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J. Biol. Chem., Vol. 280, Issue 34, 30236-30241, August 26, 2005

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Characterization of the G_s Regulator Cysteine String Protein^*

Michael Natochin, Tessa N. Campbell, Brandy Barren, Linda C. Miller, Shahid Hameed, Nikolai O. Artemyev, and Janice E. A. Braun, Recipient of a CIHR New Investigator award and an Alberta Heritage Foundation for Medical Research scholar¶

From the Hotchkiss Brain Institute, University of Calgary, Calgary, Alberta T2N 4N1, Canada and Department of Physiology and Biophysics, University of Iowa, Iowa City, Iowa 52254

Received for publication, January 20, 2005 , and in revised form, June 21, 2005.

	ABSTRACT

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Cysteine string protein (CSP) is an abundant regulated secretory vesicle protein that is composed of a string of cysteine residues, a linker domain, and an N-terminal J domain characteristic of the DnaJ/Hsp40 co-chaperone family. We have shown previously that CSP associates with heterotrimeric GTP-binding proteins (G proteins) and promotes G protein inhibition of N-type Ca²⁺ channels. To elucidate the mechanisms by which CSP modulates G protein signaling, we examined the effects of CSP_1–198 (full-length), CSP_1–112, and CSP_1–82 on the kinetics of guanine nucleotide exchange and GTP hydrolysis. In this report, we demonstrate that CSP selectively interacts with G_s and increases steady-state GTP hydrolysis. CSP_1–198 modulation of G_s was dependent on Hsc70 (70-kDa heat shock cognate protein) and SGT (small glutamine-rich tetratricopeptide repeat domain protein), whereas modulation by CSP_1–112 was Hsc70-SGT-independent. CSP_1–112 preferentially associated with the inactive GDP-bound conformation of G_s. Consistent with the stimulation of GTP hydrolysis, CSP_1–112 increased guanine nucleotide exchange of G_s. The interaction of native G_s and CSP was confirmed by coimmunoprecipitation and showed that G_s associates with CSP. Furthermore, transient expression of CSP in HEK cells increased cellular cAMP levels in the presence of the ₂ adrenergic agonist isoproterenol. Together, these results demonstrate that CSP modulates G protein function by preferentially targeting the inactive GDP-bound form of G_s and promoting GDP/GTP exchange. Our results show that the guanine nucleotide exchange activity of full-length CSP is, in turn, regulated by Hsc70-SGT.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
G proteins constitute a family of heterotrimeric GTP-binding proteins that act as transducers in a variety of transmembrane signaling systems. G proteins are composed of , , and subunits that dissociate into G and G upon activation. Activation of G proteins involves an exchange of GDP for GTP on G subunits and the release of GTP-bound G and G to interact with effector molecules. Effector molecules include adenylate cyclase, phospholipases, phosphodiesterases, and ion channels. Several modulators of G proteins have been identified and are thought to play crucial roles in the kinetics of G protein signaling (e.g. guanine nucleotide exchange factors (GEFs),¹ guanine nucleotide dissociation inhibitors, and GTPase-activating proteins). Our previous work has identified cysteine string protein (CSP) as a novel modulator of G proteins (1–3). Although the functional parallels between CSP and established G protein modulators are evident, the molecular mechanism by which CSP regulates G proteins is not yet known.

CSPs are secretory vesicle proteins of 34 kDa that contain three conserved domains: a J domain, a linker domain, and a cysteine string region (Fig. 1A). The J domain is a 70-amino acid region of homology shared by DnaJ (a well characterized bacterial co-chaperone) and many otherwise unrelated eukaryotic proteins. The linker domain is a 30-amino acid domain conserved between human and Drosophila CSP. CSP, so called because it contains a cysteine-rich domain that in rats consists of a string of nine cysteine residues flanked on the C-terminal side by three additional cysteines, is localized on synaptic vesicles (4), zymogen granules (5), and chromaffin granules (6). Most of the cysteine residues are palmitoylated and are required for membrane attachment to the secretory vesicle (7, 8). In contrast to the conserved J domain, linker domain, and cysteine string regions, the C terminus diverges among CSP isoforms (9). CSP plays a crucial role in neurotransmitter release; however, its function is controversial. Deletion of the CSP gene in Drosophila melanogaster severely impairs central and presynaptic transmission (10–12). The small number of flies that survive to adulthood is characterized by spasmic jumping, intense shaking, temperature-sensitive paralysis, and premature death. Deletion of CSP in mice results in severe impairment of synaptic transmission and neurodegeneration with no survival beyond 3 months (13). Age- and temperature-dependent deterioration of CSP null mutants suggests a chaperone role for CSP in regulating conformation and the activity of synaptic protein(s). Indeed, CSP has been proposed to function as a trimeric complex with two molecular chaperones, Hsc70 (70-kDa heat shock cognate protein) (14–16) and SGT (small glutamine-rich tetratricopeptide repeat domain protein) (17). However, the involvement of Hsc70 and SGT in the CSP modulation of G protein function has not yet been examined.

We have shown that CSP associates with G proteins and promotes G inhibition of N-type Ca²⁺ channels (1–3). N-type Ca²⁺ channels are effectors of G such that direct binding of G protein subunits to the Ca²⁺ channel ₁ subunit results in a stabilization of the deep closed states of the channel (18). The mechanism by which CSP modulates G protein function has not yet been fully evaluated. A striking feature of CSP is that it contains two G protein binding sites (Fig. 1). The region of CSP encoded by amino acids 83–112 associates with G and/or G (2). In contrast, an N-terminal site binds G subunits but not G subunits (1). These binding sites do not have homology with established modulators of G proteins. Each CSP binding site independently modulates G protein inhibition of N-type Ca²⁺ channels, and we have shown that they do this by distinct but yet to be determined molecular mechanisms (2). Here we examine GTPase activity of G_s and G_i in the presence of CSP and CSP truncations encoding one or both G protein binding sites. Our findings demonstrate that CSP stimulates GDP/GTP exchange of G_s and that modulation of G proteins by CSP is, in turn, regulated by the molecular chaperones Hsc70-SGT.

	MATERIALS AND METHODS

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Preparation of Fusion Proteins—Glutathione S-transferase (GST) fusion proteins of full-length rat CSP1, Hsc70, SGT, CSP_1–82, and CSP_1–112 were subcloned into pGEX-KG (19), expressed as GST fusion proteins in AB1899 or BL21 cells, and purified as described previously (2, 5). G_s and G_il were expressed in Escherichia coli and purified as described previously (20).

Pull-down Assays—G_i1 and G_s (30 µg) were incubated for 1 h at 25 °C in 100 µl of 20 mM Tris-HCl (pH 8.0) buffer containing 130 mM NaCl, 2 mM MgSO₄, and either 10 µM GDP or 10 µM GDP, 30 µM AlCl₃, and 10 mM NaF or 10 µM GTPS followed by the addition of 10 µg of GST, GST-CSP_1–82, or GST-CSP_1–112 and further incubation for 1 h at 25 °C. The proteins were then mixed with glutathione-agarose beads (10-µl bed volume) for 20 min at 25 °C with constant shaking. The beads were spun down and washed four times with 1 ml of the same buffer containing 10 µM GDP, or 10 µM GDP, 30 µM AlCl₃, and 10 mM NaF, or 10 µM GTPS. Bound proteins were eluted with SDS sample buffer and separated on 12% SDS-gels.

GTPase Assay—Steady-state GTPase reactions were performed using G_s or G_i1 subunits (0.3 µM) in the presence or absence of various concentrations of GST-CSP_1–82 or GST-CSP_1–112 and 0.6 µM G₁₁ (21). GTPase activity measurements were initiated by mixing proteins with 20 µM [-³²P]GTP (5 µCi) in 100 µl of 20 mM Tris-HCl (pH 8.0) buffer containing 130 mM NaCl and 10 mM MgSO₄. Aliquots (20 µl) were withdrawn at the indicated times and transferred to 100 µl of 7% perchloric acid. Nucleotides were precipitated using 700 µl of 10% (w/v) charcoal suspension in phosphate-buffered saline. ³²P_i formation was measured by liquid scintillation counting. Results were fit with linear regression.

GTPS Binding Assay—G_s and G_i1 subunits (0.3 µM) were incubated for 5 min at 25 °C in 200 µl of 20 mM Tris-HCl (pH 8.0) buffer containing 130 mM NaCl and 10 mM MgSO₄ in the presence of a 1 µM concentration of GST, GST-CSP_1–82, or GST-CSP_1–112. Binding reactions were started by the addition of 5 µM [³⁵S]GTPS (2 µCi). Aliquots of 20 µl were withdrawn at the indicated times, mixed with 1 ml of the same buffer containing 1 mM GTP and passed through Whatman cellulose nitrate filters (0.45 µm). The filters were then washed three times with 3 ml of the same buffer and counted in a liquid scintillation counter. The apparent rate constant (k_app) values for the binding reactions were calculated by fitting the data to the following equation: GTPS bound (%) = 100% · (1 - e^-kt), where t is time of reaction.

Preparation of Rat Brain Homogenate—Rat brains were homogenized in 20 mM Tris-HCl buffer (pH 7.4) containing 2 mM MgSO₄, 1 mM phenylmethylsulfonyl fluoride, and EDTA-free inhibitor mixture (Roche Applied Science). The homogenate was centrifuged at 100,000 x g for 1 h at 4 °C to obtain soluble fractions (Fig. 6, S). The pellet was washed twice with the same buffer and solubilized in the same buffer containing 1% n-dodecyl--D-maltoside (Calbiochem) for 60 min at 4 °C. The resulting supernatant (100,000 x g, 1 h, 4 °C) constituted solubilized membrane fraction (Fig. 6, M). Protein concentrations were determined by a Bio-Rad protein assay using bovine serum albumin as the standard. All of the procedures were carried out in strict accordance with a protocol approved by the University of Calgary Animal Care Committee.

Immunoprecipitations—CSP polyclonal antibodies raised against the peptide sequence corresponding to amino acids 182–198 of rat CSP and the entire CSP recombinant protein were described previously (5). The membrane fraction solubilized by 1% n-dodecyl--D-maltoside was precleared by incubation with GammaBind G-Sepharose (30-µl bed volume) in 20 mM Tris-HCl (pH7.4) buffer containing 130 mM NaCl and 2 mM MgSO₄ for 30 min at 25 °C. Immunoprecipitation was achieved by incubating the precleared membrane fraction with the indicated antibodies overnight at 4 °C followed by GammaBind G-Sepharose (Amersham Biosciences) (20-µl bed volume) for 30 min at 25 °C. The mixtures were then centrifuged, and the pellets were washed three times with 200 µl of incubation buffer containing 50 µM GDP. Samples were washed and resuspended in 30 µl of sample buffer prior to immunoblotting.

Immunoblotting—Proteins were transferred electrophoretically at 70 V for 45 min from polyacrylamide gels to 0.45 µm of nitrocellulose in 20 mM Tris, 150 mM glycine, and 12% methanol. Transferred proteins were visualized with Ponceau S. Nitrocellulose membranes were blocked for nonspecific binding using 5% milk, 0.1% Tween 20, and phosphate-buffered saline (pH 7.3) (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na₂HPO₄, 1.4 mM KH₂PO₄) prior to a 2-h incubation with primary antibody. The membranes were washed three times in the above milk/Tween/phosphate-buffered saline solution and incubated with goat anti-rabbit or goat anti-mouse IgG-coupled horseradish peroxidase. Antigen was detected using chemiluminescent horseradish peroxidase substrate (ECL, Amersham Biosciences). Immunoreactive bands were visualized following exposure of the membranes to Amersham Hyperfilm-MP.

cAMP Assays—Human embryonic kidney tsa-201 (HEK) cells were seeded into 12-well plates and grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. At 70% confluency, cells were transfected with pcDNA3/Rous sarcoma virus/His-hemagglutinin-2-adrenergic receptor (gift from M. Bouvier, University of Montreal), pMT2-CSP (1), or a combination of both using FuGENE 6 according to the manufacturer's directions. Forty-eight h post-transfection, cells were incubated for three min at 37 °C with serum-free medium, 50 µM isoproterenol, or 100 µM forskolin. Medium was then aspirated, and cells were washed with phosphate-buffered saline. Cells were lysed with 280 µl of 0.1 M HCl. Lysates were centrifuged at 600 x g, and the supernatant was collected. cAMP levels of the supernatant (100 µl) were quantified using a Direct cAMP Enzyme Immunoassay kit (Sigma) according to the manufacturer's instructions.

	RESULTS

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CSP Stimulates Steady-state Hydrolysis of GTP by G_s—To begin to investigate the mechanism by which CSP modulates G proteins, we evaluated GTPase steady-state activity in the presence of purified recombinant full-length CSP (CSP_1–198). Fig. 1B shows that the GTP hydrolysis by G_s was not altered in the presence of CSP. In contrast, GTP hydrolysis was increased 4-fold in the presence of the proposed trimeric chaperone complex CSP_1–198-Hsc70_1–646-SGT_1–314. In the absence of CSP, Hsc70-SGT did not alter GTPase activity, indicating that CSP is required for the G_s modulation. Binary complexes of CSP_1–198-Hsc70_1–646 and CSP_1–198-SGT_1–314 both triggered an increase in steady-state GTPase activity but to a lesser extent than the trimeric complex. The addition of 50 µM ATP to the assay required for Hsc70 activity did not alter GTP hydrolysis (data not shown). Neither the ATPase, Hsc70 (not shown) nor the proposed trimeric chaperone complex Hsc70-SGT-CSP was observed to stimulate GTP hydrolysis in the absence of G_s, which demonstrates that the chaperone complex does not hydrolyze GTP. Thus, stimulation of the GTPase activity of G_s by CSP is specific and requires Hsc70-SGT but cannot be attributed to a generalized folding/refolding chaperone-like action on G_s by Hsc70-SGT.

To further evaluate the modulation of G by CSP, we utilized full-length CSP and purified recombinant truncations of CSP containing either one G protein binding site (CSP_1–82) or both G protein binding sites (CSP_1–112). Fig. 2 demonstrates that CSP_1–112 triggered a large increase in the rate of GTP hydrolysis by G_s but not G_i, whereas CSP_1–82 had a modest effect on G_s. SGT and Hsc70 did not further enhance the GTP hydrolysis by G_s in the presence of either CSP_1–112 or CSP_1–82 (data not shown). In agreement with the data shown in Fig. 1, CSP did not stimulate G_s above basal levels in the absence of Hsc70-SGT. The use of CSP_1–82 and CSP_1–112 truncations rather than full-length CSP eliminates any potential influence from the cysteine string region of full-length recombinant CSP in the GTPase assay, given that interactions can occur with this region that are not characteristic of native palmitoylated CSP (22). A dose dependence of the effect of CSP_1–112 on the GTPase activity of G_s revealed a relatively strong affinity between the two proteins (EC₅₀ 415 nM) and a maximal stimulation of 8-fold (Fig. 3). Taken together, these results indicate that CSP directly and specifically modulates the GTPase activity of G_s through a region encoded by amino acids 1–112, that this region is autoinhibited in full-length CSP, and that autoinhibition is removed by Hsc70-SGT.

View larger version (17K): [in this window] [in a new window]   FIG. 1. CSP stimulates GTPase activity of Gs in the presence of Hsc70 and SGT. A, schematic representation of the G protein binding domains of CSP. B, soluble full-length CSP, Hsc70, and SGT (0.3 µm) were mixed with 10 µM [-32P]GTP (1 µCi) prior to the addition of 0.3 µM Gs. The amount of released 32P was measured as counts/min after incubation of the reaction mixture for 30 min at 25 °C. p values for two-tailed unpaired t test (95% confidence interval) were 0.0017 (**) and <0.0001 (***), respectively.

CSP Stimulates the Nucleotide Exchange of G_s—The rate of GTP hydrolysis by G protein subunits under steady-state conditions is believed to be limited by the rate of guanine-nucleotide exchange due to slow release of GDP. Because CSP_1–112 was more effective in stimulating GTPase activity of G_s, the effects of GST-CSP_1–112 on guanine nucleotide exchange of G_s and G_i were tested using the GTPS binding assay. CSP_1–112 increased the initial rate of GTPS binding to G_s but not G_i (Fig. 4).

View larger version (22K): [in this window] [in a new window]   FIG. 2. Effects of CSP1–112 and CSP1–82 on steady-state GTPase activities of Gs and Gi. Steady-state GTPase activities of Gs (A) and Gi1 (B) (0.3 µM) were measured in the presence of 0.5 µM GST (), GST-CSP1–82 (), GST-CSP1–112 (), or GST-CSP1–198 (). The assay was initiated by mixing proteins with 10 µM [-32P]GTP (5 µCi) in 100 µl of 20 mM Tris-HCl (pH 8.0) buffer containing 130 mM NaCl and 10 mM MgSO4. Data were fit with linear regression.

View larger version (13K): [in this window] [in a new window]   FIG. 3. Dose-dependent effects of CSP1–112 on steady-state GTPase activities of Gs and heterotrimeric Gs11. Steady-state GTPase activities of Gs alone (, 0.3 µM) or preincubated with G11 (, 0.6 µM) for 10 min at 25 °C were measured following the additions of indicated concentrations of GST-CSP1–112. Turnover numbers for the GTPase reactions were plotted as functions of CSP1–112 concentration and fit with the sigmoidal dose response equation. The calculated EC50 values were (mean ± S.D.): Gs, 415 ± 30 nM; Gs, 740 ± 40 nM.

View larger version (15K): [in this window] [in a new window]   FIG. 4. CSP1–112 accelerates exchange of GDP for GTPS on Gs. Gs (A) and Gi1 (B) subunits (1 µM) were incubated for 5 min at 25 °C in 200 µl of 20 mM Tris-HCl (pH 8.0) buffer containing 130 mM NaCl and 10 mM MgSO4 in the presence of a 10 µM concentration of GST () or GST-CSP1–112 (). Binding reactions were started by the addition of 5 µM [35S]GTPS (2 µCi). Aliquots of 20 µl were withdrawn at the indicated times, mixed with 1 ml of the same buffer containing 1 mM GTP, and passed through Whatman cellulose nitrate filters (0.45 µm). The apparent rate constant (kapp) values for the binding reactions were calculated by fitting the data to the following equation: GTPS bound (%) = 100% · (1 - e-kt), where t is time of reaction.

View larger version (27K): [in this window] [in a new window]   FIG. 5. Conformational selectivity of CSP binding to G. Binding of GST, GST-CSP1–82, and GST-CSP1–112 to G. Bound proteins were eluted with SDS sample buffer, fractionated by SDS-PAGE, and visualized with Coomassie Blue.

View larger version (21K): [in this window] [in a new window]   FIG. 6. Association of native CSP and Gs. Coimmunoprecipitation of Gs and CSP from solubilized rat brain homogenate was followed by immunoblot analysis with anti-G polyclonal (Calbiochem) and anti-Hsc70 monoclonal (Sigma). The lanes show results from the incubation of rat brain membrane fraction (300 µg) with nonimmune rabbit serum (lane 1), GammaBind G-Sepharose (lane 2), anti-CSP polyclonal (prepared against C-terminal peptide (amino acids 182–198)) (lane 3), anti-CSP polyclonal (entire recombinant CSP) (lane 4), anti-Gs polyclonal (Santa Cruz) (lane 5), IgG1 (Sigma) (lane 6), rat brain membrane fraction (40 µg) (lane M), and rat brain cytosol fraction (40 µg) (lane S). These results are representative of nine independent experiments.

In summary, we have provided the first evidence that the DnaJ/Hsp40 family member and secretory vesicle protein CSP modulates the GDP/GTP exchange of G_s. Data presented here indicate that upon activation by Hsc70-SGT, CSP is a direct GEF for G_s.

	DISCUSSION

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We have found that CSP, a secretory vesicle protein, is able to significantly enhance the GTP hydrolysis of G_s by stimulating GDP/GTP exchange. CSP is selective for G_s and, as such, is the first GEF to be identified for G_s. The CSP-G_s complex was detectable by immunoprecipitation and affinity chromatography. In addition, expression of CSP in HEK cells increased isoproterenol-stimulated cAMP levels, demonstrating that CSP enhances signaling through G_s.

View larger version (13K): [in this window] [in a new window]   FIG. 7. CSP promotes Gs-mediated signaling. HEK cells were transfected with 2 adrenergic receptor (2AR) cDNA, CSP1–198 cDNA, or a combination of both. Forty-eight h later, cells were incubated for 3 min with serum-free medium (Basal), 50 µM isoproterenol (2AR + I), or 100 µM forskolin, lysed, and assayed for cAMP accumulation via a Direct cAMP Enzyme Immunoassay kit. Data represent the mean of duplicate determinations that is representative of four separate experiments. p values for unpaired t tests were 0.002 (***) and not significant (ns) as indicated.

Interestingly, several functional parallels between CSP and the structurally distinct protein, Ric-8A (synembryn), are evident. First, CSP and Ric-8A are both GEFs. CSP is a GEF for G_s but not G_i1 (Figs. 1, 2, 3, 4), whereas Ric-8A is a GEF for G_i1, G_q, and G_o but not G_s (27). Second, CSP and Ric-8A are both abundant neural proteins (5, 28). Finally, deletion of either CSP or Ric-8 causes paralysis. In Drosophila, CSP null mutants that survive to adulthood (briefly) are characterized by paralytic uncoordinated sluggish movements, spasmic jumping, intense shaking, and temperature-sensitive paralysis (10). Deletion of CSP in mice causes no significant change in synaptic function during the first 2 weeks of postnatal life, but this is followed by severe impairment of synaptic transmission with no survival beyond 3 months (13). In Caenorhabditis elegans, genetic studies reveal that Ric-8 acts together with G proteins to regulate neuronal transmitter release (29, 30). Thus, it seems that CSP, like Ric-8, is responsible for integrating synaptic G protein signaling pathways. In addition, Ric-8 has been recently shown to play an essential role in the G protein receptor-1/2- and G-dependent asymmetric division of C. elegans embryos (31).

In contrast to Ric-8, CSP appears to be capable of activating heterotrimeric G_s (Fig. 3). In this respect, CSP is functionally similar to AGS1, a RAS-related G protein, which was shown to activate both heterotrimeric G proteins and free G_i/G_o subunits (32). Nonetheless, CSP is apparently a more potent modulator of monomeric G_s. In HEK cells, the activation of ₂ adrenergic receptor by isoproterenol was required for CSP to produce a large additional increase in cAMP levels. Most likely, this effect was a result of CSP targeting and activating the pool of G_sGDP generated by the G protein-coupled receptor-dependent activation of G_s and GTP-hydrolysis on G_s.

An important question concerning the function of CSP will be to determine the specific physiological pool of G_s that CSP (either membrane or intracellular) targets. When secretory vesicles are in close proximity to the plasma membrane, CSP may target and activate plasma membrane-bound G_s. An alternate and attractive hypothesis is that CSP modulates G proteins located on secretory vesicles. G proteins have been found on cholinergic synaptic vesicles from Discopyge ommata (33), rat synaptic vesicles (34), large dense core and small synaptic vesicles (35, 36), insulin secretory granules (37), bovine chromaffin granules (38), rat pituitary granules (39), and rat parotid granules (40). It is not known how these G proteins become activated or what role these vesicle G proteins play in exocytosis. Because G protein-coupled receptors and effectors appear to be absent from the vesicles, CSP is an obvious candidate to be a regulator of vesicle G proteins. In fact, although it is clear that CSP plays a significant component in neurotransmission (10, 13), its precise role is not yet established. Current reports support a role for CSP in (i) the regulation of exocytosis (41–45), (ii) the regulation of Ca²⁺ transmembrane fluxes (46–51), and (iii) the regulation of folding/refolding of synaptic proteins (13, 14). Given that the contributions of vesicular and plasma membrane G_s in the various experimental models utilized are not known, it is possible that differences in G_s signaling cascades underlie some of the differences observed among these reports.

Two distinct G protein binding sites are found in CSP (2). Deletion mutants encoding site 1 (CSP_83–198) or site 2 (CSP_1–82) both increased the G protein inhibition of N-type Ca²⁺ channels in transient expression systems (2), indicating that two separate actions occur within full-length CSP. In pull-down experiments, site 1 (CSP_83–112) associates with or subunits, whereas site 2 (CSP_1–82) binds only to the subunit. In vitro, the interaction of G proteins with site 1 is robust and stable, whereas association with site 2 is relatively weaker and sensitive to nucleotides and the purity of the G protein preparation (2). The guanine nucleotide exchange rates reported in this study are greatest for the CSP deletion mutants that encode both sites, indicating that both sites are important for G_s activation.

Because CSP has been shown to stimulate Hsc70 ATPase, it is possible that CSP activates Hsc70, which in turn modulates the conformation and activity of G_s. Two features of our data argue against this interpretation. First, CSP_1–112 stimulates G_s GTPase in the absence of Hsc70 (Fig. 2A). Second, Hsc70-SGT in the absence of CSP does not independently increase GTPase steady-state activity (Fig. 1B). Thus, although Hsc70 is known to manifest its chaperone activity in several different contexts, here we show that Hsc70 modulates the GEF activity of CSP but does not directly modulate G_s.

In this study, we confirm that the secretory vesicle protein, CSP, modulates G protein-mediated signal transduction. Furthermore, we demonstrate that the mechanism by which CSP modulates G proteins involves the binding of the region encoded by residues 1–112 of CSP to the GDP-bound conformation of G_s and the stimulation of nucleotide exchange. GEF activity of full-length CSP but not CSP_1–112 requires activation by Hsc70-SGT, suggesting that the C terminus of CSP inhibits GEF activity. CSP stimulation of guanine nucleotide exchange is selective for G_s. In this regard, CSP serves as an ideal bridge between heterotrimeric GTP protein signaling, molecular chaperones, and secretory vesicles.

	FOOTNOTES

 
^* This work was supported in part by operating grants from the Canadian Institutes of Health Research (CIHR) (to J. E. A. B.) and by National Institutes of Health Grant RO1 EY-12682 (to N. O. A.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: Hotchkiss Brain Inst., University of Calgary, 3330 Hospital Dr. N.W., Calgary, Alberta T2N 4N1, Canada.

¹ The abbreviations used are: GEF, guanine nucleotide exchange factor; CSP, cysteine string protein; GST, glutathione S-transferase; GTPS, guanosine 5'-O-(thiotriphosphate); SGT, small glutamine-rich tetratricopeptide.

	ACKNOWLEDGMENTS

 
We thank Leigh Anne Swayne for technical assistance.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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