Palmitoylation participates in G protein coupled signal transduction by affecting its oligomerization

Much in vivo and in vitro evidence has shown that the alpha subunits of heterotrimeric GTP-binding proteins (G proteins) exist as oligomers in their base state and disaggregate when being activated. In this article, the influence of palmitoylation modification of Galpha(o) on its oligomerization was explored extensively. Galpha(o) protein was expressed and purified from Escherichia coli strain JM109 cotransformed with pQE60(Galpha(o)) and pBB131(N-myristoyltransferase). Non-denaturing gel electrophoresis analysis revealed that Galpha(o) existed to a small extent as monomers but mostly as oligomers including dimers, trimers, tetramers and pentamers which could disaggregate completely into monomers by GTPgammaS stimulation. Palmitoylated Galpha(o), on the other hand, only present as oligomers that were difficult to disaggregate into monomers. The effect of palmitoylation on oligomerization of Galpha(o) was further investigated by several other biochemical and biophysical methods including gel filtration chromatography, analytical ultracentrifugation and atomic force microscopy analysis. The results consistently demonstrated that palmitoylation facilitated oligomerization of the Galpha(o) protein. Autoradiography indicated that [(14)C]-palmitoylated Galpha(o) would in no case disaggregate into monomers after treatment with GTPgammaS. [(35)S]-GTPgammaS binding activity assay showed that palmitoylated Galpha(o) was saturated at only 7.8 nmol/mg compared to 21.8 nmol/mg for non-palmitoylated Galpha(o). Fluorescent quenching studies using BODIPY FL-GTPgammaS as a probe showed that the conformation of GTP-binding domain of Galpha(o) tended to become more compact after palmitoylation. These results implied that palmitoylation may regulate the GDP/GTP exchange of Galpha(o) by influencing the oligomerization state of Galpha(o) and thereby modulate the on-off switch of the G protein in G protein-coupled signal transduction.     

G protein-coupled receptor oligomerization provides the framework for signal discrimination

The idea that G protein-coupled receptors (GPCRs) may undergo homo- or hetero-oligomerization, although highly controversial up to a few years ago, has recently gained wide acceptance. The recognition that GPCRs may exhibit either dimeric or oligomeric structures is based upon a large body of biochemical and biophysical evidence. While much effort has been spent to demonstrate the mechanism(s) by which GPCRs interact with each other, the physiological relevance of this phenomenon remains rather elusive. GPCR oligomerization has been proposed to play a role in receptor ontogeny by either chaperoning protein folding or controlling trafficking to the cell surface. However, the acquisition of these roles does not rule out the possibility that oligomeric receptors may have additional functions, once they are brought to the cell surface. Herein, we propose that protein-protein as well as protein-lipid interactions may provide the structural basis for organizing distinct cell compartments along the plasma membrane where different extracellular signals may be perceived and discriminated.     

Peptides as probes for G protein signal transduction

Triggered by agonist binding to cell surface receptors, the heterotrimeric G proteins dissociate into and βγ subunits, each activating distinct second messenger pathways. Peptides from the primary sequences of receptors, G proteins, and effectors have been used to study the molecular interactions between these proteins. Receptor-derived peptides from the second, third and fourth intracellular loops and certain naturally occurring peptides antagonize G protein interactions and can directly activate G protein. These peptides bind to G protein sites that include the N and C terminal regions of the subunit and a yet to be identified region of the β subunit. Peptides have also been useful in characterizing G protein-effector interactions. The identification of the contact sites between proteins involved in G protein signal transduction should aid in the development of non-peptide mimetic therapeutics which could specifically modify G protein-mediated cellular responses.     

植物G蛋白偶联受体及其在信号转导中的作用

G蛋白偶联受体(G protein-coupled receptors,GPCRs)是具有7个跨膜螺旋的蛋白质受体,是人体内最大的蛋白质超家族.GPCRs能调控细胞周期,参与多种植物信号通路以及影响一系列的代谢和分化活动.简要介绍了GPCR和G蛋白介导的信号转导机制,GPCRs的结构和植物GPCR及其在植物跨膜信号转导中的作用,并对GPCR的信号转导机制及植物抗病反应分子机制的研究提出展望.     

RGS与G蛋白信号转导的调节

ＲＧＳｓ（ｒｅｇｕｌａｔｏｒｓ ｏｆ Ｇ－ｐｒｏｔｅｉｎ ｓｉｇｎａｌｉｎｇ）是最近发现的Ｇ－蛋白信号转导的负调节子,大部分ＲＧＳｓ通过ＧＡＰｓ（ＧＴＰａｓｅ ａｃｔｉｖａｔｉｎｇ ｐｒｏｔｅｉｎｓ）方式发挥作用,ＲＧＳ的作用具有高度特异性,在体内受到严密的调节。对ＲＧＳ的深入研究有利于对信号转导调节的了解。     

Ghalpha/tissue transglutaminase 2: an emerging G protein in signal transduction

Gh protein is an heterodimer made up of two subunits alpha and beta. Different from the traditional monomeric and heterotrimeric G proteins, Ghalpha subunit exhibits both GTPase and transglutaminase activities whereas Ghbeta was identified as calreticulin. Activation of Gh by G protein-coupled receptors (GPCR) turns off transglutaminase activity and shifts Ghalpha to signal transducer. Thereafter, Ghalpha regulates downstream effectors. All these aspects are discussed in the present review, in order to shed new light on this atypical G protein.     

The F-BAR protein NOSTRIN participates in FGF signal transduction and vascular development

F-BAR proteins are multivalent adaptors that link plasma membrane and cytoskeleton and coordinate cellular processes such as membrane protrusion and migration. Yet, little is known about the function of F-BAR proteins in vivo. Here we report, that the F-BAR protein NOSTRIN is necessary for proper vascular development in zebrafish and postnatal retinal angiogenesis in mice. The loss of NOSTRIN impacts on the migration of endothelial tip cells and leads to a reduction of tip cell filopodia number and length. NOSTRIN forms a complex with the GTPase Rac1 and its exchange factor Sos1 and overexpression of NOSTRIN in cells induces Rac1 activation. Furthermore, NOSTRIN is required for fibroblast growth factor 2 dependent activation of Rac1 in primary endothelial cells and the angiogenic response to fibroblast growth factor 2 in the in vivo matrigel plug assay. We propose a novel regulatory circuit, in which NOSTRIN assembles a signalling complex containing FGFR1, Rac1 and Sos1 thereby facilitating the activation of Rac1 in endothelial cells during developmental angiogenesis.     

Signal transduction by plant heterotrimeric G‐protein

Heterotrimeric G‐proteins are complexes that regulate important signalling pathways essential for growth and development in both plants and animals. Although plant cells are composed of the core components (Gα, Gβ and Gγ subunits) found in animal G‐proteins, the complexities of the architecture, function and signalling mechanisms of those in animals are dissimilar to those identified in some plants. Current studies on plant G‐proteins have improved knowledge of the essential physiological and agronomic properties, which when harnessed, could potentially impact global food security. Extensive studies on the molecular mechanisms underlying these properties in diverse plant species will be imperative in improving our current understanding of G‐protein signalling pathways involved in plant growth and development. The advancement of G‐protein signalling networks in distinct plant species could significantly aid in better crop development. This review summarizes current progress, novel discoveries and future prospects for this area in potential crop improvement.     

Ectopic expression of the heterotrimeric G15 protein in pancreatic carcinoma and its potential in cancer signal transduction

G15 is a heterotrimeric G protein selectively expressed in immature cell lineages in adult tissues that feature higher cell renewal potential. It promiscuously couples a wide variety of G protein-coupled receptors (GPCRs) to phospholipase C. Intriguingly, G15 is poorly affected by GPCR desensitization. We show here that G15 α-subunit (Gα15) supports sustained stimulation of PKD1 by a constitutively desensitized GPCR co-transfected over a negative cell background.Based on the fact that PKD1 is a multifunctional protein kinase activated by PKC and known for promoting oncogenic signaling, we hypothesized that, if expressed out of its natural cell context, G15 might promote tumor growth. A screening for Gα15 mRNA expression pointed to pancreatic carcinoma among different human cancer cell types and revealed significant expression in human tumor biopsies xenografted in mice.In addition, G15 ectopic presence could functionally contribute to the transformation process since siRNA-induced depletion of Gα15 in pancreatic carcinoma cell lines dramatically inhibited anchorage-independent growth and resistance to the lack of nutrients.Altogether, our findings suggest that G15 supports tumorigenic signaling in pancreas and hence it may be considered as a novel potential target for the therapy of this form of cancer.     

The CSPalpha/G protein complex in PC12 cells

Cysteine string proteinalpha (CSPalpha) is a regulated vesicle protein and molecular chaperone that has been found to be critical for continuous synaptic transmission and is implicated in the defense against neurodegeneration. Previous work has revealed links between CSPalpha and heterotrimeric GTP binding protein (G protein) signal transduction pathways. We have shown that CSPalpha is a guanine nucleotide exchange factor (GEF) for Galphas. In vitro Hsc70 (70 kDa heat shock cognate protein) and SGT (small glutamine-rich tetratricopeptide repeat domain protein) switch CSPalpha from an inactive GEF to an active GEF. Here we have examined the cellular distribution of the CSPalpha system in the PC12 neuroendocrine cell line. CSPalpha, an established secretory vesicle protein, was found to concentrate in the processes of NGF-differentiated PC12 cells as expected. Gbeta subunits co-localized and Galphas subunits partially co-localized with CSPalpha. However, under the conditions examined, the GEF activity of CSPalpha is expected to be inactive, in that Hsc70 was not found in PC12 processes. These results indicate that CSPalpha activity is subject to regulation by factors that alter Hsc70 distribution and translocation within the cell.     

Reviews in Molecular Biology and Biotechnology: Transmembrane Signaling by G Protein-Coupled Receptors

As the most diverse type of cell surface receptor, the importance heptahelical G protein-coupled receptors (GPCRs) to clinical medicine cannot be overestimated. Visual, olfactory and gustatory sensation, intermediary metabolism, cell growth and differentiation are all influenced by GPCR signals. The basic receptor-G protein-effector mechanism of GPCR signaling is tuned by a complex interplay of positive and negative regulatory events that amplify the effect of a hormone binding the receptor or that dampen cellular responsiveness. The association of heptahelical receptors with a variety of intracellular partners other than G proteins has led to the discovery of potential mechanisms of GPCR signaling that extend beyond the classical paradigms. While the physiologic relevance of many of these novel mechanisms of GPCR signaling remains to be established, their existence suggests that the mechanisms of GPCR signaling are even more diverse than previously imagined.     

Coordinate expression of the alpha and beta subunits of heterotrimeric G proteins involves regulation of protein degradation in CHO cells

Individual cell types express a characteristic balance between heterotrimeric G protein alpha and betagamma subunits, but little is known about the regulatory mechanism. We systemically examined the regulatory mechanism in CHO cells. We found that expression of Galphas, Galphai2, and Galphaq proteins increased in direct proportion to the increase of Gbeta1gamma2 overexpressed transiently. Expression of Gbeta protein also increased following overexpression of Galphas, Galphai2, and Galphaq. The Gbetagamma overexpression stimulated degradation of Gbeta in contrast to reduction of Galphas degradation. We conclude that coordinate expression of the G protein subunits involves regulation of protein degradation via proteasome in CHO cells.     

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.     

Activation of the adhesion G protein–coupled receptor GPR133 by antibodies targeting its N-terminus

We recently demonstrated that GPR133 (ADGRD1), an adhesion G protein–coupled receptor involved in raising cytosolic cAMP levels, is necessary for growth of glioblastoma (GBM) and is de novo expressed in GBM relative to normal brain tissue. Our previous work suggested that dissociation of autoproteolytically generated N-terminal and C-terminal fragments of GPR133 at the plasma membrane correlates with receptor activation and signaling. To promote the goal of developing biologics that modulate GPR133 function, we investigated the effects of antibodies against the N-terminus of GPR133 on receptor signaling. Here, we show that treatment of HEK293T cells overexpressing GPR133 with these antibodies increased cAMP levels in a concentration-dependent manner. Analysis of culture medium following antibody treatment further indicated the presence of complexes of these antibodies with the autoproteolytically cleaved N-terminal fragments of GPR133. In addition, cells expressing a cleavage-deficient mutant of GPR133 (H543R) did not respond to antibody stimulation, suggesting that the effect is cleavage dependent. Finally, we demonstrate the antibody-mediated stimulation of WT GPR133, but not the cleavage-deficient H543R mutant, was reproducible in patient-derived GBM cells. These findings provide a paradigm for modulation of GPR133 function with biologics and support the hypothesis that the intramolecular cleavage in the N-terminus modulates receptor activation and signaling.     

Concerted stimulation and deactivation of pertussis toxin-sensitive G proteins by chimeric G protein-coupled receptor-regulator of G protein signaling 4 fusion proteins: analysis of the contribution of palmitoylated cysteine residues to the GAP activity of RGS4

Agonists stimulated high-affinity GTPase activity in membranes of HEK293 cells following coexpression of the alpha 2A-adrenoceptor and a pertussis toxin-resistant mutant of Go1 alpha. Enzyme kinetic analysis of Vmax and Km failed to detect regulation of the effect of agonist by a GTPase activating protein. This did occur, however, when cells were also transfected to express RGS4. Both elements of a fusion protein in which the N-terminus of RGS4 was linked to the C-terminal tail of the alpha 2A-adrenoceptor were functional, as it was able to provide concerted stimulation and deactivation of the G protein. By contrast, the alpha 2A-adrenoceptor-RGS4 fusion protein stimulated but did not enhance deactivation of a form of Go1 alpha that is resistant to the effects of regulator of G protein signaling (RGS) proteins. Employing this model system, mutation of Asn128 but not Asn88 eliminated detectable GTPase activating protein activity of RGS4 against Go1 alpha. Mutation of all three cysteine residues that are sites of post-translational acylation in RGS4 also eliminated GTPase activating protein activity but this was not achieved by less concerted mutation of these sites. These studies demonstrate that a fusion protein between a G protein-coupled receptor and an RGS protein is fully functional in providing both enhanced guanine nucleotide exchange and GTP hydrolysis of a coexpressed G protein. They also provide a direct means to assess, in mammalian cells, the effects of mutation of the RGS protein on function in circumstances in which the spatial relationship and orientation of the RGS to its target G protein is defined and maintained.     

Structural and dynamic analysis of G558R mutation in chicken TSHR gene shows altered signal transduction and corroborates its role as a domestication gene

The thyroid-stimulating hormone receptor (TSHR) has been indicated as a putative domestication gene in chicken. Comparison of WGS identified a variant in residue 558 of the transmembrane domain (TM) of TSHR, where the domestic chicken (GGD) presents an arginine, whereas the red jungle fowl (RJF) shares a conserved glycine with other vertebrates. This variant has been demonstrated to be associated with phenotypes that are important for domestication and related to thyroid regulation, such as less fearful behavior, reduced aggressive behavior and reduced dependence on seasonal reproduction in GGD as compared with RJF. By means of molecular dynamics simulations, we highlighted the structural and dynamic differences of variant Gly558Arg in the TSHR TM domain. Alterations in TM helix flexibility, structure and protein overall motion are described. The so-called ‘arginine snorkeling’ of residue 568 in GGD is observed and we hypothesize it as the originating force that produces the observed whole-protein perturbation in the helix bundle dynamics, capable of altering the TSHR signal transduction. The results are discussed in the context of their implications for a better understanding of biological mechanisms in chicken under control of the thyroid, such as body metabolism, as well as for their usefulness in biomedical research.