Function of phosducin-like proteins in G protein signaling and chaperone-assisted protein folding

Members of the phosducin gene family were initially proposed to act as down-regulators of G protein signaling by binding G protein βγ dimers (Gβγ) and inhibiting their ability to interact with G protein subunits (G) and effectors. However, recent findings have over-turned this hypothesis by showing that most members of the phosducin family act as co-chaperones with the cytosolic chaperonin complex (CCT) to assist in the folding of a variety of proteins from their nascent polypeptides. In fact rather than inhibiting G protein pathways, phosducin-like protein 1 (PhLP1) has been shown to be essential for G protein signaling by catalyzing the folding and assembly of the Gβγ dimer. PhLP2 and PhLP3 have no role in G protein signaling, but they appear to assist in the folding of proteins essential in regulating cell cycle progression as well as actin and tubulin. Phosducin itself is the only family member that does not participate with CCT in protein folding, but it is believed to have a specific role in visual signal transduction to chaperone Gβγ subunits as they translocate to and from the outer and inner segments of photoreceptor cells during light-adaptation.     

Functional reconstitution of an atypical G protein heterotrimer and regulator of G protein signaling protein (RGS1) from Arabidopsis thaliana

It has long been known that animal heterotrimeric Gαβγ proteins are activated by cell-surface receptors that promote GTP binding to the Gα subunit and dissociation of the heterotrimer. In contrast, the Gα protein from Arabidopsis thaliana (AtGPA1) can activate itself without a receptor or other exchange factor. It is unknown how AtGPA1 is regulated by Gβγ and the RGS (regulator of G protein signaling) protein AtRGS1, which is comprised of an RGS domain fused to a receptor-like domain. To better understand the cycle of G protein activation and inactivation in plants, we purified and reconstituted AtGPA1, full-length AtRGS1, and two putative Gβγ dimers. We show that the Arabidopsis Gα protein binds to its cognate Gβγ dimer directly and in a nucleotide-dependent manner. Although animal Gβγ dimers inhibit GTP binding to the Gα subunit, AtGPA1 retains fast activation in the presence of its cognate Gβγ dimer. We show further that the full-length AtRGS1 protein accelerates GTP hydrolysis and thereby counteracts the fast nucleotide exchange rate of AtGPA1. Finally, we show that AtGPA1 is less stable in complex with GDP than in complex with GTP or the Gβγ dimer. Molecular dynamics simulations and biophysical studies reveal that altered stability is likely due to increased dynamic motion in the N-terminal α-helix and Switch II of AtGPA1. Thus, despite profound differences in the mechanisms of activation, the Arabidopsis G protein is readily inactivated by its cognate RGS protein and forms a stable, GDP-bound, heterotrimeric complex similar to that found in animals.     

D(2)-Dopamine receptors target regulator of G protein signaling 9-2 to detergent-resistant membrane fractions

Detergent-resistant membranes (DRM) are thought to contain structures such as lipid rafts that are involved in compartmentalizing cell membranes. We report that the majority of D(2)-dopamine receptors (D(2)R) expressed endogenously in mouse striatum or expressed in immortalized cell-lines is found in DRM. In addition, exogenous co-expression of D(2)R in a cell line shifted the expression of regulator of G protein signaling 9-2 (RGS9-2) into DRM. RGS9-2 is a protein that is highly enriched in the striatum and specifically regulates striatal D(2)R. In the striatum, RGS9-2 is mostly associated with DRMs but when expressed in cell lines, RGS9-2 is present in the soluble cytoplasmic fraction. In contrast, the majority of mu opioid receptors and delta opioid receptors are found in detergent-soluble membrane and there was no shift of RGS9-2 into DRM after co-expression of mu opioid receptor. These data suggest that the targeting of RGS9-2 to DRM in the striatum is mediated by D(2)R and that DRM is involved in the formation of a D(2)R signaling complex. D(2)R-mediated targeting of RGS9-2 to DRM was blocked by the deletion of the RGS9-2 DEP domain or by a point mutation that abolishes the GTPase accelerating protein function of RGS9-2.     

Regulation of longevity by regulator of G-protein signaling protein, Loco

Regulator of G-protein signaling (RGS) proteins contribute to G-protein signaling pathways as activators or repressors with GTPase-activating protein (GAP) activity. To characterize whether regulation of RGS proteins influences longevity in several species, we measured stress responses and lifespan of RGS-overexpressing and RGS-lacking mutants. Reduced expression of Loco, a RGS protein of Drosophila melanogaster, resulted in a longer lifespan for both male and female flies, also exhibiting stronger resistance to three different stressors (starvation, oxidation, and heat) and higher manganese-containing superoxide dismutase (MnSOD) activity. In addition, this reduction in Loco expression increased fat content and diminished cAMP levels. In contrast, overexpression of both genomic and cDNA loco gene significantly shortened the lifespan with weaker stress resistance and lower fat content. Deletion analysis of the Loco demonstrated that its RGS domain is required for the regulation of longevity. Consistently, when expression of RGS14, mammalian homologue of Loco, was reduced in rat fibroblast cells, the resistance to oxidative stress increased with higher MnSOD expression. The changes of yeast Rgs2 expression, which shares a conserved RGS domain with the fly Loco protein, also altered lifespan and stress resistance in Saccharomyces cerevisiae. Here, we provide the first evidence that RGS proteins with GAP activity affect both stress resistance and longevity in several species.     

How regulators of G protein signaling achieve selective regulation

The regulators of G protein signaling (RGS) are a family of cellular proteins that play an essential regulatory role in G protein-mediated signal transduction. There are multiple RGS subfamilies consisting of over 20 different RGS proteins. They are basically the guanosine triphosphatase (GTPase)-accelerating proteins that specifically interact with G protein alpha subunits. RGS proteins display remarkable selectivity and specificity in their regulation of receptors, ion channels, and other G protein-mediated physiological events. The molecular and cellular mechanisms underlying such selectivity are complex and cooperate at many different levels. Recent research data have provided strong evidence that the spatiotemporal-specific expression of RGS proteins and their target components, as well as the specific protein-protein recognition and interaction through their characteristic structural domains and functional motifs, are determinants for RGS selectivity and specificity. Other molecular mechanisms, such as alternative splicing and scaffold proteins, also significantly contribute to RGS selectivity. To pursue a thorough understanding of the mechanisms of RGS selective regulation will be of great significance for the advancement of our knowledge of molecular and cellular signal transduction.     

新型抗体结合蛋白CBD—SPG的设计与表达

设计并表达可用于纯化IgG的新型高栽量抗体结合蛋白CBD—SPG。利用基因重组技术将纤维素结合结构域（Cellulose Binding Domain,CBD）基因插入到表达载体pET28a—SPG中,获得重组质粒pET28a—CBD—SPG,并转化大肠杆菌曰位J（DE3）。IPTG诱导CBD—SPG融合蛋白表达,并用SDS—PAGE和Westernblot对表达产物进行鉴定。重组表达质粒pET28a—CBD-SPG经双酶切及测序验证无误;表达产物经SDS．PAGE和WesternBlot分析表明融合蛋白的表观分子量约为40kD;CBD—SPG具有良好的结合纤维素和抗体的能力,晶体纤维素Avicelphl01对CBD—SPG的载量可达11．61mg／g（w／w）。成功构建并运用原核系统表达CBD-SPG;CBD—SPG在保持良好抗体结合能力的同时,更具有了结合纤维素的能力,有望成为一种新型的亲和材料。     

In vivo interaction between RGS4 and calmodulin visualized with FRET techniques: possible involvement of lipid raft

Regulators of G-protein signaling (RGS) are a family of proteins which accelerate intrinsic GTP-hydrolysis on heterotrimeric G-protein-alpha-subunits. Although it has been suggested that the function of RGS4 is reciprocally regulated by competitive binding of the membrane phospholipid, phosphatidylinositol-3,4,5,-trisphosphate(PtdIns(3,4,5)P(3)), and Ca(2+)/calmodulin (CaM), it remains to be shown that these interactions occur in vivo. Here, using fluorescence resonance energy transfer (FRET) techniques, we show that an elevation of intracellular Ca(2+) concentration by ionomycin increased the FRET efficiency from ECFP (a variant of cyan fluorescent protein)-labeled calmodulin to Venus (a variant of yellow fluorescent protein)-labeled RGS4. The increase in FRET efficiency was greatly attenuated by pre-treating the cells with methyl-beta-cyclodextrin, which depletes membrane cholesterol and thus disrupts lipid rafts. These results provide the first demonstration of a Ca(2+)-dependent interaction between RGS4 and CaM in vivo and show that association in lipid rafts of the plasma membrane might be involved in this physiological regulation of RGS proteins.     

RDJ2 (DNAJA2) chaperones neural G protein signaling pathways

A number of structurally divergent proteins with J domains, called J proteins, interact with and activate the ATPase of Hsp70s, thereby harnessing the ATPase activity for conformational work on target proteins. The precise role of most mammalian J proteins remains undefined. In this paper, we demonstrate that transient expression of the J protein, Rdj2, in HEK 293 cells increased cellular cyclic adenosine monophosphate (cAMP) levels in the presence of the β-adrenergic agonist isoproterenol. In CNS-derived catecholaminergic neuronal cell line (CAD) neuroblastoma cells, expression of Rdj2 increased isoproterenol-stimulated phosphorylation of cAMP response element binding protein (CREB). Moreover, we have characterized the binding properties of Rdj2 and observed a direct interaction between Rdj2 and receptor-coupled trimeric GTP-binding proteins (G proteins). We further show that the composition of the Rdj2-chaperone complex and the cysteine string protein (CSPα)-chaperone complex, another J protein, is distinct. Our data demonstrate that Rdj2 modulates G protein signaling and further suggest that chaperoning G proteins is an emerging theme of the J protein network.     

Phosducin-like protein acts as a molecular chaperone for G protein betagamma dimer assembly

Phosducin-like protein (PhLP) is a widely expressed binding partner of the G protein betagamma subunit dimer (Gbetagamma). However, its physiological role is poorly understood. To investigate PhLP function, its cellular expression was blocked using RNA interference, resulting in inhibition of Gbetagamma expression and G protein signaling. This inhibition was caused by an inability of nascent Gbetagamma to form dimers. Phosphorylation of PhLP at serines 18-20 by protein kinase CK2 was required for Gbetagamma formation, while a high-affinity interaction of PhLP with the cytosolic chaperonin complex appeared unnecessary. PhLP bound nascent Gbeta in the absence of Ggamma, and S18-20 phosphorylation was required for Ggamma to associate with the PhLP-Gbeta complex. Once Ggamma bound, PhLP was released. These results suggest a mechanism for Gbetagamma assembly in which PhLP stabilizes the nascent Gbeta polypeptide until Ggamma can associate, resulting in membrane binding of Gbetagamma and release of PhLP to catalyze another round of assembly.     

MAU-8 is a Phosducin-like Protein required for G protein signaling in C. elegans

The mau-8(qm57) mutation inhibits the function of GPB-2, a heterotrimeric G protein beta subunit, and profoundly affects behavior through the Galphaq/Galphao signaling network in C. elegans. mau-8 encodes a nematode Phosducin-like Protein (PhLP), and the qm57 mutation leads to the loss of a predicted phosphorylation site in the C-terminal domain of PhLP that binds the Gbetagamma surface implicated in membrane interactions. In developing embryos, MAU-8/PhLP localizes to the cortical region, concentrates at the centrosomes of mitotic cells and remains associated with the germline blastomere. In adult animals, MAU-8/PhLP is ubiquitously expressed in somatic tissues and germline cells. MAU-8/PhLP interacts with the PAR-5/14.3.3 protein and with the Gbeta subunit GPB-1. In mau-8 mutants, the disruption of MAU-8/PhLP stabilizes the association of GPB-1 with the microtubules of centrosomes. Our results indicate that MAU-8/PhLP modulates G protein signaling, stability and subcellular location to regulate various physiological functions, and they suggest that MAU-8 might not be limited to the Galphaq/Galphao network.     

Cooperative binding of dominant-negative prion protein to kringle domains

Conversion of the cellular prion protein (PrP(C)) to the pathogenic isoform (PrP(Sc)) is a major biochemical alteration in the progression of prion disease. This conversion process is thought to require interaction between PrP(C) and an as yet unidentified auxiliary factor, provisionally designated protein X. In searching for protein X, we screened a phage display cDNA expression library constructed from prion-infected neuroblastoma (ScN2a) cells and identified a kringle protein domain using full-length recombinant mouse PrP (recMoPrP(23-231), hereafter recMoPrP) expressing a dominant-negative mutation at codon 218 (recMoPrP(Q218K)). In vitro binding analysis using ELISA verified specific interaction of recMoPrP to kringle domains (K(1+2+3)) with higher binding by recMoPrP(Q218K) than by full-length recMoPrP without the mutation. This interaction was confirmed by competitive binding analysis, in which the addition of either a specific anti-kringle antibody or L-lysine abolished the interaction. Biochemical studies of the interactions between K(1+2+3) and various concentrations of both recMoPrP molecules demonstrated binding in a dose-dependent manner. A Hill plot analysis of the data indicates positive cooperative binding of both recMoPrP(Q218K) and recMoPrP to K(1+2+3) with stronger binding by recMoPrP(Q218K). Using full-length and an N-terminally truncated MoPrP(89-231), we demonstrate that N-terminal sequences enable PrP to bind strongly to K(1+2+3). Further characterization with truncated MoPrP(89-231) refolded in different conformations revealed that both alpha-helical and beta-sheet conformations bind to K(1+2+3). Our data demonstrate specific, high-affinity binding of a dominant-negative PrP as well as binding of other PrPs to K(1+2+3). The relevance of such interactions during prion pathogenesis remains to be established.     

Central and C-terminal domains of heterotrimeric G protein gamma subunits differentially influence the signaling necessary for primordial germ cell migration

Heterotrimeric G protein signaling is involved in many pathways essential to development including those controlling cell migration, proliferation, differentiation and apoptosis. One key developmental event known to rely on proper heterotrimeric G protein signaling is primordial germ cell (PGC) migration. We previously developed an in vivo PGC migration assay that identified differences in the signaling capacity of G protein gamma subunits. In this study we developed Gγ subunit chimeras to determine the regions of Gγ isoforms that are responsible for these differences. The central section of the Gγ subunit was found to be necessary for the ability of a Gγ subunit to mediate signaling involved in PGC migration. Residues found in the carboxy-terminal segment of Gγ transducin (gngt1) were found to be responsible for the ability of this subunit to disrupt PGC migration. The type of prenylation did not affect the ability of a Gγ subunit to reverse prenylation-deficient-Gγ-induced PGC migration defects. However, a version of gng2, engineered to be farnesylated instead of geranylgeranylated, still lacks the ability to reverse PGC migration defects known to result from treatment of zebrafish with geranylgeranyl transferase inhibitors (GGTI), supporting the notion that Gγ subunits are one of several protein targets that need to be geranylgeranylated to orchestrate the proper long-range migration of PGCs.     

Structural domains of ribosomal protein S8 and their relationship to ribosomal RNA binding

Escherichia coli ribosomal protein S8 has been subjected to mild proteolytic digestion in order to search for structural domains within the protein [1]. A characteristic fragment produced in high yield after chymotrypsin treatment has been located with the protein sequence. Circular dichroism has shown this domain to be rich in α helix. However, the fragment loses its ability to bind to 16 S rRNA as does a similar fragment produced by trypsin cleavage. The intact protein is required for rRNA binding and is highly protected against proteolytic digestion when bound to the RNA.     

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.     

In vivo functional analysis of the daughter of sevenless protein in receptor tyrosine kinase signaling

One mechanism used by receptor tyrosine kinases to relay a signal to different downstream effector molecules is to use adaptor proteins that provide docking sites for a variety of proteins. The daughter of sevenless (dos) gene was isolated in a genetic screen for components acting downstream of the Sevenless (Sev) receptor tyrosine kinase. Dos contains a N-terminally located PH domain and several tyrosine residues within consensus binding sites for a number of SH2 domain containing proteins. The structural features of Dos and experiments demonstrating tyrosine phosphorylation of Dos upon Sev activation suggested that Dos belongs to the family of multisite adaptor proteins that include the Insulin Receptor Substrate (IRS) proteins, Gab1, and Gab2. Here, we studied the structural requirements for Dos function in receptor tyrosine kinase mediated signaling processes by expressing mutated dos transgenes in the fly. We show that mutant Dos proteins lacking the putative binding sites for the SH2 domains of Shc, PhospholipaseC-γ (PLC-γ) and the regulatory subunit of Phosphoinositide 3-kinase (PI3-K) can substitute the loss of endogenous Dos function during development. In contrast, tyrosine 801, corresponding to a predicted Corkscrew (Csw) tyrosine phosphatase SH2 domain binding site, is essential for Dos function. Furthermore, we assayed whether the Pleckstrin homology (PH) domain is required for Dos function and localization. Evidence is provided that deletion or mutation of the PH domain interferes with the function but not with localization of the Dos protein. The Dos PH domain can be replaced by the Gab1 PH domain but not by a heterologous membrane anchor, suggesting a specific function of the PH domain in regulating signal transduction.