Resistance to inhibitors of cholinesterase-8A (Ric-8A) is critical for growth factor receptor-induced actin cytoskeletal reorganization

Heterotrimeric G proteins are critical transducers of cellular signaling. In addition to their classic roles in relaying signals from G protein-coupled receptors (GPCRs), heterotrimeric G proteins also mediate physiological functions from non-GPCRs. Previously, we have shown that Gα(13), a member of the heterotrimeric G proteins, is essential for growth factor receptor-induced actin cytoskeletal reorganization such as dynamic dorsal ruffle turnover and cell migration. These Gα(13)-mediated dorsal ruffle turnover and cell migration by growth factors acting on their receptor tyrosine kinases (RTKs) are independent of GPCRs. However, the mechanism by which RTKs signal to Gα(13) is not known. Here, we show that cholinesterase-8A (Ric-8A), a nonreceptor guanine nucleotide exchange factor for some heterotrimeric G proteins, is critical for coupling RTKs to Gα(13). Down-regulation of Ric-8A protein levels in cells by RNA interference slowed down platelet-derived growth factor (PDGF)-induced dorsal ruffle turnover and inhibited PDGF-initiated cell migration. PDGF was able to increase the activity of Ric-8A in cells. Furthermore, purified Ric-8A proteins interact directly with purified Gα(13) protein in a nucleotide-dependent manner. Deficiency of Ric-8A prevented the translocation of Gα(13) to the cell cortex. Hence, Ric-8A is critical for growth factor receptor-induced actin cytoskeletal reorganization.     

Integrins regulate the linkage between upstream and downstream events in G protein-coupled receptor signaling to mitogen-activated protein kinase

Receptor tyrosine kinases (RTKs) and G protein-coupled receptors (GPCRs) can both activate mitogen-activated protein kinase (MAPK), a critical intermediate in the transduction of proliferative signals. Numerous observations have demonstrated that integrin-mediated cell anchorage can regulate the efficiency of signaling from RTKs to MAPK. Recently, a relationship between integrins and GPCR signaling has also emerged; however, little is understood concerning the mechanisms involved. Here, we investigate integrin regulation of GPCR signaling to MAPK, focusing on the P2Y class of GPCRs that function through activation of phospholipase Cbeta. P2Y receptor signaling to the downstream components mitogen-activated protein kinase kinase and MAPK is highly dependent on integrin-mediated cell anchorage. However, activation of upstream events, including inositol phosphate production and generation of calcium transients, is completely independent of cell anchorage. This indicates that integrins regulate the linkage between upstream and downstream events in this GPCR pathway, just as they do in some aspects of RTK signaling. However, the P2Y pathway does not involve cross-activation of a RTK, nor a role for Shc or c-Raf; thus, it is quite distinct from the classical RTK-Ras-Raf-MAPK cascade. Rather, integrin-modulated P2Y receptor stimulation of MAPK depends on calcium and on the activation of protein kinase C.     

Protein tyrosine kinase-mediated pathways in G protein-coupled receptor signaling

Abundant evidence has indicated that protein tyrosine kinases (PTKs) convey signals from G protein-coupled receptors (GPCRs) to regulate cell proliferation, migration, adhesion, and potentialy cellular transformation. Molecular mechanisms by which PTKs regulate such diverse effects in GPCR signaling are not well understood. Recently, an unifying theme has emerged where both growth factors and GPCRs utilize protein tyrosine kinase activity and the highly conserved Ras/MAP kinase pathway to control mitogenic signals. Additionally, PTKs are also involved in the regulation of signal transmission from GPCRs to activation of the JNK/SAPK kinase pathway. Furthermore novel insights in chemokine receptor-activated PTKs and their role in mediating cell functions are discussed in this review.     

Pertussis toxin-sensitive and -insensitive thrombin stimulation of Shc phosphorylation and mitogenesis are mediated through distinct pathways

Activation of both receptor tyrosine kinases (RTKs) and G protein-coupled receptors (GPCRs) result in phosphorylation of the adaptor protein Shc, providing sites of interaction for proteins in downstream signal transduction cascades. The mechanism of Shc phosphorylation and its function in G protein signaling pathways is still unclear. By examining Shc phosphorylation in response to thrombin in two cell lines, we have defined distinct pertussis toxin (PTX)-sensitive and -insensitive mechanisms by which GPCRs can stimulate tyrosine phosphorylation of Shc. By mutating the tyrosines in Shc, we show that the three sites of tyrosine phosphorylation, Y239, Y240, and Y317, are necessary for thrombin signaling in both systems. The SH2 (src homology 2) domain of Shc is also critical for signaling, but not required for phosphorylation of Shc. In both cell types, inhibition of src family member kinases by chemical inhibitors or microinjection block Shc phosphorylation and bromodeoxyuridine (BrdU) incorporation in response to thrombin. However, in the PTX-sensitive thrombin pathway, both betagamma function and the epidermal growth factor receptor (EGFR) are necessary for Shc phosphorylation and BrdU incorporation. In contrast, signaling in the PTX-insensitive pathway is not mediated through betagamma or the EGFR. Thus, while phosphorylation and function of Shc appear to be the same in both thrombin pathways, the mechanism of tyrosine kinase activation proximal to Shc is different. The differences in signaling between the two thrombin pathways may be representative of mechanisms used by other PTX-sensitive and -insensitive GPCRs to mediate specific responses. In addition, transactivation of RTKs may be a manner by which GPCRs can amplify their signal.     

Platelet-derived growth factor (PDGF)-induced chemotaxis does not require the G protein-coupled receptor S1P1 in murine embryonic fibroblasts and vascular smooth muscle cells

Sphingosine 1-phosphate (S1P), a bioactive lipid mediator, signals via G protein-coupled receptors (GPCR). The prototypical S1P receptor, S1P1 (also known as EDG-1), a Gi-linked receptor, is critical for vascular maturation during development. Recent work suggested that platelet-derived growth factor (PDGF)-induced cell migration required the S1P1 receptor, representing a novel mechanism for cross-talk between receptor tyrosine kinases and GPCRs. Since both S1P and PDGF are implicated in vascular smooth muscle cell (VSMC) pathobiology and development, we investigated this issue in rat VSMC and in embryonic fibroblasts derived from S1P1 null mice. Our data suggest that the S1P1 receptor is critical for S1P-induced, Gi-dependent migration but not for PDGF-BB-induced, receptor tyrosine kinase-dependent chemotaxis in VSMC. In addition, lack of S1P1 receptor in mouse embryonic fibroblasts did not significantly affect PDGF-induced cell migration. These data question the generality of the concept that S1P1 GPCR is a critical downstream component of PDGF-induced chemotaxis.     

When a G protein-coupled receptor does not couple to a G protein

Classically, G protein-coupled receptors (GPCRs) relay signals by directly activating heterotrimeric guanine nucleotide-binding proteins (G proteins). Increasing evidence indicates that GPCRs may also signal through G protein-independent pathways. JAK/STATs, Src-family tyrosine kinases, GRKs/beta-arrestins, and PDZ domain-containing proteins have been suggested to directly relay signals from GPCRs independent of G proteins. In addition, our laboratory recently reported that the beta(2) adrenergic receptor (beta(2)AR) could switch from G protein-coupled to G protein-independent ERK (extracellular signal-regulated kinase) activation in an agonist dosage-dependent manner. This finding provides a novel mechanism for G protein-independent GPCR signaling. This review focuses on recent progress in understanding the mechanisms by which G protein-independent GPCR signaling occurs.     

与癌症相关的G 蛋白偶联受体及通路的研究进展

G蛋白偶联受体(G protein-coupled receptors,GPCRs)是一类重要的细胞膜表面跨膜蛋白受体超家族,具有7个跨膜螺旋结构。GPCRs的细胞内信号由G蛋白介导,可将激素、神经递质、药物、趋化因子等多种物理和化学的细胞外刺激穿过细胞膜转导到细胞内不同的效应分子,激活相应的信号级联系统进而影响恶性肿瘤的生长迁移过程。虽然目前药物市场上有很多治疗癌症的小分子药物属于G蛋白受体相关药物,但所作用的靶点集中于少数特定G蛋白偶联受体。因此,新的具有成药性的G蛋白偶联受体的开发具有很大的研究价值和市场潜力。本文主要以在癌症发生、发展中起重要作用的溶血磷脂酸(LPA),G蛋白偶联受体30(GPR30)、内皮素A受体(ETAR)等不同G蛋白偶联受体为分类依据,综述其与相关的信号通路在癌症进程中的作用,并对相应的小分子药物的临床应用和研究进展进行展望。     

A concept for G protein activation by G protein-coupled receptor dimers: the transducin/rhodopsin interface.

G protein-coupled receptors (GPCRs) are ubiquitous and essential in modulating virtually all physiological processes. These receptors share a similar structural design consisting of the seven-transmembrane alpha-helical segments. The active conformations of the receptors are stabilized by an agonist and couple to structurally highly conserved heterotrimeric G proteins. One of the most important unanswered questions is how GPCRs couple to their cognate G proteins. Phototransduction represents an excellent model system for understanding G protein signaling, owing to the high expression of rhodopsin in rod photoreceptors and the multidisciplinary experimental approaches used to study this GPCR. Here, we describe how a G protein (transducin) docks on to an oligomeric GPCR (rhodopsin), revealing structural details of this critical interface in the signal transduction process. This conceptual model takes into account recent structural information on the receptor and G protein, as well as oligomeric states of GPCRs.     

Dosage-dependent switch from G protein-coupled to G protein-independent signaling by a GPCR

G-protein-coupled receptors (GPCRs) mostly signal through heterotrimeric G proteins. Increasing evidence suggests that GPCRs could function in a G-protein-independent manner. Here, we show that at low concentrations of an agonist, beta(2)-adrenergic receptors (beta(2)-ARs) signal through Galpha(s) to activate the mitogen-activated protein kinase pathway in mouse embryonic fibroblast cells. At high agonist concentrations, signals are also transduced through beta(2)-ARs via an additional pathway that is G-protein-independent but tyrosine kinase Src-dependent. This new dosage-dependent switch of signaling modes of GPCRs has significant implications for GPCR intrinsic properties and desensitization.     

Clathrin-dependent mechanisms of G protein-coupled receptor endocytosis

The heptahelical G protein-coupled receptor (GPCR) family includes approximately 900 members and is the largest family of signaling receptors encoded in the mammalian genome. G protein-coupled receptors elicit cellular responses to diverse extracellular stimuli at the plasma membrane and some internalized receptors continue to signal from intracellular compartments. In addition to rapid desensitization, receptor trafficking is critical for regulation of the temporal and spatial aspects of GPCR signaling. Indeed, GPCR internalization functions to control signal termination and propagation as well as receptor resensitization. Our knowledge of the mechanisms that regulate mammalian GPCR endocytosis is based predominantly on arrestin regulation of receptors through a clathrin- and dynamin-dependent pathway. However, multiple clathrin adaptors, which recognize distinct endocytic signals, are now known to function in clathrin-mediated endocytosis of diverse cargo. Given the vast number and diversity of GPCRs, the complexity of clathrin-mediated endocytosis and the discovery of multiple clathrin adaptors, a single universal mechanism controlling endocytosis of all mammalian GPCRs is unlikely. Indeed, several recent studies now suggest that endocytosis of different GPCRs is regulated by distinct mechanisms and clathrin adaptors. In this review, we discuss the diverse mechanisms that regulate clathrin-dependent GPCR endocytosis.     

G protein-coupled receptors stimulation and the control of cell migration

Cell migration is a fundamental biological process involved in normal physiology. Altered motile phenotypes are however often associated with the development and progression of diseases such as cancer and atherosclerosis. Remodeling of the actin cytoskeleton is required for cell shape changes and is controlled by a broad variety of cellular proteins. Interestingly, several extracellular stimuli can promote actin reorganization and result in enhanced cell migration. Namely, G protein-coupled receptors (GPCRs), which are activated by factors ranging from small amines, to hormones, and chemokines, initiate signalling cascades resulting in cell shape changes, formation of a migrating front (leading edge) and altered adhesion. GPCRs are heptahelical membrane proteins, which classically transmit signal via the activation of heterotrimeric G proteins. Sustained stimulation leads to the activation of G protein-coupled receptor kinases (GRKs) and the recruitment of arrestin proteins, which engage alternative signalling pathways. In this review, we will discuss the role of GPCR mediated signal transduction and review their importance in the regulation of actin remodeling leading to cell migration.     

Mitogenic signaling pathways induced by G protein-coupled receptors

G protein-coupled receptor (GPCR) agonists, including neurotransmitters, hormones, chemokines, and bioactive lipids, act as potent cellular growth factors and have been implicated in a variety of normal and abnormal processes, including development, inflammation, and malignant transformation. Typically, the binding of an agonistic ligand to its cognate GPCR triggers the activation of multiple signal transduction pathways that act in a synergistic and combinatorial fashion to relay the mitogenic signal to the nucleus and promote cell proliferation. A rapid increase in the activity of phospholipases C, D, and A2 leading to the synthesis of lipid-derived second messengers, Ca2+ fluxes and subsequent activation of protein phosphorylation cascades, including PKC/PKD, Raf/MEK/ERK, and Akt/mTOR/p70S6K is an important early response to mitogenic GPCR agonists. The EGF receptor (EGFR) tyrosine kinase has emerged as a transducer in the signaling by GPCRs, a process termed transactivation. GPCR signal transduction also induces striking morphological changes and rapid tyrosine phosphorylation of multiple cellular proteins, including the non-receptor tyrosine kinases Src, focal adhesion kinase (FAK), and the adaptor proteins CAS and paxillin. The pathways stimulated by GPCRs are extensively interconnected by synergistic and antagonistic crosstalks that play a critical role in signal transmission, integration, and dissemination. The purpose of this article is to review recent advances in defining the pathways that play a role in transducing mitogenic responses induced by GPCR agonists.     

Signaling through G protein coupled receptors

Heterotrimeric G proteins (Gα, Gβ/Gγ subunits) constitute one of the most important components of cell signaling cascade. G Protein Coupled Receptors (GPCRs) perceive many extracellular signals and transduce them to heterotrimeric G proteins, which further transduce these signals intracellular to appropriate downstream effectors and thereby play an important role in various signaling pathways. GPCRs exist as a superfamily of integral membrane protein receptors that contain seven transmembrane α-helical regions, which bind to a wide range of ligands. Upon activation by a ligand, the GPCR undergoes a conformational change and then activate the G proteins by promoting the exchange of GDP/GTP associated with the Gα subunit. This leads to the dissociation of Gβ/Gγ dimer from Gα. Both these moieties then become free to act upon their downstream effectors and thereby initiate unique intracellular signaling responses. After the signal propagation, the GTP of Gα-GTP is hydrolyzed to GDP and Gα becomes inactive (Gα-GDP), which leads to its re-association with the Gβ/Gγ dimer to form the inactive heterotrimeric complex. The GPCR can also transduce the signal through G protein independent pathway. GPCRs also regulate cell cycle progression. Till to date thousands of GPCRs are known from animal kingdom with little homology among them, but only single GPCR has been identified in plant system. The Arabidopsis GPCR was reported to be cell cycle regulated and also involved in ABA and in stress signaling. Here I have described a general mechanism of signal transduction through GPCR/G proteins, structure of GPCRs, family of GPCRs and plant GPCR and its role.Key words: heterotrimeric G proteins, GPCRs, seven-transmembrane receptors, signal transduction, stress signaling     

ADAMs as mediators of EGF receptor transactivation by G protein-coupled receptors

A disintegrin and metalloprotease (ADAM) is a membrane-anchored metalloprotease implicated in the ectodomain shedding of cell surface proteins, including the ligands for epidermal growth factor (EGF) receptors (EGFR)/ErbB. It has been well documented that the transactivation of the EGFR plays critical roles for many cellular functions, such as proliferation and migration mediated through multiple G protein-coupled receptors (GPCRs). Recent accumulating evidence has suggested that ADAMs are the key metalloproteases activated by several GPCR agonists to produce a mature EGFR ligand leading to the EGFR transactivation. In this review, we describe the current knowledge on ADAMs implicated in mediating EGFR transactivation. The major focus of the review will be on the possible upstream mechanisms of ADAM activation by GPCRs as well as downstream signal transduction and the pathophysiological significances of ADAM-dependent EGFR transactivation. ectodomain shedding; angiotensin II     

G protein-coupled receptor systems and their lipid environment in health disorders during aging

Cells, tissues and organs undergo phenotypic changes and deteriorate as they age. Cell growth arrest and hyporesponsiveness to extrinsic stimuli are all hallmarks of senescent cells. Most such external stimuli received by a cell are processed by two different cell membrane systems: receptor tyrosine kinases (RTKs) and G protein-coupled receptors (GPCRs). GPCRs form the largest gene family in the human genome and they are involved in most relevant physiological functions. Given the changes observed in the expression and activity of GPCRs during aging, it is possible that these receptors are directly involved in aging and certain age-related pathologies. On the other hand, both GPCRs and G proteins are associated with the plasma membrane and since lipid-protein interactions regulate their activity, they can both be considered to be sensitive to the lipid environment. Changes in membrane lipid composition and structure have been described in aged cells and furthermore, these membrane changes have been associated with alterations in GPCR mediated signaling in some of the main health disorders in elderly subjects. Although senescence could be considered a physiologic process, not all aging humans develop the same health disorders. Here, we review the involvement of GPCRs and their lipid environment in the development of the major human pathologies associated with aging such as cancer, neurodegenerative disorders and cardiovascular pathologies.     

G protein-coupled receptor systems and their lipid environment in health disorders during aging

Cells, tissues and organs undergo phenotypic changes and deteriorate as they age. Cell growth arrest and hyporesponsiveness to extrinsic stimuli are all hallmarks of senescent cells. Most such external stimuli received by a cell are processed by two different cell membrane systems: receptor tyrosine kinases (RTKs) and G protein-coupled receptors (GPCRs). GPCRs form the largest gene family in the human genome and they are involved in most relevant physiological functions. Given the changes observed in the expression and activity of GPCRs during aging, it is possible that these receptors are directly involved in aging and certain age-related pathologies. On the other hand, both GPCRs and G proteins are associated with the plasma membrane and since lipid-protein interactions regulate their activity, they can both be considered to be sensitive to the lipid environment. Changes in membrane lipid composition and structure have been described in aged cells and furthermore, these membrane changes have been associated with alterations in GPCR mediated signaling in some of the main health disorders in elderly subjects. Although senescence could be considered a physiologic process, not all aging humans develop the same health disorders. Here, we review the involvement of GPCRs and their lipid environment in the development of the major human pathologies associated with aging such as cancer, neurodegenerative disorders and cardiovascular pathologies.     

G protein mediated signaling pathways in lysophospholipid induced cell proliferation and survival

Agonist activation of a subset of G protein coupled receptors (GPCRs) stimulates cell proliferation, mimicking the better known effects of tyrosine kinase growth factors. Cell survival or apoptosis is also regulated via pathways initiated by stimulation of these same GPCRs. This review focuses on aspects of signaling by the lysophospholipid mediators, lysophosphatidic acid (LPA), and sphingosine 1 phosphate (S1P), which make these agonists uniquely capable of modulating cell growth and survival. The general features of GPCR coupling to specific G proteins, downstream effectors and signaling cascades are first reviewed. GPCR coupling to G(i) and Ras/MAPK or to G(q) and phospholipase generated second messengers are insufficient to regulate cell proliferation while G(12/13)/Rho engagement provides additional complementary signals required for cell proliferation. Survival is best predicted by coupling to G(i) pathways that regulate PI3K and Akt, but other signals generated through different G protein pathways are also implicated. The unique ability of LPA and S1P to concomitantly stimulate G(i), G(q), and G(12/13) pathways, given the proper complement of expressed LPA or S1P receptors, allows these receptors to support cell survival and proliferation. In pathophysiological situations, e.g., vascular disease, cancer, brain injury, and inflammation, components of the signaling cascade downstream of lysophospholipid receptors, in particular those involving Ras or Rho, may be altered. In addition, up or downregulation of LPA or S1P receptor subtypes, altering their ratio, and increased availability of the lysophospholipid ligands at sites of injury or inflammation, likely contribute to disease and may be important targets for therapeutic intervention.     

Reversible translocation of p115-RhoGEF by G(12/13)-coupled receptors

G protein-coupled receptors (GPCRs) are important targets for medicinal agents. Four different G protein families, G(s), G(i), G(q), and G(12), engage in their linkage to activation of receptor-specific signal transduction pathways. G(12) proteins were more recently studied, and upon activation by GPCRs they mediate activation of RhoGTPase guanine nucleotide exchange factors (RhoGEFs), which in turn activate the small GTPase RhoA. RhoA is involved in many cellular and physiological aspects, and a dysfunction of the G(12/13)-Rho pathway can lead to hypertension, cardiovascular diseases, stroke, impaired wound healing and immune cell functions, cancer progression and metastasis, or asthma. In this study, regulator of G protein signaling (RGS) domain-containing RhoGEFs were tagged with enhanced green fluorescent protein (EGFP) to detect their subcellular localization and translocation upon receptor activation. Constitutively active Galpha(12) and Galpha(13) mutants induced redistribution of these RhoGEFs from the cytosol to the plasma membrane. Furthermore, a pronounced and rapid translocation of p115-RhoGEF from the cytosol to the plasma membrane was observed upon activation of several G(12/13)-coupled GPCRs in a cell type-independent fashion. Plasma membrane translocation of p115-RhoGEF stimulated by a GPCR agonist could be completely and rapidly reversed by subsequent application of an antagonist for the respective GPCR, that is, p115-RhoGEF relocated back to the cytosol. The translocation of RhoGEF by G(12/13)-linked GPCRs can be quantified and therefore used for pharmacological studies of the pathway, and to discover active compounds in a G(12/13)-related disease context.     

G protein-coupled receptor-induced sensitization of phospholipase C stimulation by receptor tyrosine kinases

Activation of stably expressed M(2) and M(3) muscarinic acetylcholine receptors (mAChRs) as well as of endogenously expressed lysophosphatidic acid and purinergic receptors in HEK-293 cells can induce a long lasting potentiation of phospholipase C (PLC) stimulation by these and other G protein-coupled receptors (GPCRs). Here, we report that GPCRs can induce an up-regulation of PLC stimulation by receptor tyrosine kinases (RTKs) as well and provide essential mechanistic characteristics of this sensitization process. Pretreatment of HEK-293 cells for 2 min with carbachol, a mAChR agonist, lysophosphatidic acid, or ATP, followed by agonist washout, strongly increased (by 2-3-fold) maximal PLC stimulation (measured >/=40 min later) by epidermal growth factor and platelet-derived growth factor, but not insulin, and largely enhanced PLC sensitivity to these RTK agonists. The up-regulation of RTK-induced PLC stimulation was cycloheximide-insensitive and was observed for up to approximately 90 min after removal of the GPCR agonist. Sensitization of receptor-induced PLC stimulation caused by prior M(2) mAChR activation was fully prevented by pertussis toxin and strongly reduced by expression of Gbetagamma scavengers. Furthermore, inhibition of conventional protein kinase C (PKC) isoenzymes and chelation of intracellular Ca(2+) suppressed the sensitization process, while overexpression of PKC-alpha, but not PKC-betaI, further enhanced the M(2) mAChR-induced sensitization of PLC stimulation. None of these treatments affected acute PLC stimulation by either GPCR or RTK agonists. Taken together, short term activation of GPCRs can induce a strong and long lasting sensitization of PLC stimulation by RTKs, a process apparently involving G(i)-derived Gbetagammas as well as increases in intracellular Ca(2+) and activation of a PKC isoenzyme, most likely PKC-alpha.