo most MAP3K activation is independent of heterotrimeric G proteins, being initiated by receptor tyrosine kinases and/or integrins. As far as MAP kinases are concerned, the best-established role of arrestins is scaffolding of the three-tiered cascades(MAP3K-MAP2 K-MAPK). Thus, it seems likely that arrestins, GPCRbound and free, facilitate the propagation of signals in these cascades, whereas signal initiation via MAP3K activation may be independent of arrestins. Different MAP3Ks are activated by various inputs, some of which are mediated by G proteins, particularly in cell culture, where we artificially prevent signaling by receptor tyrosine kinases and integrins, thereby favoring GPCR-induced signaling. Thus, there is no reason to change the paradigm: Arrestins and G proteins play distinct non-overlapping roles in cell signaling.     

G protein regulation of phospholipase A2

Many neurotransmitters and hormones activate receptors that are known to be coupled to their effectors by GTP-binding regulatory proteins, G proteins. Activation of many of these same receptors elicits arachidonate release and metabolism. During the past few years, novel experimental techniques have revealed that in many cells arachidonate release is independent of generation of other second messengers, including inositol phosphates, diacylglycerols, and elevation in free intracellular calcium. Much evidence has accumulated to implicate phospholipase A2 as the enzyme catalyzing arachidonate release, and suggesting that this effector enzyme, too, is activated by G proteins. In neural tissues as well as epithelium, endothelium, contractile and connective tissues, and blood cells, G proteins coupled to receptors for a variety of peptide and nonpeptide neurotransmitters and hormones have been shown to directly activate phospholipase A2. In retinal rod outer segments, transducin is the coupling G protein, but the G proteins coupling receptor activation to phospholipase A2 in other cell types is less clear. Some are pertussis toxin-sensitive, whereas others are not, and evidence exists that the ras gene product G protein may also be coupled to and regulate phospholipase A2.     

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.     

The membrane-proximal portion of CD3 epsilon associates with the serine/threonine kinase GRK2

The activation of protein kinases is one of the primary mechanisms whereby T cell receptors (TCR) propagate intracellular signals. To date, the majority of kinases known to be involved in the early stages of TCR signaling are protein-tyrosine kinases such as Lck, Fyn, and ZAP-70. Here we report a constitutive association between the TCR and a serine/threonine kinase, which was mediated through the membrane-proximal portion of CD3 epsilon. Mass spectrometry analysis of CD3 epsilon-associated proteins identified G protein-coupled receptor kinase 2 (GRK2) as a candidate Ser/Thr kinase. Transient transfection assays and Western blot analysis verified the ability of GRK2 to interact with the cytoplasmic domain of CD3 epsilon within a cell. These findings are consistent with recent reports demonstrating the ability of certain G protein-coupled receptors (GPCR) and G proteins to physically associate with the alpha/beta TCR. Because GRK2 is primarily involved in arresting GPCR signals, its interaction with CD3 epsilon may provide a novel means whereby the TCR can negatively regulate signals generated through GPCRs.     

RGS proteins have a signalling complex: interactions between RGS proteins and GPCRs, effectors, and auxiliary proteins

The intracellular regulator of G protein signalling (RGS) proteins were first identified as GTPase activating proteins (GAPs) for heterotrimeric G proteins, however, it was later found that they can also regulate G protein-effector interactions in other ways that are still not well understood. There is increasing evidence that some of the effects of RGS proteins occur due to their ability to interact with multiprotein signalling complexes. In this review, we will discuss recent evidence that supports the idea that RGS proteins can bind to proteins other than Galpha, such as G protein coupled receptors (GPCRs, e.g. muscarinic, dopaminergic, adrenergic, angiotensin, interleukin and opioid receptors) and effectors (e.g. adenylyl cyclase, GIRK channels, PDEgamma, PLC-beta and Ca(2+) channels). Furthermore, we will investigate novel RGS binding partners (e.g. GIPC, spinophilin, 14-3-3) that underlie the formation of signalling scaffolds or govern RGS protein availability and/or activity.     

Detecting and imaging protein-protein interactions during G protein-mediated signal transduction in vivo and In situ by using fluorescence-based techniques

An important goal in cell biology has been to observe dynamic interactions between protein molecules within a living cell as they execute the reactions of a particular biochemical pathway. An important step toward achieving this goal has been the development of noninvasive fluorescence-based detection and imaging techniques for determining whether and when specific biomolecules in a cell become associated with one another. Furthermore, these techniques, which take advantage of phenomena known as bioluminescence- and fluorescence resonance energy transfer (BRET and FRET, respectively) as well as biomolecular fluorescence complementation (BiFC), can provide information about where and when protein-protein interactions occur in the cell. Increasingly BRET, FRET, and BiFC are being used to probe interactions between components involved in G protein-mediated signal transduction. Heptahelical (7TM) receptors, heterotrimeric guanine nucleotide binding proteins (G proteins) and their proximal downstream effectors constitute the core components of these ubiquitous signaling pathways. Signal transduction is initiated by the binding of agonist to heptahelical (7TM) receptors that in turn activate their cognate G proteins. The activated G protein subsequently regulates the activity of specific effectors. 7TM receptors, G proteins, and effectors are all membrane-associated proteins, and for decades two opposing hypotheses have vied for acceptance. The predominant hypothesis has been that these proteins move about independently of one another in membranes and that signal trandduction occurs when they encounter each other as the result of random collisions. The contending hypothesis is that signaling is propagated by organized complexes of these proteins. Until recently, the data supporting these hypotheses came from studying signaling proteins in solution, in isolated membranes, or in fixed cells. Although the former hypothesis has been favored, recent studies using BRET and FRET have generally supported the latter hypothesis as being the most likely scenario operating in living cells. In addition to the core components, there are many other proteins involved in G protein signaling, and BRET and FRET studies have been used to investigate their interactions as well. This review describes various BRET, FRET, and BiFC techniques, how they have been or can be applied to the study of G protein signaling, what caveats are involved in interpreting the results, and what has been learned about G protein signaling from the published studies.     

Diversity among the beta subunits of heterotrimeric GTP-binding proteins: characterization of a novel beta-subunit cDNA.

Heterotrimeric guanine nucleotide binding proteins transduce signals from cell surface receptors to intracellular effectors. The alpha subunit is believed to confer receptor and effector specificity on the G protein. This role is reflected in the diversity of genes that encode these subunits. The beta and gamma subunits are thought to have a more passive role in G protein function; biochemical data suggests that beta-gamma dimers are shared among the alpha subunits. However, there is growing evidence for active participation of beta-gamma dimers in some G protein mediated signaling systems. To further investigate this role, we examined the diversity of the beta subunit family in mouse. Using the polymerase chain reaction, we uncovered a new member of this family, G beta 4, which is expressed at widely varying levels in a variety of tissues. The predicted amino acid sequence of G beta 4 is 79% to 89% identical to the three previously known beta subunits. The diversity of beta gene products may be an important corollary to the functional diversity of G proteins.     

Recognition in the face of diversity: interactions of heterotrimeric G proteins and G protein-coupled receptor (GPCR) kinases with activated GPCRs

G protein-coupled receptors (GPCRs) represent the largest class of integral membrane protein receptors in the human genome. Despite the great diversity of ligands that activate these GPCRs, they interact with a relatively small number of intracellular proteins to induce profound physiological change. Both heterotrimeric G proteins and GPCR kinases are well known for their ability to specifically recognize GPCRs in their active state. Recent structural studies now suggest that heterotrimeric G proteins and GPCR kinases identify activated receptors via a common molecular mechanism despite having completely different folds.     

G proteins control diverse pathways of transmembrane signaling

Hormones, neurotransmitters, and autacoids interact with specific receptors and thereby trigger a series of molecular events that ultimately produce their biological effects. These receptors, localized in the plasma membrane, carry binding sites for ligands as diverse as peptides (e.g., glucagon, neuropeptides), lipids (e.g., prostaglandins), nucleosides and nucleotides (e.g., adenosine), and amines (e.g., catecholamines, serotonin). These receptors do not interest directly with their respective downstream effector (i.e., an ion channel and/or an enzyme that synthesizes a second messenger); rather, they control one or several target systems via the activation of an intermediary guanine nucleotide-binding regulatory protein or G protein. G proteins serve as signal transducers, linking extracellularly oriented receptors to membrane-bound effectors. Traffic in these pathways is regulated by a GTP (on)-GDP (off) switch, which is regulated by the receptor. The combination of classical biochemistry and recombinant DNA technology has resulted in the discovery of many members of the G protein family. These approaches, complemented in particular by electrophysiological experiments, have also identified several effectors that are regulated by G proteins. We can safely assume that current lists of G proteins and the functions that they control are incomplete.