G蛋白偶联受体激酶活性调控与细胞炎性损伤

G蛋白偶联受体激酶 (Gprotein coupledreceptorki nases,GRKs)不仅调节G蛋白偶联受体 (GPCR)磷酸化、介导受体脱敏 ,使信号效应降低或消失 ,而且也调节G蛋白和靶细胞骨架 ,同时它还受到蛋白激酶A(PKA)、蛋白激酶C(PKC)、肌动蛋白和细胞内第二信使钙离子等调节。组织细胞表面存在多种GPCR如血小板活化因子 (PAF)受体、组胺受体、凝血酶受体等 ,介导炎性介质所致细胞损伤的信号转导作用。GRKs磷酸化GPCR ,在炎症诱导细胞损伤过程中起一定调控作用     

G蛋白信号调节因子在心血管系统中的作用

G蛋白偶联受体（G-Protein-Coupled-Receptors,GPCRs）在体内分布广泛,几乎参与所有生理活动的调节。G蛋白调节因子（Regulator of G protein Signaling,RGS）参与了G蛋白失活的调节。目前研究已证明,参与心血管系统生理和病理活动的很多递质和激素都是通过GPCR信号转导通路发挥作用的。RGS蛋白通过调节GPCR通路信号转导和非GPCR依赖性途径影响多种心血管疾病的发生,其在心脏血管结构和功能中的地位已逐渐引起重视,有望成为相关疾病治疗的新靶点。本文将就RGS蛋白及其在心血管系统中的作用作一综述。     

靶向抑制G蛋白偶联受体激酶2在心力衰竭基因治疗中的研究进展

G蛋白偶联受体激酶2(G protein coupled receptor kinases, GRK2)在β-肾上腺素受体信号转导途径中具有重要的作用。慢性心力衰竭心肌细胞的GRK2的活性及表达水平显著升高,导致βAR通路减敏及心脏功能降低。研究发现通过基因治疗调控GRK2活性可对多种心脏疾病起到良好的治疗作用,为心血管疾病的治疗开辟了一个新途径。本文就GRK2在正常心脏及心力衰竭中的作用机制,及其在心力衰竭基因治疗研究中的新进展做一综述。     

δ阿片受体激动剂DPDPE和TIPP不同的通过转激活表皮生长因子受体激活ERK的机制研究

G蛋白偶联受体(GPCR)可通过多种信号途径激活ERK。其中通过转激活酪氨酸受体是GPCR激活ERK的重要途径之一。我们以前的研究发现阿片受体激动剂DPDPE,TIPP和吗啡能够通过不同的信号途径激活ERK。本研究探讨这些阿片受体激动剂不同的激活ERK是否与它们不同的转激活受体酪氨酸激酶有关及其转激活相关的机制。本研究发现DPDPE,TIPP和吗啡对δ阿片受体转移激活表皮生长因子受体(EGFR)的作用上存在不同的调节效应,发现TIPP和吗啡激动δ阿片受体可引起对EGFR的转移激活,而DPDPE则不能;TIPP和吗啡激动δ阿片受体引起对EGFR的转移激活过程中有PKCδ的参与;GRK磷酸化δ阿片受体的位点丝氨酸363突变可以使原本不引起转移激活的激动剂DPDPE,引起转移激活作用;并且,RNA干扰β-arrestin可以影响EGF刺激EGFR引起的磷酸化ERK信号,故β-arres-tin可能参与转移激活作用。本课题的研究对于深入探索δ阿片受体信号转导的机制有重要的意义。     

磷脂酰乙醇胺结合蛋白对阿片受体信号转导及阿片依赖的调节作用

药物成瘾是一种慢性、反复发作性脑疾病,其机制尚未阐明。磷脂酰乙醇胺结合蛋白(PEBP)是近来发现的一种多功能蛋白,能够调节信号转导功能,也能通过水解产生的海马胆碱能神经刺激肽(HCNP)增强胆碱乙酰基转移酶(ChAT)活性而影响隔-海马胆碱能系统功能。但是,PEBP在药物成瘾中有何作用尚未见文献报道。我们采用蛋白质组学研究技术首次发现吗啡慢性处理能够上调大鼠海马内PEBP的表达,提示其与阿片成瘾可能有一定的相关性。鉴于PEBP可通过磷酸化与非磷酸化形式的转化而调节ERK和GRK的活性,我们首先研究了PEBP对μ阿片受体信号转导的影响。结果发现,μ阿片受体激活不影响PEBP磷酸化与非磷酸化之间的转化;过表达PEBP不影响μ阿片受体与激动剂处理诱导的G蛋白的偶联,也不影响μ阿片受体的磷酸化和脱敏,但是降低μ阿片受体激活后对腺苷酸环化酶活性的抑制和对ERK的激活,提示PEBP对μ阿片受体的信号转导有一定的调节作用。整体动物水平的研究发现,吗啡慢性处理导致大鼠海马内PEBP蛋白的表达水平特异性的上调,并呈现时间及剂量依赖性;而戒断3 d时海马内PEBP表达水平恢复至接近基础水平,之后其表达水平逐渐上调并维持至28 d。采用反义核酸的策略下调海马内PEBP表达,能够加重吗啡依赖的形成。进一步的调节机制研究发现,吗啡依赖及戒断引起海马内ChAT活性出现变化,其变化趋势与PEBP表达水平的变化完全一致,提示PEBP可能通过剪切成HCNP影响胆碱能系统功能而调节阿片依赖。因此,本文研究不仅发现PEBP对μ阿片受体信号转导具有一定的调节作用,而且发现PEBP参与了阿片依赖过程,机制可能与影响海马内ChAT活性、进而影响胆碱能神经系统功能有关。     

与类风湿关节炎相关的G蛋白偶联受体及治疗药物作用靶点

很多胞外信号直接或间接通过G蛋白偶联受体向胞内传输。多种G蛋白偶联受体包括趋化因子受体、前列腺素(prostaglandins,PGs)受体、β2-肾上腺素受体(β2-adrener-gicreceptor)、致炎肽P物质(proinflammatorypeptidesub-stanceP,SP)受体、蛋白酶活化受体2(protease-activatedre-ceptor2,PAR-2)等在免疫应答调节中起至关重要的作用。该文综述了与类风湿关节炎相关的G蛋白偶联受体(Gpro-tein-coupledreceptors,GPCRs)信号转导的一些蛋白作用,包括G蛋白偶联受体激酶、视紫红质抑制蛋白(arrestin)、G蛋白信号转导调节因子、G蛋白偶联致炎受体等。作用于这些信号或其转导过程的药物正成为类风湿关节炎治疗的新策略之一。     

G蛋白偶联受体在肝细胞癌中调控作用研究进展

G蛋白偶联受体(GPCR),又称为7-α螺旋跨膜蛋白受体,是己知的3类涉及跨膜信号转导的膜受体之一。GPCR与G蛋白结合产生生物学效应,对机体生理功能和病理过程有广泛的调控作用。大量研究表明,GPCR通过调节下游某些信号的转导途径影响肝癌细胞的增殖、侵袭和转移过程,参与肝细胞癌(HCC)的发生和发展。本文就趋化因子受体、前列腺素受体、肾上腺素受体和血管紧张素受体等GPCR及其相关的信号通路在HCC发生发展进程中的作用进行综述,并对靶向GPCR的HCC治疗前景予以展望。     

β-arrestins介导的信号通路在恶性肿瘤病理过程中的作用

β-arrestins是介导受体脱敏的重要的蛋白质家族,在G蛋白偶联受体的脱敏、内化和复敏中都有重要的作用。作为一类多功能的蛋白,β-arrestins对绝大多数由G蛋白偶联受体介导的信号通路有调节作用,并且参与调节一些非七次跨膜受体的信号转导。在多种恶性肿瘤中,β-arrestins通过调节G蛋白偶联受体或其他一些信号转导途径影响肿瘤细胞的增殖、侵袭和转移过程,参与了恶性肿瘤的病理过程。该文就β-arrestins在恶性肿瘤发生发展过程中所起到的作用及目前的研究状况作一综述。     

G蛋白偶联受体固有活性研究进展与新药开发

G蛋白偶联受体(G-prote in-coup led receptor,GPCR)是与G蛋白有信号连接的一大类受体家族,是人体内最大的膜受体蛋白家族,是一类具有7个跨膜螺旋的跨膜蛋白受体。GPCR的结构特征和在信号传导中的重要作用决定了其可以作为很好的药物靶标。目前世界药物市场上有三分之一的小分子药物是GPCR的激活剂(agon ist)或拮抗剂(antagon ist)。以其为靶点的药物在医药产业中占据显著地位。在当今前50种最畅销的上市药物中,20%属于G蛋白受体相关药物。近来的研究发现,大多数G蛋白偶联受体具有一个很重要的特性,就是具有固有活性(Constitutive ac-tivity),即无激动剂条件受体自发的维持激活并维持下游信号传导通路的活性。固有活性涉及受体、G蛋白及下游信号通路之间的关系。该文就G蛋白偶联受体固有活性概念、研究进展、反相激动剂与固有活性研究、固有活性与新药开发4个方面,进行以下论述。     

与糖尿病肾病相关的G蛋白偶联受体及相关治疗药物的研究进展

目的:了解糖尿病肾病(DN)的病理机制,从而为治疗DN提供新途径。方法:根据文献,综述了在DN发生、发展中起重要作用的G蛋白偶联受体(GPCR)的结构特点及其主要的信号转导机制,以及与DN进展相关的GPCR类型及相关治疗药物等内容。结果与结论:GPCR是一种与三聚体G蛋白相偶联的细胞表面受体,由1条多肽链构成;其信号转导途径由细胞膜受体、G蛋白、第二信使和效应器4部分组成,包括腺苷酸环化酶系统和磷脂酶C系统。与DN进展相关的GPCR有5-羟色胺2A受体(相关药物:酮色林、盐酸沙格雷酯等)、前列腺素E2受体、血管紧张素Ⅱ受体(相关药物:替米沙坦等)、生长抑素受体(相关药物:奥曲肽)。作用于GPCR的药物,正成为研发治疗DN药物的新方向。     

Regulatory effects of GRK2 on GPCRs and possible use as a drug target

G protein-coupled receptor kinase 2(GRK2),as a key Ser/Thr protein kinase,belong to the member of the G protein-coupled receptor kinase(GRK)family.The C-terminus of GRK2 including a plekstrin homology domain and the N-terminus of GRK2 including the RGS homology domain with binding sites for several proteins and lipids such as G protein-coupled receptors(GPCRs),G protein,phospholipase C,phosphatidylinositol 4,5-bisphosphate,extracellular signal-regulated kinase,protein kinase A and Gβγ,which can regulate the activity of GRK2.GRK2 can regulate GPCR desensitization and internalization by phosphorylating the GPCR,promoting the affinity of binding to arrestins,and uncoupling the receptors from G proteins,which play an important role in maintaining the balance between the receptors and signal transduction.Previous studies have indicated that cardiac GRK2overexpression can promote the phosphorylation ofβ-adrenergic receptor(βAR)leading toβAR desensitization and internalization,which play a pivotal role in inducing heart failure(HF)-related dysfunction and myocyte death.GRK2,as a regulator of cell function,is overexpression in hypertension.Overexpression GRK2 can inhibit Akt/e NOS signaling pathway and decreased the production and activation of e NOS leading to endothelial dysfunction.Collagen-induced arthritis induces the upregulation of GRK2 expression in fibroblast-like synoviocytes.In this review,we mainly discussed the evidence for the association between GRK2 overexpression and various diseases,which suggests that GRK2 may be an effective drug target for preventing and treating heart failure,hypertension and inflammatory disease.     

The complex G protein-coupled receptor kinase 2 (GRK2) interactome unveils new physiopathological targets

GRK2 is a ubiquitous member of the G protein-coupled receptor kinase (GRK) family that appears to play a central, integrative role in signal transduction cascades. GRKs participate together with arrestins in the regulation of G protein-coupled receptors (GPCR), a family of hundreds of membrane proteins of key physiological and pharmacological importance, by triggering receptor desensitization from G proteins and GPCR internalization, and also by helping assemble macromolecular signalosomes in the receptor environment acting as agonist-regulated adaptor scaffolds, thus contributing to signal propagation. In addition, emerging evidence indicates that GRK2 can phosphorylate a growing number of non-GPCR substrates and associate with a variety of proteins related to signal transduction, thus suggesting that this kinase could also have diverse ‘effector’ functions. We discuss herein the increasing complexity of such GRK2 ‘interactome’, with emphasis on the recently reported roles of this kinase in cell migration and cell cycle progression and on the functional impact of the altered GRK2 levels observed in several relevant cardiovascular, inflammatory or tumour pathologies. Deciphering how the different networks of potential GRK2 functional interactions are orchestrated in a stimulus, cell type or context-specific way is critical to unveil the contribution of GRK2 to basic cellular processes, to understand how alterations in GRK2 levels or functionality may participate in the onset or development of several cardiovascular, tumour or inflammatory diseases, and to assess the feasibility of new therapeutic strategies based on the modulation of the activity, levels or specific interactions of GRK2.     

The structure of GRK2-G beta gamma complex: intimate association of G-protein signaling modules

G-protein-mediated signaling is the most widely used signaling mechanism in cells and its regulation is crucial for various physiological functions. G-protein-coupled receptor (GPCR) kinases (GRKs) are involved in the desensitization of GPCR signals. Recently, the X-ray crystal structure of GRK2 complexed with G beta gamma was demonstrated and revealed the intimate association of three important signaling modules with G beta gamma to regulate GRK2 activity.     

Agonist-dependent modulation of G protein-coupled receptor kinase 2 by mitogen-activated protein kinases

A variety of G protein-coupled receptors (GPCRs) are phosphorylated by G protein-coupled receptor kinase 2 (GRK2). This event promotes the binding of regulatory proteins termed beta-arrestins to GPCRs, leading to uncoupling from G proteins and receptor internalization. Recent data indicate that GRK2 and beta-arrestins also play an important role in the stimulation of the extracellular signal-regulated kinases (ERK)/mitogen-activated protein kinase (MAPK) cascade by GPCRs. In this report, we have investigated the existence of functional interactions between GRK2 and MAPK. We show that activation of beta(2)-adrenergic receptors (beta(2)-AR) promotes the rapid association of GRK2 and MAPK in living cells, as assessed by coimmunoprecipitation experiments in COS-7 cells transfected with beta(2)-AR, GRK2, and an epitope-tagged MAPK. Coimmunoprecipitation of MAPK and GRK2 is blocked by inhibition of the MAPK cascade and is not observed upon activation of MAPK in the absence of beta(2)-AR stimulation, thus indicating that both an active MAPK and agonist occupancy of GPCR are required for the association to occur. Interestingly, we have found that purified ERK1/MAPK can directly phosphorylate the C-terminal domain of GRK2, and that the phosphorylation process is favored by the presence of Gbetagamma-subunits or an activated receptor. Furthermore, GRK2 phosphorylation by MAPK leads to a decreased activity of GRK2 toward GPCR. Taken together, our results suggest that stimulation of GPCRs promotes the rapid association of GRK2 and MAPK leading to modulation of GRK2 functionality, thus putting forward a new feedback mechanism for the regulation of GPCR signaling.     

Phosphorylation-independent attenuation of GPCR signalling

The uncoupling of G-protein-coupled receptors (GPCRs) from their cognate heterotrimeric G proteins provides an essential physiological 'feedback' mechanism that protects against both acute and chronic overstimulation of receptors. The primary mechanism by which GPCR activity is regulated is the feedback phosphorylation of activated GPCRs by kinases that are dependent on second messengers, GPCR kinases (GRKs) and arrestins. It has recently become apparent, however, that GRK2-mediated regulation of GPCR responsiveness also involves a phosphorylation-independent component that requires both heterotrimeric G-protein alpha-subunit interactions and GPCR binding. Moreover, in addition to GRK2, a growing number of GPCR-interacting proteins might contribute to the phosphorylation-independent G-protein uncoupling of GPCRs. Here, new information about the mechanisms underlying this phosphorylation-independent regulation of receptor and G-protein coupling is reviewed.     

Evolving concepts in G protein-coupled receptor endocytosis: the role in receptor desensitization and signaling

G protein-coupled receptors (GPCRs) are seven transmembrane proteins that form the largest single family of integral membrane receptors. GPCRs transduce information provided by extracellular stimuli into intracellular second messengers via their coupling to heterotrimeric G proteins and the subsequent regulation of a diverse variety of effector systems. Agonist activation of GPCRs also initiates processes that are involved in the feedback desensitization of GPCR responsiveness, the internalization of GPCRs, and the coupling of GPCRs to heterotrimeric G protein-independent signal transduction pathways. GPCR desensitization occurs as a consequence of G protein uncoupling in response to phosphorylation by both second messenger-dependent protein kinases and G protein-coupled receptor kinases (GRKs). GRK-mediated receptor phosphorylation promotes the binding of beta-arrestins, which not only uncouple receptors from heterotrimeric G proteins but also target many GPCRs for internalization in clathrin-coated vesicles. beta-Arrestin-dependent endocytosis of GPCRs involves the direct interaction of the carboxyl-terminal tail domain of beta-arrestins with both beta-adaptin and clathrin. The focus of this review is the current and evolving understanding of the contribution of GRKs, beta-arrestins, and endocytosis to GPCR-specific patterns of desensitization and resensitization. In addition to their role as GPCR-specific endocytic adaptor proteins, beta-arrestins also serve as molecular scaffolds that foster the formation of alternative, heterotrimeric G protein-independent signal transduction complexes. Similar to what is observed for GPCR desensitization and resensitization, beta-arrestin-dependent GPCR internalization is involved in the intracellular compartmentalization of these protein complexes.     

GRK2: multiple roles beyond G protein-coupled receptor desensitization

G protein-coupled receptor kinases (GRKs) regulate numerous G protein-coupled receptors (GPCRs) by phosphorylating the intracellular domain of the active receptor, resulting in receptor desensitization and internalization. GRKs also regulate GPCR trafficking in a phosphorylation-independent manner via direct protein-protein interactions. Emerging evidence suggests that GRK2, the most widely studied member of this family of kinases, modulates multiple cellular responses in various physiological contexts by either phosphorylating non-receptor substrates or interacting directly with signaling molecules. In this review, we discuss traditional and newly discovered roles of GRK2 in receptor internalization and signaling as well as its impact on non-receptor substrates. We also discuss novel exciting roles of GRK2 in the regulation of dopamine receptor signaling and in the activation and trafficking of the atypical GPCR, Smoothened (Smo).     

Substrate specificities of g protein-coupled receptor kinase-2 and -3 at cardiac myocyte receptors provide basis for distinct roles in regulation of myocardial function

The closely related G protein-coupled receptor kinases GRK2 and GRK3 are both expressed in cardiac myocytes. Although GRK2 has been extensively investigated in terms of regulation of cardiac beta-adrenergic receptors, the substrate specificities of the two GRK isoforms at G protein-coupled receptors (GPCR) are poorly understood. In this study, the substrate specificities of GRK2 and GRK3 at GPCRs that control cardiac myocyte function were determined in fully differentiated adult cardiac myocytes. Concentration-effect relationships of GRK2, GRK3, and their respective competitive inhibitors, GRK2ct and GRK3ct, at endogenous endothelin, alpha(1)-adrenergic, and beta(1)-adrenergic receptor-generated responses in cardiac myocytes were achieved by adenovirus gene transduction. GRK3 and GRK3ct were highly potent and efficient at the endothelin receptors (IC(50) for GRK3, 5 +/- 0.7 pmol/mg of protein; EC(50) for GRK3ct, 2 +/- 0.2 pmol/mg of protein). The alpha(1)-adrenergic receptor was also a preferred substrate of GRK3 (IC(50),7 +/- 0.4 pmol/mg of protein). GRK2 lacked efficacy at both endothelin and alpha(1)-adrenergic receptors despite massive overexpression. On the contrary, both GRK2ct and GRK3ct enhanced beta(1)-adrenergic receptor-induced cAMP production with comparable potencies. However, the potency of GRK3ct at beta(1)-adrenergic receptors was at least 20-fold lower than that at endothelin receptors. In conclusion, this study demonstrates distinct substrate specificities of GRK2 and GRK3 at different GPCRs in fully differentiated adult cardiac myocytes. As inferred from the above findings, GRK2 may play its primary role in regulation of cardiac contractility and chronotropy by controlling beta(1)-adrenergic receptors, whereas GRK3 may play important roles in regulation of cardiac growth and hypertrophy by selectively controlling endothelin and alpha(1)-adrenergic receptors.     

Are we beta-ARKing up the wrong tree? Casein kinase 1 alpha provides an additional pathway for GPCR phosphorylation

Although originally linked to receptor desensitization, G-protein-coupled receptor (GPCR) phosphorylation has now been implicated in coupling receptors to specific signalling pathways. Generally, this phosphorylation event is thought to be mediated by one of the members of the GPCR kinase (GRK) family. However, recent studies have indicated that protein kinases distinct from the GRK family might also be involved in agonist-mediated GPCR phosphorylation. This review analyses the approaches employed to investigate the nature of GPCR phosphorylation and discusses recent developments implicating other kinases, particularly casein kinase 1 alpha, in the phosphorylation of GPCRs.     

Involvement of intramolecular interactions in the regulation of G protein-coupled receptor kinase 2

The G protein-coupled receptor (GPCR) kinase GRK2 phosphorylates G protein-coupled receptors in an agonist-dependent manner. GRK2 activity is modulated through interactions of diverse domains of the kinase with G protein betagamma subunits, several lipids, anchoring proteins, and activated receptors. We report that kinase activity toward either GPCR (rhodopsin) or a synthetic peptide substrate is enhanced in the presence of GST-GRK2 fusion proteins or peptides corresponding to either N- or C-terminal sequences of GRK2. This direct stimulatory action of intrinsic domains on GRK2 activity does not add to the effect of other regulators, such as Gbetagamma subunits, and strongly suggests the existence of some mode of autoregulation. The existence of regulatory intramolecular interactions in GRK2 is supported by the facts that a C-terminal peptide protects the N-terminal region from proteolytic cleavage and that two domains of GRK2 independently coexpressed in cells associate as assessed by immunoprecipitation. Molecular modeling suggests that intramolecular interactions among the N-terminal, C-terminal and kinase domains would keep GRK2 in a constrained conformation characteristic of an inactive, basal state. Our model proposes that disruption of such intramolecular contacts by intermolecular interactions with regulatory proteins (mimicked by exogenously added kinase fragments in vitro) would promote the conformational changes required to bring about GRK2 translocation and activation.