共查询到20条相似文献,搜索用时 15 毫秒
1.
Previously we reported that the G-protein Galphai3 localized in pancreatic zymogen granule (ZG) membrane participates in vesicular fusion at the cell plasma membrane (PM). In the present study, the presence of cytosolic phosholipase A2 (cPLA2) in rat ZGs was demonstrated and its potential role in G-protein-mediated ZG-PM fusion was investigated. In vitro fusion assays utilizing both enzymatic and fluorimetric techniques demonstrate that ZGs fuse with PM with a greater potency in the presence of GTP. Arachidonyl trifluoromethyl ketone (AACOCF3) at 40 microM reduces GTP-induced ZG-PM fusion by 25-50%. Anti-cPLA2 antibody reduces ZG-PM fusion in a dose-dependent manner and a 50% reduction of the fusion takes place in the range of 0.48-0.64 ratios of cPLA2 antibody to ZG proteins. PLAP, a cPLA2 activator synthetic peptide increases ZG-PM fusion in a limited dose-dependent manner and tends to inhibit at higher concentrations. Exogenous arachidonic acid inhibits GTP-induced ZG-PM fusion in a dose-dependent manner. Furthermore, a non-hydrolysable GTP analogue, Gpp(NH)p, reduces PLAP effect in ZG-PM fusion; and the net effect of Gpp(NH)p and PLAP differs significantly from the net effect of GTP and PLAP on ZG-PM fusion suggesting that cPLA2 is involved in G-protein-mediated secretory vesicle fusion. 相似文献
2.
Faust F Gomez-Lazaro M Borta H Agricola B Schrader M 《Traffic (Copenhagen, Denmark)》2008,9(6):964-979
Zymogen granules (ZGs) are specialized storage organelles in the exocrine pancreas, which allow digestive enzyme storage and regulated apical secretion. To understand the function of these important organelles, we are conducting studies to identify and characterize ZG membrane proteins. Small guanosine triphosphatases (GTPases) of the Rab family are key protein components involved in vesicular/granular trafficking and membrane fusion in eukaryotic cells. In this study, we show by morphological studies that Rab8 (Rab8A) localizes to ZGs in acinar cells of the pancreas. We find that Rab8 is present on isolated ZGs from rat pancreas and in the ZG membrane fraction obtained after granule subfractionation. To address a putative role of Rab8 in granule biogenesis, we conducted RNA interference experiments to 'knock down' the expression of Rab8 in pancreatic AR42J cells. Silencing of Rab8 (but not of Rab3) resulted in a decrease in the number of ZGs and in an accumulation of granule marker proteins within the Golgi complex. By contrast, the trafficking of lysosomal and plasma membrane proteins was not affected. These data provide first evidence for a role of Rab8 early on in ZG formation at the Golgi complex and thus, apical trafficking of digestive enzymes in acinar cells of the pancreas. 相似文献
3.
Regulation of the water channel aquaporin-1: isolation and reconstitution of the regulatory complex 总被引:4,自引:0,他引:4
Abu-Hamdah R Cho WJ Cho SJ Jeremic A Kelly M Ilie AE Jena BP 《Cell biology international》2004,28(1):7-17
Aquaporins (AQP) are involved in rapid and active gating of water across biological membranes. The molecular regulation of AQP is unknown. Here we report the isolation, identification and reconstitution of the regulatory complex of AQP-1. AQP-1 and Galphai3 have been implicated in GTP-induced gating of water in zymogen granules (ZG), the secretory vesicles in exocrine pancreas. In the present study, detergent-solubilized ZGs immunoprecipitated with monoclonal AQP-1 antibody, co-isolates AQP-1, PLA2, Galphai3, potassium channel IRK-8, and the chloride channel ClC-2. Exposure of ZGs to either the potassium channel blocker glyburide, or the PLA2 inhibitor ONO-RS-082, blocked GTP-induced ZG swelling. RBC known to possess AQP-1 at the plasma membrane, swell on exposure to the Galphai-agonist mastoparan, and respond similarly to ONO-RS-082 and glyburide, as ZGs. Liposomes reconstituted with the AQP-1 immunoisolated complex from solubilized ZG, also swell in response to GTP. Glyburide or ONO-RS-082 abolished the GTP effect. Immunoisolate-reconstituted planar lipid bilayers demonstrate conductance, which is sensitive to glyburide and an AQP-1 specific antibody. Our results demonstrate a Galphai3-PLA2 mediated pathway and potassium channel involvement in AQP-1 regulation. 相似文献
4.
Ogier-Denis E Pattingre S El Benna J Codogno P 《The Journal of biological chemistry》2000,275(50):39090-39095
Galpha-interacting protein (GAIP) is a regulator of G protein signaling (RGS) that accelerates the rate of GTP hydrolysis by the alpha-subunit of the trimeric G(i3) protein. Both proteins are part of a signaling pathway that controls lysosomal-autophagic catabolism in human colon cancer HT-29 cells. Here we show that GAIP is phosphorylated by an extracellular signal-regulated (Erk1/2) MAP kinase-dependent pathway sensitive to amino acids, MEK1/2 (PD098059), and protein kinase C (GF109203X) inhibitors. An in vitro phosphorylation assay demonstrates that Erk2-dependent phosphorylation of GAIP stimulates its GTPase-activating protein activity toward the Galpha(i3) protein (k = 0.187 +/- 0.001 s(-)(1), EC(50) = 1.12 +/- 0.10 microm) when compared with unphosphorylated recombinant GAIP (k = 0.145 +/- 0.003 s(-)(1), EC(50) = 3.16 +/- 0. 12 microm) or to GAIP phosphorylated by other Ser/Thr protein kinases (protein kinase C, casein kinase II). This stimulation and the phosphorylation of GAIP by Erk2 were abrogated when serine at position 151 in the RGS domain was substituted by an alanine residue using site-directed mutagenesis. Furthermore, the lysosomal-autophagic pathway was not stimulated in S151A-GAIP mutant-expressing cells when compared with wild-type GAIP-expressing cells. These results demonstrate that the GTPase-activating protein activity of GAIP is stimulated by Erk2 phosphorylation. They also suggested that Erk1/2 and GAIP are engaged in the signaling control of a major catabolic pathway in intestinal derived cells. 相似文献
5.
RGS-GAIP, a GTPase-activating Protein for Gαi Heterotrimeric G Proteins, Is Located on Clathrin-coated Vesicles
下载免费PDF全文
![点击此处可从《Molecular biology of the cell》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Luc De Vries Eric Elenko J. Michael McCaffery Thierry Fischer Laura Hubler Tammie McQuistan Nicki Watson Marilyn G. Farquhar 《Molecular biology of the cell》1998,9(5):1123-1134
RGS-GAIP (Gα-interacting protein) is a member of the RGS (regulator of G protein signaling) family of proteins that functions to down-regulate Gαi/Gαq-linked signaling. GAIP is a GAP or guanosine triphosphatase-activating protein that was initially discovered by virtue of its ability to bind to the heterotrimeric G protein Gαi3, which is found on both the plasma membrane (PM) and Golgi membranes. Previously, we demonstrated that, in contrast to most other GAPs, GAIP is membrane anchored and palmitoylated. In this work we used cell fractionation and immunocytochemistry to determine with what particular membranes GAIP is associated. In pituitary cells we found that GAIP fractionated with intracellular membranes, not the PM; by immunogold labeling GAIP was found on clathrin-coated buds or vesicles (CCVs) in the Golgi region. In rat liver GAIP was concentrated in vesicular carrier fractions; it was not found in either Golgi- or PM-enriched fractions. By immunogold labeling it was detected on clathrin-coated pits or CCVs located near the sinusoidal PM. These results suggest that GAIP may be associated with both TGN-derived and PM-derived CCVs. GAIP represents the first GAP found on CCVs or any other intracellular membranes. The presence of GAIP on CCVs suggests a model whereby a GAP is separated in space from its target G protein with the two coming into contact at the time of vesicle fusion. 相似文献
6.
GAIP is a regulator of G protein signaling (RGS) that accelerates the rate of GTP hydrolysis by some G protein alpha subunits. In the present studies, we have examined the structural basis for the ability of GAIP to discriminate among members of the Galphai family. Galphai1, Galphai3, and Galphao interacted strongly with GAIP, whereas Galphai2 interacted weakly and Galphas did not interact at all. A chimeric G protein composed of a Galphai2 N terminus and a Galphai1 C terminus interacted as strongly with GAIP as native Galphai1, whereas a chimeric N-terminal Galphai1 with a Galphai2 C terminus did not interact. These results suggest that the determinants responsible for GAIP selectivity between these two Galphais reside within the C-terminal GTPase domain of the G protein. To further localize residues contributing to G protein-GAIP selectivity, a panel of 15 site-directed Galphai1 and Galphai2 mutants were assayed. Of the Galphai1 mutants tested, only that containing a mutation at aspartate 229 located at the N terminus of Switch 3 did not interact with GAIP. Furthermore, the only Galphai2 variant that interacted strongly with GAIP contained a replacement of the corresponding Galphai2 Switch 3 residue (Ala230) with aspartate. To determine whether GAIP showed functional preferences for Galpha subunits that correlate with the binding data, the ability of GAIP to enhance the GTPase activity of purified alpha subunits was tested. GAIP catalyzed a 3-5-fold increase in the rate of GTP hydrolysis by Galphai1 and Galphai2(A230D) but no increase in the rate of Galphai2 and less than a 2-fold increase in the rate of Galphai1(D229A) under the same conditions. Thus, GAIP was able to discriminate between Galphai1 and Galphai2 in both binding and functional assays, and in both cases residue 229/230 played a critical role in selective recognition. 相似文献
7.
Kimple RJ De Vries L Tronchère H Behe CI Morris RA Gist Farquhar M Siderovski DP 《The Journal of biological chemistry》2001,276(31):29275-29281
The regulators of G-protein signaling (RGS) proteins accelerate the intrinsic guanosine triphosphatase activity of heterotrimeric G-protein alpha subunits and are thus recognized as key modulators of G-protein-coupled receptor signaling. RGS12 and RGS14 contain not only the hallmark RGS box responsible for GTPase-accelerating activity but also a single G alpha(i/o)-Loco (GoLoco) motif predicted to represent a second G alpha interaction site. Here, we describe functional characterization of the GoLoco motif regions of RGS12 and RGS14. Both regions interact exclusively with G alpha(i1), G alpha(i2), and G alpha(i3) in their GDP-bound forms. In GTP gamma S binding assays, both regions exhibit guanine nucleotide dissociation inhibitor (GDI) activity, inhibiting the rate of exchange of GDP for GTP by G alpha(i1). Both regions also stabilize G alpha(i1) in its GDP-bound form, inhibiting the increase in intrinsic tryptophan fluorescence stimulated by AlF(4)(-). Our results indicate that both RGS12 and RGS14 harbor two distinctly different G alpha interaction sites: a previously recognized N-terminal RGS box possessing G alpha(i/o) GAP activity and a C-terminal GoLoco region exhibiting G alpha(i) GDI activity. The presence of two, independent G alpha interaction sites suggests that RGS12 and RGS14 participate in a complex coordination of G-protein signaling beyond simple G alpha GAP activity. 相似文献
8.
Signal-transducing guanine-nucleotide-binding regulatory proteins (G proteins) are heterotrimers, composed of the nucleotide-binding alpha subunit and a beta gamma dimer. The influence of beta gamma dimer preparations of the retinal G protein transducin (TD) was studied on formylpeptide-receptor--G-protein interactions in membranes of differentiated HL 60 cells. For this, TD was prepared from bovine rod outer segment (ROS) membranes with either GTP or its analogs, guanosine 5'-[gamma-thio]triphosphate (GTP[S]) and guanosine 5'-[beta gamma-imino]triphosphate (Gpp[NH]p). After removal of free nucleotides, TD beta gamma was separated from TD alpha and its function analyzed. Addition of TD beta gamma isolated from TD prepared with GTP[S] (TD beta gamma GTP[S]) to HL 60 membranes abolished high-affinity binding of fMet-Leu-[3H]Phe (fMet, N-formylmethionine) to its receptor. In contrast, TD beta gamma isolated from TD prepared with GTP (TD beta gamma GTP), boiled TD beta gamma GTP[S] and TD alpha prepared with GTP[S] had no or only slight effects. The inhibitory effect of TD beta gamma GTP[S] on fMet-Leu-[3H]Phe receptor binding was potentiated by GDP at low concentrations but not by GTP[S]. Furthermore, TD beta gamma GTP[S], but not TD beta gamma GTP or TD beta gamma isolated from TD prepared with Gpp[NH]p (TD beta gamma Gpp[NH]p), prevented fMet-Leu-Phe-stimulated binding of [35S]GTP[S] to G proteins in HL 60 membranes, measured in the presence of GDP. When TD beta gamma GTP was incubated with GTP [S] and TD-depleted illuminated ROS membranes, and subsequently separated from the membranes and free GTP[S], this TD beta gamma GTP, similar to TD beta gamma GTP[S], abolished high-affinity binding of fMet-Leu-[3H]Phe to its receptor, fMet-Leu-Phe-stimulated binding of [35S]GTP[S], and fMet-Leu-Phe-stimulated GTP hydrolysis in HL 60 membranes. Inhibition of [35S]GTP[S] binding by TD beta gamma was not seen in the presence of the metabolically stable GDP analog, guanosine 5'-[beta-thio]diphosphate. In order to obtain an insight into the modification of TD beta gamma apparently caused by GTP[S], and into its mechanism of action in HL 60 membranes, TD, TD alpha and TD beta gamma, all prepared in the presence of GTP, were incubated with [35S]GTP[S] and TD-depleted illuminated ROS membranes. Fluorographic analysis of the supernatant proteins revealed 35S labelling of the beta band of the G protein. When apparently thiophosphorylated TD beta gamma was incubated with [3H]GDP in the presence of HL 60 membranes, [3H]GTP[S] was rapidly formed.(ABSTRACT TRUNCATED AT 400 WORDS) 相似文献
9.
We have studied the expression and localization of several H(+) and HCO(3)(-) transporters, whose presence in the rat pancreas is still unclear. The Cl(-)/HCO(3)(-) exchanger AE2, the Na(+)/H(+) exchangers NHE1 and NHE4, and the 31-kD and 70-kD vacuolar H(+)-ATPase (V-ATPase) subunits were detected by immunoblotting and immunocytochemical techniques. Immunoblotting of plasma membranes with transporter-specific antibodies revealed protein bands at approximately 160 kD for AE2, at approximately 90 kD and approximately 103 kD for NHE1 and NHE4, respectively, and at 31 kD and 70 kD for V-ATPase. NHE1 and NHE4 were further identified by amplification of isoform-specific cDNA using RT-PCR. Immunohistochemistry revealed a basolateral location of AE2, NHE1, and NHE4 in acinar cells. In ducts, NHE1 and NHE4 were basolaterally located but no AE2 expression was detected. V-ATPase was detected in zymogen granules (ZGs) by immunogold labeling, and basolaterally in duct cells by immunohistochemistry. The data indicate that NHE1 and NHE4 are co-expressed in rat pancreatic acini and ducts. Basolateral acinar AE2 could contribute to Cl(-) uptake and/or pH regulation. V-ATPase may be involved in ZG fusion/exocytosis and ductal HCO(3)(-) secretion. The molecular identity of the ductal Cl(-)/HCO(3)(-) exchanger remains unclear. 相似文献
10.
Manuel Antonio Manso Andres C. Garcia-Montero Ana Isabel Rodriguez Alberto Orfao Isabel De Dios 《The Journal of steroid biochemistry and molecular biology》1997,62(5-6):439-448
This study was performed to evaluate the effects of different doses of hydrocortisone (1, 10 and 25 mg/kg/day) administered for 1, 3 and 8 days on pancreatic enzyme storage in rats. The enzyme content in both pancreas homogenates and in individual isolated zymogen granules (ZGs) was measured using standard biochemical assays and flow cytometry, respectively. Hydrocortisone did not alter the total amount of pancreatic DNA but increased the pancreas enzyme content in a time-dose-dependent way. Amylase activity was significantly increased after hydrocortisone administration at day +8 when 10 mg/kg/day was used, and from the first day of treatment when 25 mg/kg/day was administered. A significant increase in trypsin activity was also observed in response to 25 mg/kg/day of hydrocortisone but only from the third day of treatment onwards. As compared with control rats, chronic administration of either 1 or 10 mg/kg/day of hydrocortisone did not alter significantly either the size or the percentage of the two ZG subpopulations (Z1 and Z2) identified in the pancreas by flow cytometry; in addition, no significant changes were observed in the mean amylase content per individual granule, although its mean concentration increased in rats treated with 10 mg/kg/day for 3 and 8 days. Nevertheless, when 25 mg/kg/day of hydrocortisone were administered for 1 and 3 days, a significant increase in the proportion of Z1 ZGs was observed, which may be related to the formation of new and smaller ZGs. When a very high dose of hydrocortisone (25 mg/kg/day) was used, an overall increase in the pancreatic enzyme content related to an increase in the mean amylase content per individual ZG was observed; this effect was apparent from the first day of treatment in the Z1 subset of ZGs and from day +3 in the Z2 subpopulation. Only a high concentration of hydrocortisone was able to alter the enzyme storage process in individual zymogen granules, but they maintain a normal enzyme load at lower hydrocortisone doses. 相似文献
11.
Regulators of G protein signaling (RGS) proteins bind to active G alpha subunits and accelerate the rate of GTP hydrolysis and/or block interaction with effector molecules, thereby decreasing signal duration and strength. RGS proteins are defined by the presence of a conserved 120-residue region termed the RGS domain. Recently, it was shown that the G protein-coupled receptor kinase 2 (GRK2) contains an RGS domain that binds to the active form of G alpha(q). Here, the ability of GRK2 to interact with other members of the G alpha(q) family, G alpha(11), G alpha(14), and G alpha(16), was tested. The signaling of all members of the G alpha(q) family, with the exception of G alpha(16), was inhibited by GRK2. Immunoprecipitation of full-length GRK2 or pull down of GST-GRK2-(45-178) resulted in the detection of G alpha(q), but not G alpha(16), in an activation-dependent manner. Moreover, activated G alpha(16) failed to promote plasma membrane (PM) recruitment of a GRK2-(45-178)-GFP fusion protein. Assays with chimeric G alpha(q)(-)(16) subunits indicated that the C-terminus of G alpha(q) mediates binding to GRK2. Despite showing no interaction with GRK2, G alpha(16) does interact with RGS2, in both inositol phosphate and PM recruitment assays. Thus, GRK2 is the first identified RGS protein that discriminates between members of the G alpha(q) family, while another RGS protein, RGS2, binds to both G alpha(q) and G alpha(16). 相似文献
12.
Agonist-stimulated high affinity GTPase activity of fusion proteins between the alpha(2A)-adrenoreceptor and the alpha subunits of forms of the G proteins G(i1), G(i2), G(i3), and G(o1), modified to render them insensitive to the action of pertussis toxin, was measured following transient expression in COS-7 cells. Addition of a recombinant regulator of G protein signaling protein, RGS4, did not significantly affect basal GTPase activity nor agonist stimulation of the fusion proteins containing Galpha(i1) and Galpha(i3) but markedly enhanced agonist-stimulation of the proteins containing Galpha(i2) and Galpha(o1.) The effect of RGS4 on the alpha(2A)-adrenoreceptor-Galpha(o1) fusion protein was concentration-dependent with EC(50) of 30 +/- 3 nm and the potency of the receptor agonist UK14304 was reduced 3-fold by 100 nm RGS4. Equivalent reconstitution with Asn(88)-Ser RGS4 failed to enhance agonist function on the alpha(2A)-adrenoreceptor-Galpha(o1) or alpha(2A)-adrenoreceptor-Galpha(i2) fusion proteins. Enzyme kinetic analysis of the GTPase activity of the alpha(2A)-adrenoreceptor-Galpha(o1) and alpha(2A)-adrenoreceptor-Galpha(i2) fusion proteins demonstrated that RGS4 both substantially increased GTPase V(max) and significantly increased K(m) of the fusion proteins for GTP. The increase in K(m) for GTP was dependent upon RGS4 amount and is consistent with previously proposed mechanisms of RGS function. Agonist-stimulated GTPase turnover number in the presence of 100 nm RGS4 was substantially higher for alpha(2A)-adrenoreceptor-Galpha(o1) than for alpha(2A)-adrenoreceptor-Galpha(i2). These studies demonstrate that although RGS4 has been described as a generic stimulator of the GTPase activity of G(i)-family G proteins, selectivity of this interaction and quantitative variation in its function can be monitored in the presence of receptor activation of the G proteins. 相似文献
13.
V K Turmukhambetova M P Danilenko M P Panchenko V B Ritov O V Esyrev V A Tkachuk 《Biokhimii?a (Moscow, Russia)》1992,57(2):163-173
The effects of the cholinergic agonist carbachol (Cch) and guanine nucleotides on the Na,K-ATPase and K-dependent p-nitrophenylphosphatase (K-p-NPPase) activities in rabbit and dog myocardial sarcolemma vesicles in the presence of the pore-forming antibiotic alamethicin (20 micrograms/ml), was studied. Cch (0.01-100 microM) inhibited the both enzymatic activities by 40-45% (IC50 = 0.3-0.5 microM) only after addition of GTP (50 microM) or its analogs: GTP gamma S (0.1-1.0 microM) and Gpp(NH)p (10 microM). The muscarinic acetylcholine receptor (mAchR) antagonist atropine (10 microM) blocked the effect of Cch. GTP gamma S alone produced a concentration-dependent decrease in the both Na,K-ATPase and K-p-NPPase activities by 40-45% (IC50 = 1-2 microM) with a lag period of about 3 minutes; this lag disappeared in the presence of the agonist. The GDP analog GDP beta S (0.01-100 microM) neither affected these activities nor promoted the inhibiting effect of Cch. Pretreatment of sarcolemmal vesicles with 20 micrograms/ml of pertussis toxin in the presence of 100 microM NAD abolished the inhibiting effect of Cch on the Na,K-ATPase and phosphatase activities. Under these conditions pertussis toxin catalyzed the ADP-ribosylation of alpha-subunits of the inhibitory GTP-binding protein (G1) which were identified immunochemically as alpha i2, alpha i3 and, possibly, alpha i1. The data obtained testify to the involvement of G1 in the mAchR-mediated inhibition of myocardial sarcolemmal Na,K-ATPase as well as in the signal transduction from the receptor to the enzyme. 相似文献
14.
Moon HE Cavalli A Bahia DS Hoffmann M Massotte D Milligan G 《Journal of neurochemistry》2001,76(6):1805-1813
To assess the relative capacity of the human delta opioid receptor to activate closely related G proteins, fusion proteins were constructed in which the alpha-subunits of either G(i1) or G(o1), containing point mutations to render them insensitive to the actions of pertussis toxin, were linked in-frame with the C-terminus of the receptor. Following transient and stable expression in HEK 293 cells, both constructs bound the antagonist [(3)H]naltrindole with high affinity. D-ala(2),D-leu(5) Enkephalin effectively inhibited forskolin-stimulated adenylyl cyclase activity in intact cells in a concentration-dependent, but pertussis toxin-insensitive, manner. The high-affinity GTPase activity of both constructs was also stimulated by D-ala(2),D-leu(5) enkephalin with similar potency. However, enzyme kinetic analysis of agonist stimulation of GTPase activity demonstrated that the GTP turnover number produced in response to D-ala(2),D-leu(5) enkephalin was more than three times greater for G(i1)alpha than for G(o1)alpha. As the effect of agonist in both cases was to increase V:(max) without increasing the observed K:(m) for GTP, this is consistent with receptor promoting greater guanine nucleotide exchange, and thus activation, of G(i1)alpha compared with G(o1)alpha. An equivalent fusion protein between the human mu opioid receptor-1 and G(i1)alpha produced a similar D-ala(2),D-leu(5) enkephalin-induced GTP turnover number as the delta opioid receptor-G(i1)alpha fusion construct, consistent with agonist occupation of these two opioid receptor subtypes being equally efficiently coupled to activation of G(i1)alpha. 相似文献
15.
In membranes of undifferentiated neuroblastoma x glioma hybrid cell line NG108-15, the apparent specific binding of [3H]yohimbine measured in the presence of 1 microM noradrenaline, was increased substantially by the presence of the poorly hydrolysed analogue of GTP, guanylyl-imidodiphosphate (Gpp[NH]p) or by preincubation of membranes with antibodies against the C-terminal decapeptide of the alpha subunit of the G-protein Gi2. Such an effect was not produced by antibodies against the equivalent region of Go alpha Gi3 alpha or Gs alpha or from non-immune serum. By contrast, total specific binding of [3H]yohimbine was not modified by co-incubation with Gpp[NH]p or by preincubation with the antibodies from any of the anti-G protein antisera. These results demonstrate a direct interaction of the alpha 2B adrenergic receptor of NG108-15 cells with Gi2. 相似文献
16.
Five G protein-coupled receptors (GPCRs) for the lysophospholipid sphingosine 1-phosphate (S1P) have been cloned and characterized so far. We report here about the identification of gpr3, gpr6 and gpr12 as additional members of the S1P-GPCR family. When expressed transiently in HEK293 cells, gpr3, gpr6 and gpr12 confer constitutive activation of adenylate cyclase (AC) similar in amplitude to that seen with fully activated G(alpha)(s)-coupled receptors. Culturing the transfected cells in medium with charcoal-stripped serum (devoid of lipids) significantly reduces cyclic adenosine monophosphate (cAMP) levels, suggesting a lipid-like ligand. A library containing 200 bioactive lipids was applied in functional assays recording intracellular Ca(2+) mobilization. S1P and dihydrosphingosine 1-phosphate (DHS1P) were identified as functional activators exhibiting nanomolar EC(50) values. In the presence of the S1P and LPA receptor antagonist suramin, gpr3-, gpr6- and gpr12-mediated intracellular Ca(2+) mobilization via S1P is enhanced. Besides constitutive activation of G(alpha)(s) type of G proteins, all three receptors are capable of constitutively activating inhibitory G(alpha)(i/o) proteins: (i) in the presence of pertussis toxin, gpr3-, gpr6- and gpr12-mediated stimulation of AC is enhanced; and (ii) overexpression of G(alpha)(i) significantly reduces the stimulatory action on intracellular cAMP levels. Agonist (S1P)-mediated internalization can be visualized in intact HEK293 cells using a gpr6 green fluorescent protein (GFP) fusion protein. In summary, our data suggest that gpr3, gpr6 and gpr12 are a family of constitutively active receptors with dual coupling to G(alpha)(s) and G(alpha)(i) type of G proteins. Constitutive activation of AC and mobilization of [Ca(2+)](i) can be modulated by the sphingophospholipids S1P and DHS1P, adding three additional members to the family of S1P receptors. 相似文献
17.
18.
Differential distribution of alpha subunits and beta gamma subunits of heterotrimeric G proteins on Golgi membranes of the exocrine pancreas 总被引:3,自引:0,他引:3
下载免费PDF全文
![点击此处可从《The Journal of cell biology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
《The Journal of cell biology》1996,133(5):1027-1040
Heterotrimeric G proteins are well known to be involved in signaling via plasma membrane (PM) receptors. Recent data indicate that heterotrimeric G proteins are also present on intracellular membranes and may regulate vesicular transport along the exocytic pathway. We have used subcellular fractionation and immunocytochemical localization to investigate the distribution of G alpha and G beta gamma subunits in the rat exocrine pancreas which is highly specialized for protein secretion. We show that G alpha s, G alpha i3 and G alpha q/11 are present in Golgi fractions which are > 95% devoid of PM. Removal of residual PM by absorption on wheat germ agglutinin (WGA) did not deplete G alpha subunits. G alpha s was largely restricted to TGN- enriched fractions by immunoblotting, whereas G alpha i3 and G alpha q/11 were broadly distributed across Golgi fractions. G alpha s did not colocalize with TGN38 or caveolin, suggesting that G alpha s is associated with a distinct population of membranes. G beta subunits were barely detectable in purified Golgi fractions. By immunofluorescence and immunogold labeling, G beta subunits were detected on PM but not on Golgi membranes, whereas G alpha s and G alpha i3 were readily detected on both Golgi and PM. G alpha and G beta subunits were not found on membranes of zymogen granules. These data indicate that G alpha s, G alpha q/11, and G alpha i3 associate with Golgi membranes independent of G beta subunits and have distinctive distributions within the Golgi stack. G beta subunits are thought to lock G alpha in the GDP-bound form, prevent it from activating its effector, and assist in anchoring it to the PM. Therefore the presence of free G alpha subunits on Golgi membranes has several important functional implications: it suggests that G alpha subunits associated with Golgi membranes are in the active, GTP-bound form or are bound to some other unidentified protein(s) which can substitute for G beta gamma subunits. It further implies that G alpha subunits are tethered to Golgi membranes by posttranslational modifications (e.g., palmitoylation) or by binding to another protein(s). 相似文献
19.
2'(3')-O-(N-Methylanthraniloyl)-(MANT)-substituted nucleotides are fluorescent and widely used for the kinetic analysis of enzymes and signaling proteins. We studied the effects of MANT-guanosine 5'-[gamma-thio]triphosphate (MANT-GTP gamma S) and MANT-guanosine 5'-[beta,gamma-imido]triphosphate (MANT-GppNHp) on G alpha(s)- and G alpha(i)-protein-mediated signaling. MANT-GTP gamma S/MANT-GppNHp had lower affinities for G alpha(s) and G alpha(i) than GTP gamma S/GppNHp as assessed by inhibition of GTP hydrolysis of receptor-G alpha fusion proteins. MANT-GTP gamma S was much less effective than GTP gamma S at disrupting the ternary complex between the formyl peptide receptor and G alpha(i2). MANT-GTP gamma S/MANT-GppNHp non-competitively inhibited GTP gamma S/GppNHp-, AlF(4)(-)-, beta(2)-adrenoceptor plus GTP-, cholera toxin plus GTP-, and forskolin-stimulated adenylyl cyclase (AC) in G alpha(s)-expressing Sf9 insect cell membranes and S49 wild-type lymphoma cell membranes. AC inhibition by MANT-GTP gamma S/MANT-GppNHp was not due to G alpha(s) inhibition because it was also observed in G alpha(s)-deficient S49 cyc(-) lymphoma cell membranes. Mn(2+) blocked AC inhibition by GTP gamma S/GppNHp in S49 cyc(-) membranes but enhanced the potency of MANT-GTP gamma S/MANT-GppNHp at inhibiting AC by approximately 4-8-fold. MANT-GTP gamma S and MANT-GppNHp competitively inhibited forskolin/Mn(2+)-stimulated AC in S49 cyc(-) membranes with K(i) values of 53 and 160 nm, respectively. The K(i) value for MANT-GppNHp at insect cell AC was 155 nm. Collectively, MANT-GTP gamma S/MANT-GppNHp bind to G alpha(s)- and G alpha(i)-proteins with low affinity and are ineffective at activating G alpha. Instead, MANT-GTP gamma S/MANT-GppNHp constitute a novel class of potent competitive AC inhibitors. 相似文献
20.
Regulators of G-protein signaling (RGS) proteins constitute a large family of GTPase-activating proteins for heterotrimeric G proteins. More than 20 RGS genes have been identified in mammals. One of these, the Galpha-interacting protein (GAIP), preferentially interacts with members of the G(i)/G(o) subfamily of G proteins in mammalian cells, but its selectivity among members of this subfamily in vitro is limited. Here we report the cloning and functional characterization of a unique cDNA isoform of GAIP, derived from embryonic chicken dorsal root ganglion neurons. Chick GAIP is composed of 199 amino acids, organized into a conserved RGS domain (85% identical to human GAIP), and a unique, short N terminus (only 41% identical, 50% homologous to known mammalian orthologues). Consistent with this unique primary structure, chick GAIP has physiological properties that distinguish it from mammalian GAIPs. We have explored the selectivity of chick GAIP in electrophysiological assays of two G(o)-mediated forms of Ca(2+) channel inhibition produced by gamma-aminobutyric acid in chick dorsal root ganglion neurons, voltage-independent inhibition (mediated by G(o)alpha) and voltage-dependent inhibition (mediated by G(o)betagamma). Dialyzing recombinant chick GAIP in these cells selectively reduced voltage-independent inhibition without affecting voltage-dependent inhibition. Mammalian GAIP, tested under identical conditions in previous studies, demonstrated no selectivity between these two inhibitory processes; thus, our results suggest that the functional specificity of chick GAIP is likely to be determined by its unique N terminus. 相似文献