共查询到20条相似文献,搜索用时 31 毫秒
1.
Lishko PV Martemyanov KA Hopp JA Arshavsky VY 《The Journal of biological chemistry》2002,277(27):24376-24381
The complex between the short splice variant of the ninth member of the RGS protein family and the long splice variant of type 5 G protein beta subunit (RGS9-Gbeta5L) plays a critical role in regulating the duration of the light response in vertebrate photoreceptors by activating the GTPase activity of the photoreceptor-specific G protein, transducin. RGS9-Gbeta5L is tightly associated with the membranes of photoreceptor outer segments; however, the nature of this association remains unknown. Here we demonstrate that rod outer segment membranes contain a limited number of sites for high affinity RGS9-Gbeta5L binding, which are highly sensitive to proteolysis. In membranes isolated from bovine rod outer segments, all of these sites are occupied by the endogenous RGS9-Gbeta5L, which prevents the binding of exogenous recombinant RGS9-Gbeta5L to these sites. However, treating membranes with urea or high pH buffers causes either removal or denaturation of the endogenous RGS9-Gbeta5L, allowing for high affinity binding of recombinant RGS9-Gbeta5L to these sites. This binding results in a striking approximately 70-fold increase in the RGS9-Gbeta5L ability to activate transducin GTPase. The DEP (disheveled/EGL-10/pleckstrin) domain of RGS9 plays a crucial role in the RGS9-Gbeta5L membrane attachment, as evident from the analysis of membrane-binding properties of deletion mutants lacking either N- or C-terminal parts of the RGS9 molecule. Our data indicate that specific association of RGS9-Gbeta5L with photoreceptor disc membranes serves not only as a means of targeting it to an appropriate subcellular compartment but also serves as an important determinant of its catalytic activity. 相似文献
2.
RGS9-1 is a GTPase-accelerating protein (GAP) required for rapid recovery of the light response in vertebrate rod and cone photoreceptors. Similar to its phototransduction partners transducin (G(t)) and cGMP phosphodiesterase, it is a peripheral protein of the disc membranes, but it binds membranes much more tightly. It lacks the lipid modifications found on G(t) and cGMP phosphodiesterase, and the mechanism for membrane attachment is unknown. We have used limited proteolysis to generate a fragment of RGS9-1 that is readily removed from membranes under moderate salt conditions. Immunoblots reveal that this soluble fragment lacks a 3-kDa fragment from the C-terminal domain, the only domain within RGS9-1 that differs in sequence from the brain-specific isoform RGS9-2. Recombinant fragments of RGS9-1 with or without the partner subunit G beta(5L) were constructed with or without the C-terminal domain. Those lacking the C-terminal domain bound to photoreceptor membranes much less tightly than those containing it. Removal by urea of G beta(5L) from endogenous or recombinant RGS9-1 bound to rod outer segment membranes left RGS9-1 tightly membrane-bound, and recombinant RGS9-1 was urea-soluble in the absence of membranes. Thus the C-terminal domain of RGS9-1 is critical for membrane binding, whereas G beta(5L) does not play an important role in membrane attachment. 相似文献
3.
Hiol A Davey PC Osterhout JL Waheed AA Fischer ER Chen CK Milligan G Druey KM Jones TL 《The Journal of biological chemistry》2003,278(21):19301-19308
Regulators of G-protein signaling (RGS) proteins down-regulate signaling by heterotrimeric G-proteins by accelerating GTP hydrolysis on the G alpha subunits. Palmitoylation, the reversible addition of palmitate to cysteine residues, occurs on several RGS proteins and is critical for their activity. For RGS16, mutation of Cys-2 and Cys-12 blocks its incorporation of [3H]palmitate and ability to turn-off Gi and Gq signaling and significantly inhibited its GTPase activating protein activity toward aG alpha subunit fused to the 5-hydroxytryptamine receptor 1A, but did not reduce its plasma membrane localization based on cell fractionation studies and immunoelectron microscopy. Palmitoylation can target proteins, including many signaling proteins, to membrane microdomains, called lipid rafts. A subpopulation of endogenous RGS16 in rat liver membranes and overexpressed RGS16 in COS cells, but not the nonpalmitoylated cysteine mutant of RGS16, localized to lipid rafts. However, disruption of lipid rafts by treatment with methyl-beta-cyclodextrin did not decrease the GTPase activating protein activity of RGS16. The lipid raft fractions were enriched in protein acyltransferase activity, and RGS16 incorporated [3H]palmitate into a peptide fragment containing Cys-98, a highly conserved cysteine within the RGS box. These results suggest that the amino-terminal palmitoylation of an RGS protein promotes its lipid raft targeting that allows palmitoylation of a poorly accessible cysteine residue that we show in the accompanying article (Osterhout, J. L., Waheed, A. A., Hiol, A., Ward, R. J., Davey, P. C., Nini, L., Wang, J., Milligan, G., Jones, T. L. Z., and Druey, K. M. (2003) J. Biol. Chem. 278, 19309-19316) was critical for RGS16 and RGS4 GAP activity. 相似文献
4.
Seno K Kishimoto M Abe M Higuchi Y Mieda M Owada Y Yoshiyama W Liu H Hayashi F 《The Journal of biological chemistry》2001,276(24):20813-20816
Detergent-resistant membrane microdomains in the plasma membrane, known as lipid rafts, have been implicated in various cellular processes. We report here that a low-density Triton X-100-insoluble membrane (detergent-resistant membrane; DRM) fraction is present in bovine rod photoreceptor outer segments (ROS). In dark-adapted ROS, transducin and most of cGMP-phosphodiesterase (PDE) were detergent-soluble. When ROS membranes were exposed to light, however, a large portion of transducin localized in the DRM fraction. Furthermore, on addition of guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) to light-bleached ROS, transducin became detergent-soluble again. PDE was not recruited to the DRM fraction after light stimulus alone, but simultaneous stimulation by light and GTPgammaS induced a massive translocation of all PDE subunits to the DRM. A cholesterol-removing reagent, methyl-beta-cyclodextrin, selectively but partially solubilized PDE from the DRM, suggesting that cholesterol contributes, at least in part, to the association of PDE with the DRM. By contrast, transducin was not extracted by the depletion of cholesterol. These data suggest that transducin and PDE are likely to perform their functions in phototransduction by changing their localization between two distinct lipid phases, rafts and surrounding fluid membrane, on disc membranes in an activation-dependent manner. 相似文献
5.
The Epstein-Barr virus (EBV) is an important human pathogen that is associated with multiple cancers. The major oncoprotein of the virus, latent membrane protein 1 (LMP1), is essential for EBV B-cell immortalization and is sufficient to transform rodent fibroblasts. This viral transmembrane protein activates multiple cellular signaling pathways by engaging critical effector molecules and thus acts as a ligand-independent growth factor receptor. LMP1 is thought to signal from internal lipid raft containing membranes; however, the mechanisms through which these events occur remain largely unknown. Lipid rafts are microdomains within membranes that are rich in cholesterol and sphingolipids. Lipid rafts act as organization centers for biological processes, including signal transduction, protein trafficking, and pathogen entry and egress. In this study, the recruitment of key signaling components to lipid raft microdomains by LMP1 was analyzed. LMP1 increased the localization of phosphatidylinositol 3-kinase (PI3K) and its activated downstream target, Akt, to lipid rafts. In addition, mass spectrometry analyses identified elevated vimentin in rafts isolated from LMP1 expressing NPC cells. Disruption of lipid rafts through cholesterol depletion inhibited PI3K localization to membranes and decreased both Akt and ERK activation. Reduction of vimentin levels or disruption of its organization also decreased LMP1-mediated Akt and ERK activation and inhibited transformation of rodent fibroblasts. These findings indicate that LMP1 reorganizes membrane and cytoskeleton microdomains to modulate signal transduction. 相似文献
6.
Compartmentalization of the exocyst complex in lipid rafts controls Glut4 vesicle tethering
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Lipid raft microdomains act as organizing centers for signal transduction. We report here that the exocyst complex, consisting of Exo70, Sec6, and Sec8, regulates the compartmentalization of Glut4-containing vesicles at lipid raft domains in adipocytes. Exo70 is recruited by the G protein TC10 after activation by insulin and brings with it Sec6 and Sec8. Knockdowns of these proteins block insulin-stimulated glucose uptake. Moreover, their targeting to lipid rafts is required for glucose uptake and Glut4 docking at the plasma membrane. The assembly of this complex also requires the PDZ domain protein SAP97, a member of the MAGUKs family, which binds to Sec8 upon its translocation to the lipid raft. Exocyst assembly at lipid rafts sets up targeting sites for Glut4 vesicles, which transiently associate with these microdomains upon stimulation of cells with insulin. These results suggest that the TC10/exocyst complex/SAP97 axis plays an important role in the tethering of Glut4 vesicles to the plasma membrane in adipocytes. 相似文献
7.
Heliothis virescens and Manduca sexta lipid rafts are involved in Cry1A toxin binding to the midgut epithelium and subsequent pore formation 总被引:1,自引:0,他引:1
Zhuang M Oltean DI Gómez I Pullikuth AK Soberón M Bravo A Gill SS 《The Journal of biological chemistry》2002,277(16):13863-13872
Lipid rafts are characterized by their insolubility in nonionic detergents such as Triton X-100 at 4 degrees C. They have been studied in mammals, where they play critical roles in protein sorting and signal transduction. To understand the potential role of lipid rafts in lepidopteran insects, we isolated and analyzed the protein and lipid components of these lipid raft microdomains from the midgut epithelial membrane of Heliothis virescens and Manduca sexta. Like their mammalian counterparts, H. virescens and M. sexta lipid rafts are enriched in cholesterol, sphingolipids, and glycosylphosphatidylinositol-anchored proteins. In H. virescens and M. sexta, pretreatment of membranes with the cholesterol-depleting reagent saponin and methyl-beta-cyclodextrin differentially disrupted the formation of lipid rafts, indicating an important role for cholesterol in lepidopteran lipid rafts structure. We showed that several putative Bacillus thuringiensis Cry1A receptors, including the 120- and 170-kDa aminopeptidases from H. virescens and the 120-kDa aminopeptidase from M. sexta, were preferentially partitioned into lipid rafts. Additionally, the leucine aminopeptidase activity was enriched approximately 2-3-fold in these rafts compared with brush border membrane vesicles. We also demonstrated that Cry1A toxins were associated with lipid rafts, and that lipid raft integrity was essential for in vitro Cry1Ab pore forming activity. Our study strongly suggests that these microdomains might be involved in Cry1A toxin aggregation and pore formation. 相似文献
8.
Cell extraction with cold nonionic detergents or alkaline carbonate prepares an insoluble membrane fraction whose buoyant density permits its flotation in discontinuous sucrose gradients. These lipid "rafts" are implicated in protein sorting and are attractive candidates as platforms that coordinate signal transduction pathways with intracellular substrates. Gap junctions form a direct molecular signaling pathway by end-to-end apposition of hemichannels containing one (homomeric) or more (heteromeric) connexin isoforms. Residency of channels composed of Cx26 and/or Cx32 in lipid rafts was assessed by membrane insolubility in alkaline carbonate or different concentrations of Triton X100, Nonidet P40 and Brij-58 nonionic detergents. Using Triton X100, insoluble raft membranes contained homomeric Cx32 channels, but Cx26-containing channels only when low detergent concentrations were used. Results were similar using Nonidet P40, except that Cx26-containing channels were excluded from raft membranes at all detergent concentrations. In contrast, homomeric Cx26 channels were enriched within Brij-58-insoluble rafts, whereas Cx32-containing channels partitioned between raft and nonraft membranes. Immunofluorescence microscopy showed prominent colocalization only of nonjunctional connexin channels with raft plasma membrane; junctional plaques were not lipid rafts. Rafts prepared by different extraction methods had considerable quantitative and qualitative differences in their lipid compositions. That functionally different nonjunctional connexin channels partition among rafts with distinct lipid compositions suggests that unpaired Cx26 and/or Cx32 channels exist in membrane domains of slightly different physicochemical character. Rafts may be involved in trafficking of plasma membrane connexin channels to gap junctions. 相似文献
9.
Lipid rafts are specialized cholesterol-enriched membrane domains that participate in cellular signaling processes. Caveolae are related domains that become invaginated due to the presence of the structural protein, caveolin-1. In this paper, we use electrospray ionization mass spectrometry (ESI/MS) to quantitatively compare the phospholipids present in plasma membranes and nondetergent lipid rafts from caveolin-1-expressing and nonexpressing cells. Lipid rafts are enriched in cholesterol and sphingomyelin as compared to the plasma membrane fraction. Expression of caveolin-1 increases the amount of cholesterol recovered in the lipid raft fraction but does not affect the relative proportions of the various phospholipid classes. Surprisingly, ESI/MS demonstrated that lipid rafts are enriched in plasmenylethanolamines, particularly those containing arachidonic acid. While the total content of anionic phospholipids was similar in plasma membranes and nondetergent lipid rafts, the latter were highly enriched in phosphatidylserine but relatively depleted in phosphatidylinositol. Detergent-resistant membranes made from the same cells showed a higher cholesterol content than nondetergent lipid rafts but were depleted in anionic phospholipids. In addition, these detergent-resistant membranes were not enriched in arachidonic acid-containing ethanolamine plasmalogens. These data provide insight into the structure of lipid rafts and identify potential new roles for these domains in signal transduction. 相似文献
10.
Lipid rafts are detergent-resistant, liquid-ordered microdomains in plasma membranes that are enriched in cholesterol and sphingolipids and involved in intracellular signal transduction, membrane trafficking, and molecular sorting. In this study, we investigated the possibility that lipid rafts on Eimeria tenella sporozoites may act as platforms for host cell invasion. Flotillin-1, a resident protein of lipid rafts, was identified on E. tenella sporozoites and was prominently expressed at the apex of the cells, a region mediating host cell invasion. Pretreatment of sporozoites with antibody against flotillin-1 blocked parasite invasion. Furthermore, the anticoccidial drug, monensin, disrupted the localization of flotillin-1 within raft structures resulting in loss of invasion. We conclude that Eimeria sporozoites utilize lipid rafts containing flotillin-1 for internalization into host cells. 相似文献
11.
Visualizing lipid raft dynamics and early signaling events during antigen receptor-mediated B-lymphocyte activation 总被引:10,自引:0,他引:10
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Recent biochemical evidence indicates that an early event in signal transduction by the B-cell antigen receptor (BCR) is its translocation to specialized membrane subdomains known as lipid rafts. We have taken a microscopic approach to image lipid rafts and early events associated with BCR signal transduction. Lipid rafts were visualized on primary splenic B lymphocytes from wild-type or anti-hen egg lysozyme BCR transgenic mice, and on a mature mouse B-cell line Bal 17 by using fluorescent conjugates of cholera toxin B subunit or a Lyn-based chimeric protein, which targets green fluorescent protein to the lipid raft compartment. Time-lapse imaging of B cells stimulated via the BCR with the antigen hen egg lysozyme, or surrogate for antigen anti-IgM, demonstrated that lipid rafts are highly dynamic entities, which move laterally on the surface of these cells and coalesce into large regions. These regions of aggregated lipid rafts colocalized with the BCR and tyrosine-phosphorylated proteins. Microscopic imaging of live B cells also revealed an inducible colocalization of lipid rafts with the tyrosine kinase Syk and the receptor tyrosine phosphatase CD45. These two proteins play indispensable roles in BCR-mediated signaling but are not detectable in biochemically purified lipid raft fractions. Strikingly, BCR stimulation also induced the formation of long, thread-like filopodial projections, similar to previously described structures called cytonemes. These B-cell cytonemes are rich in lipid rafts and actin filaments, suggesting that they might play a role in long-range communication and/or transportation of signaling molecules during an immune response. These results provide a window into the morphological and molecular organization of the B-cell membrane during the early phase of BCR signaling. 相似文献
12.
Scarlata S 《Biochemistry》2002,41(22):7092-7099
One function of membrane domains of liquid-ordered lipids or "rafts" may be to stabilize complexes of signaling proteins, thereby playing a role in the transduction of cellular signals. Here, we have used fluorescence methods to directly test this idea by assessing the ability of phospholipase Cbeta2 (PLCbeta2) to associate with G protein subunits on model membranes in the fluid phase and on membranes that contain domains of lipids in the liquid-ordered phase (rafts). We find that the apparent dissociation constant for the equilibrium between PLCbeta2 and Galpha(q)(GTPgammaS) was identical on both types of membrane surfaces. However, the degree of association between PLCbeta2 and Gbetagamma subunits was significantly reduced on the surfaces containing rafts. Time studies indicate that this phenomenon is a dynamic process. Incorporating the lipid substrate of PLCbeta2 into membranes that forms rafts, we find that its basal activity is unaffected. However, its activation by Gbetagamma subunits is inhibited, supporting a reduced degree of interaction between these two proteins when rafts are present. Since lipid rafts affected PLCbeta2-Gbetagamma association and not PLCbeta2-Galpha(q)(GTPgammaS) association, we explored the possibility that the membrane interaction of Gbetagamma differed when rafts are present. We find that although the membrane partition coefficient of Gbetagamma is not significantly changed in the presence of rafts, proteolysis of Gbetagamma by trypsin increases and the ability of Gbetagamma Tyr/Trp fluorescence to be quenched by iodide ions decreases when rafts are present. These results suggest a model in which lipid rafts occlude the PLCbeta2 interaction site on Gbetagamma subunits by localizing these subunits at the domain interface. 相似文献
13.
Human immunodeficiency virus type 1 Nef associates with lipid rafts to downmodulate cell surface CD4 and class I major histocompatibility complex expression and to increase viral infectivity 总被引:2,自引:0,他引:2
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Alexander M Bor YC Ravichandran KS Hammarskjöld ML Rekosh D 《Journal of virology》2004,78(4):1685-1696
Lipid rafts are membrane microdomains that are functionally distinct from other membrane regions. We have shown that 10% of human immunodeficiency virus type 1 (HIV-1) Nef expressed in SupT1 cells is present in lipid rafts and that this represents virtually all of the membrane-associated Nef. To determine whether raft targeting, rather than simply membrane localization, has functional significance, we created a Nef fusion protein (LAT-Nef) containing the N-terminal 35 amino acids from LAT, a protein that is exclusively localized to rafts. Greater than 90% of the LAT-Nef protein was found in the raft fraction. In contrast, a mutated form, lacking two cysteine palmitoylation sites, showed less than 5% raft localization. Both proteins were equally expressed and targeted nearly exclusively to membranes. The LAT-Nef protein was more efficient than its nonraft mutant counterpart at downmodulating both cell surface CD4 and class I major histocompatibility complex (MHC) expression, as well as in enhancing first-round infectivity and being incorporated into virus particles. This demonstrates that targeting of Nef to lipid rafts is mechanistically important for all of these functions. Compared to wild-type Nef, LAT-Nef downmodulated class I MHC nearly as effectively as the wild-type Nef protein, but was only about 60% as effective for CD4 downmodulation and 30% as effective for infectivity enhancement. Since the LAT-Nef protein was found entirely in rafts while the wild-type Nef protein was distributed 10% in rafts and 90% in the soluble fraction, our results suggest that class I MHC downmodulation by Nef may be performed exclusively by raft-bound Nef. In contrast, CD4 downmodulation and infectivity enhancement may require a non-membrane-bound Nef component as well as the membrane-bound form. 相似文献
14.
The duration of the photoreceptor's response to a light stimulus determines the speed at which an animal adjusts to ever-changing conditions of the visual environment. One critical component which regulates the photoresponse duration on the molecular level is the complex between the ninth member of the regulators of G protein signaling family (RGS9-1) and its partner, type 5 G protein beta-subunit (Gbeta5L). RGS9-1.Gbeta5L is responsible for the activation of the GTPase activity of the photoreceptor-specific G protein, transducin. Importantly, this function of RGS9-1.Gbeta5L is regulated by its membrane anchor, R9AP, which drastically potentiates the ability of RGS9-1.Gbeta5L to activate transducin GTPase. In this study, we address the kinetic mechanism of R9AP action and find that it consists primarily of a direct increase in the RGS9-1.Gbeta5L activity. We further showed that the binding site for RGS9-1.Gbeta5L is located within the N-terminal putative trihelical domain of R9AP, and even though this domain is sufficient for binding, it takes the entire R9AP molecule to potentiate the activity of RGS9-1.Gbeta5L. The mechanism revealed in this study is different from and complements another well-established mechanism of regulation of RGS9-1.Gbeta5L by the effector enzyme, cGMP phosphodiesterase, which is based entirely on the enhancement in the affinity between RGS9-1.Gbeta5L and transducin. Together, these mechanisms ensure timely transducin inactivation in the course of the photoresponse, a requisite for normal vision. 相似文献
15.
Recent epidemiological studies show a reduced prevalence of Alzheimer's disease (AD) in patients treated with inhibitors of cholesterol biosynthesis. Moreover, the cholesterol-transport protein, apolipoprotein E4, and elevated cholesterol are important risk factors for AD. Additionally, in vitro and in vivo studies show that intracellular cholesterol levels can modulate the processing of amyloid precursor protein (APP) to beta-amyloid, the major constituent of senile plaques. Cholesterol plays a crucial role in maintaining lipid rafts in a functional state. Lipid rafts are cholesterol-enriched membrane microdomains implicated in signal transduction, protein trafficking, and proteolytic processing. Since APP, beta-amyloid, and the putative gamma-secretase, presenilin-1 (PS-1), have all been found in lipid rafts, we hypothesized that the recently identified beta-secretase, Asp2 (BACE1), might also be present in rafts. Here, we report that recombinant Asp2 expressed in three distinct cell lines is raft associated. Using both detergent and nondetergent methods, Asp2 protein and activity were found in a light membrane raft fraction that also contained other components of the amyloidogenic pathway. Immunoisolation of caveolin-containing vesicles indicated that Asp2 was present in a unique raft population distinct from caveolae. Finally, depletion of raft cholesterol abrogated association of Asp2 with the light membrane fraction. These observations are consistent with the raft localization of APP processing and suggest that the partitioning of Asp2 into lipid rafts may underlie the cholesterol sensitivity of beta-amyloid production. 相似文献
16.
Rhodopsin is a prototypical G protein-coupled receptor (GPCR) - a member of the superfamily that shares a similar structural architecture consisting of seven-transmembrane helices and propagates various signals across biological membranes. Rhodopsin is embedded in the lipid bilayer of specialized disk membranes in the outer segments of retinal rod photoreceptor cells where it transmits a light-stimulated signal. Photoactivated rhodopsin then activates a visual signaling cascade through its cognate G protein, transducin or Gt, that results in a neuronal response in the brain. Interestingly, the lipid composition of ROS membranes not only differs from that of the photoreceptor plasma membrane but is critical for visual transduction. Specifically, lipids can modulate structural changes in rhodopsin that occur after photoactivation and influence binding of transducin. Thus, altering the lipid organization of ROS membranes can result in visual dysfunction and blindness. 相似文献
17.
We examined the role of BCR cell membrane redistribution in anti-IgM-induced apoptosis in three human B cell lines, RA#1, 2G6, and MC116, that differ in their relative levels of sIgM expression. The apoptotic response was found to be dependent on the nature of the anti-IgM and the cell line. In the cell lines, RA#1 and MC116, sIgM aggregated into patches that were insensitive to the disruption of cholesterol-rich membrane microdomains by nystatin or beta-MCD. The B cell line 2G6 was able to reorganize sIgM into a tight coalescent cap upon anti-IgM treatment. However, in this case, the lipid raft inhibitors nystatin and beta-MCD disrupted the patching. In 2G6 cells, BCR-mediated apoptosis was not affected by nystatin treatment, whereas it increased in beta-MCD pretreated cells. Thus, no evident correlation was found between apoptosis and BCR cell membrane redistribution or lipid raft formation in either of the three cell lines. The data indicate that the apoptotic signal transduction pathway is independent of BCR translocation into lipid rafts and/or aggregation. 相似文献
18.
Sato M Moroi K Nishiyama M Zhou J Usui H Kasuya Y Fukuda M Kohara Y Komuro I Kimura S 《Life sciences》2003,73(7):917-932
RGS (regulator of G protein signaling) proteins are GTPase-activating proteins (GAPs) for heterotrimeric G protein alpha subunits and negatively regulate G protein-mediated signal transduction. In this study, we determined the cDNA sequence of a novel Caenorhabditis elegans (C. elegans) RGS protein. The predicted protein, termed C2-RGS, consists of 782 amino acids, and contains a C2 domain and an RGS domain. C2 domains are typically known to be Ca(2+) and phospholipid binding sites, found in many proteins involved in membrane traffic or signal transduction, and most of their biological roles are not identified. To study the function of C2-RGS protein, a series of six truncated versions of C2-RGS were constructed. When the full-length protein of C2-RGS was expressed transiently in AT1a-293T cells, ET-1-induced Ca(2+) responses were strongly suppressed. When each of the mutants with either RGS domain or C2 domain was expressed, the Ca(2+) responses were suppressed moderately. Furthermore, we found that C2 domain of PLC-beta1 also had a similar moderate inhibitory effect. RGS domain of C2-RGS bound to mammalian and C. elegans Galphai/o and Galphaq subunits only in the presence of GDP/AlF(4)(-), and had GAP activity to Galphai3. On the other hand, C2 domains of C2-RGS and PLC-beta1 also bound strongly to Galphaq subunit, in the presence of GDP, GDP/AlF(4)(-), and GTPgammaS, suggesting the stable persistent association between these C2 domains and Galphaq subunit at any stage during GTPase cycle. These results indicate that both the RGS domain and the C2 domain are responsible for the inhibitory effect of the full-length C2-RGS protein on Galphaq-mediated signaling, and suggest that C2 domains of C2-RGS and PLC-beta1 may act as a scaffold module to organize Galphaq and the respective whole protein molecule in a stable signaling complex, both in the absence and presence of stimulus. 相似文献
19.
Wang Q Zhang X Zhang L He F Zhang G Jamrich M Wensel TG 《The Journal of biological chemistry》2008,283(44):30015-30024
The dynamics of G protein-mediated signal transduction depend on the two-dimensional diffusion of membrane-bound G proteins and receptors, which has been suggested to be rate-limiting for vertebrate phototransduction, a highly amplified G protein-coupled signaling pathway. Using fluorescence recovery after photobleaching (FRAP), we measured the diffusion of the G protein transducin alpha-subunit (Galpha(t)) and the G protein-coupled receptor rhodopsin on disk membranes of living rod photoreceptors from transgenic Xenopus laevis. Treatment with either methyl-beta-cyclodextrin or filipin III to disrupt cholesterol-containing lipid microdomains dramatically accelerated diffusion of Galpha(t) in its GTP-bound state and of the rhodopsin-Galphabetagamma(t) complex but not of rhodopsin or inactive GDP-bound Galphabetagamma. These results imply an activity-dependent sequestration of G proteins into cholesterol-dependent lipid microdomains, which limits diffusion and exclude the majority of free rhodopsin and the free G protein heterotrimer. Our data offer a novel demonstration of lipid microdomains in the internal membranes of living sensory neurons. 相似文献
20.
The complex between the photoreceptor-specific regulator of G protein signaling (RGS) protein, RGS9-1, and type 5 G protein beta-subunit, Gbeta5L, regulates the duration of the cellular response to light by stimulating the GTPase activity of G protein, transducin. An important property of RGS9-1.Gbeta5L is that it interacts specifically with transducin bound to its effector, cGMP phosphodiesterase, rather than with transducin alone. The minimal structure within the RGS9-1.Gbeta5L complex capable of activating transducin GTPase is the catalytic domain of RGS9. This domain itself is also able to discriminate between free and effector-bound transducin but to a lesser degree than RGS9-1.Gbeta5L. The goal of this study was to determine whether other, noncatalytic domains of RGS9-1.Gbeta5L enhance the intrinsic specificity of the catalytic domain or whether they set the specificity of RGS9-1.Gbeta5L regardless of the specificity of its catalytic domain. We found that a double L353E/R360P amino acid substitution reversed the specificity of the recombinant catalytic domain but did not reverse the specificity of RGS9-1.Gbeta5L. However, the degree of discrimination between free and effector-bound transducin was reduced. Therefore, noncatalytic domains of RGS9-1.Gbeta5L play a decisive role in establishing its substrate specificity, yet the high degree of this specificity observed under physiological conditions requires an additional contribution from the catalytic domain. 相似文献