Analysis of the Complex between Ca2+ Channel β-Subunit and the Rem GTPase
2006; Elsevier BV; Volume: 281; Issue: 33 Linguagem: Inglês
10.1074/jbc.m604867200
ISSN1083-351X
AutoresBrian S. Finlin, Robert N. Correll, Chunyan Pang, Shawn M. Crump, Jonathan Satin, Douglas Andres,
Tópico(s)Neuroscience and Neuropharmacology Research
ResumoVoltage-gated calcium channels are multiprotein complexes that regulate calcium influx and are important contributors to cardiac excitability and contractility. The auxiliary β-subunit (CaVβ) binds a conserved domain (the α-interaction domain (AID)) of the pore-forming CaVα1 subunit to modulate channel gating properties and promote cell surface trafficking. Recently, members of the RGK family of small GTPases (Rem, Rem2, Rad, Gem/Kir) have been identified as novel contributors to the regulation of L-type calcium channel activity. Here, we describe the Rem-association domain within CaVβ2a. The Rem interaction module is located in a ∼130-residue region within the highly conserved guanylate kinase domain that also directs AID binding. Importantly, CaVβ mutants were identified that lost the ability to bind AID but retained their association with Rem, indicating that the AID and Rem association sites of CaVβ2a are structurally distinct. In vitro binding studies indicate that the affinity of Rem for CaVβ2a interaction is lower than that of AID for CaVβ2a. Furthermore, in vitro binding studies indicate that Rem association does not inhibit the interaction of CaVβ2a with AID. Instead, CaVβ can simultaneously associate with both Rem and CaVα1-AID. Previous studies had suggested that RGK proteins may regulate Ca2+ channel activity by blocking the association of CaVβ subunits with CaVα1 to inhibit plasma membrane trafficking. However, surface biotinylation studies in HIT-T15 cells indicate that Rem can acutely modulate channel function without decreasing the density of L-type channels at the plasma membrane. Together these data suggest that Rem-dependent Ca2+ channel modulation involves formation of a Rem·CaVβ·AID regulatory complex without the need to disrupt CaVα1·CaVβ association or alter CaVα1 expression at the plasma membrane. Voltage-gated calcium channels are multiprotein complexes that regulate calcium influx and are important contributors to cardiac excitability and contractility. The auxiliary β-subunit (CaVβ) binds a conserved domain (the α-interaction domain (AID)) of the pore-forming CaVα1 subunit to modulate channel gating properties and promote cell surface trafficking. Recently, members of the RGK family of small GTPases (Rem, Rem2, Rad, Gem/Kir) have been identified as novel contributors to the regulation of L-type calcium channel activity. Here, we describe the Rem-association domain within CaVβ2a. The Rem interaction module is located in a ∼130-residue region within the highly conserved guanylate kinase domain that also directs AID binding. Importantly, CaVβ mutants were identified that lost the ability to bind AID but retained their association with Rem, indicating that the AID and Rem association sites of CaVβ2a are structurally distinct. In vitro binding studies indicate that the affinity of Rem for CaVβ2a interaction is lower than that of AID for CaVβ2a. Furthermore, in vitro binding studies indicate that Rem association does not inhibit the interaction of CaVβ2a with AID. Instead, CaVβ can simultaneously associate with both Rem and CaVα1-AID. Previous studies had suggested that RGK proteins may regulate Ca2+ channel activity by blocking the association of CaVβ subunits with CaVα1 to inhibit plasma membrane trafficking. However, surface biotinylation studies in HIT-T15 cells indicate that Rem can acutely modulate channel function without decreasing the density of L-type channels at the plasma membrane. Together these data suggest that Rem-dependent Ca2+ channel modulation involves formation of a Rem·CaVβ·AID regulatory complex without the need to disrupt CaVα1·CaVβ association or alter CaVα1 expression at the plasma membrane. Voltage-dependent calcium channels regulate Ca2+ entry to control numerous cellular functions, including muscle contraction, secretion, and gene expression (1Catterall W.A. Annu. Rev. Cell Dev. Biol. 2000; 16: 521-555Crossref PubMed Scopus (1985) Google Scholar). L-type voltage-activated calcium channels are comprised of a pore-forming α1-subunit and regulatory α2δ and -β subunits. The CaVα1 subunit allows calcium entry in response to membrane depolarization, but CaVβ subunits modulate the functional properties of the mature channel complex by regulating both the electrophysiological properties and trafficking of the CaVα1 subunit (2Richards M.W. Butcher A.J. Dolphin A.C. Trends Pharmacol. Sci. 2004; 25: 626-632Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). A conserved α1-interaction domain (AID) 4The abbreviations used are: AID, α-interaction domain; RGK proteins, Rem, Rem2, Rad, and Gem/Kir GTPases; GK, guanylate kinase domain; SH3, Src homology 3 domain; ABP, α-subunit binding pocket; GST, glutathione S-transferase; HEK, human embryonic kidney; GFP, green fluorescent protein; BID, β-interaction domain; HA, hemagglutinin; GTPαS, guanosine 5′-3-O-(thio)triphosphate or guanosine 5′-O-(thiotriphosphate); WT, wild type; pF, picofarad. located between the first and second repeats of the CaVα1-subunit (loop I-II) serves as a high affinity binding site for all CaVβ subunits (3Pragnell M. De Waard M. Mori Y. Tanabe T. Snutch T.P. Campbell K.P. Nature. 1994; 368: 67-70Crossref PubMed Scopus (562) Google Scholar). AID association promotes plasma membrane trafficking of the CaVβ·CaVα1 complex and results in higher current densities (2Richards M.W. Butcher A.J. Dolphin A.C. Trends Pharmacol. 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Although several recent studies have indicated that the expression of RGK GTPases inhibits the trafficking of co-transfected epitope-tagged CaVα1 subunits to the plasma membrane (21Beguin P. Nagashima K. Gonoi T. Shibasaki T. Takahashi K. Kashima Y. Ozaki N. Geering K. Iwanaga T. Seino S. Nature. 2001; 411: 701-706Crossref PubMed Scopus (254) Google Scholar, 26Beguin P. Mahalakshmi R.N. Nagashima K. Cher D.H. Kuwamura N. Yamada Y. Seino Y. Hunziker W. Biochem. J. 2005; 390: 67-75Crossref PubMed Scopus (65) Google Scholar, 34Sasaki T. Shibasaki T. Beguin P. Nagashima K. Miyazaki M. Seino S. J. Biol. Chem. 2005; 280: 9308-9312Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar), we have shown that expression of Rem2 can modulate both endogenous Ca2+ channel activity and glucose-dependent insulin release in insulinoma cells without obviously altering membrane expression of the endogenous channel (23Finlin B.S. Mosley A.L. Crump S.M. Correll R.N. Ozcan S. Satin J. Andres D.A. J. Biol. Chem. 2005; 280: 41864-41871Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). Moreover, overexpression of Rem2 had no effect on the surface density of N-type Ca2+ channels stably expressed in tsA201 cells while potently inhibiting channel function (32Chen H. Puhl 3rd, H.L. Niu S.L. Mitchell D.C. Ikeda S.R. J. Neurosci. 2005; 25: 9762-9772Crossref PubMed Scopus (81) Google Scholar). Thus, questions concerning the nature of the RGK-β-subunit association and its role in RGK-dependent regulation of Ca2+ channel activity and trafficking remain to be addressed. In the present study we define a region located within the conserved GK domain as being critically involved in the interaction of β subunits with Rem. Biochemical characterization indicates that the Rem-β-subunit interaction is of relatively low affinity, with competitive binding studies demonstrating that Rem fails to effectively compete with AID for β-subunit association. Moreover, Rem can enter into a CaVβ2a·AID complex, establishing that CaVβ·Rem binding is mechanistically and structurally distinct from CaVβ·AID binding. Taken together, these data indicate that Rem does not inhibit channel activity by competing with CaVα1 subunits for a limiting intracellular pool of uncomplexed CaVβ subunits. In support of this hypothesis, surface biotinylation studies indicate no change in the number of surface-exposed Ca2+ channels after Rem co-expression at a time when channel activity is greatly inhibited. These studies suggest that Rem-mediated regulation of Ca2+ channel activity involves direct regulation of the plasma membrane-located channel complex and can occur without the need to disrupt the steady-state levels of surface-expressed Ca2+ channels. Plasmids—Mammalian expression vectors for CaV1.2α-subunit, β2a-subunit, FLAG epitope-tagged β2a-subunit, and HA epitope-tagged Rem have been described previously (22Finlin B.S. Crump S.M. Satin J. Andres D.A. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 14469-14474Crossref PubMed Scopus (178) Google Scholar). CaVβ2a was subcloned into pCite 4 (Novagen) for production of in vitro translated protein. Truncation mutations were generated by PCR using CaVβ2a as the template and fully sequenced. The CaVα1.2 loop I-II and loop II-III domains were generated by PCR and subcloned into pGex KG for production of recombinant proteins. Protein Production—GST Rem and thrombin-cleaved Rem were produced as described previously (17Finlin B.S. Andres D.A. J. Biol. Chem. 1997; 272: 21982-21988Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). GST CaVα1.2 loop I-II (AID) was made in BL21DE3 and affinity-purified on glutathione beads (Sigma). The protein was then dialyzed into 50 mm Tris, 150 mm NaCl, 1 mm DTT, 10% glycerol and used in binding assays. When needed, thrombin was used to cleave the CaVα1.2 loop I-II from GST by washing GST-CaVα1.2AID immobilized on glutathione beads with thrombin cleavage buffer (17Finlin B.S. Andres D.A. J. Biol. Chem. 1997; 272: 21982-21988Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar) and then incubating with thrombin for 1 h with rotation. Thrombin was then removed with benzamidine-Sepharose (17Finlin B.S. Andres D.A. J. Biol. Chem. 1997; 272: 21982-21988Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar), and the protein concentration was determined by the Bradford assay. To ensure that thrombin was removed by benzamidine-Sepharose, GST immobilized on glutathione beads was incubated with thrombin and then subjected to treatment with benzamidine-Sepharose. This treatment labeled Buffer in Fig. 2B indicates that thrombin was efficiently removed. In Vitro Pulldown Assays—Radiolabeled full-length CaV β2a-subunit or the indicated CaV β2a truncations were prepared by in vitro transcription and translation in the presence of [35S]methionine using the Single Tube Protein System 3 (STP3) kit (Novagen) according to the manufacturers' instructions. CaVβ2a or the indicated CaVβ2a truncation mutants cloned into the pCITE vector were used as the template. Binding of radiolabeled β2a to Rem was assessed as follows. All manipulations were carried out at 4 °C. Ten μl glutathione-Sepharose beads (GE Healthcare) were washed 2 times with 500 μl of EDTA buffer (50 mm Tris, pH 7.5, 100 mm NaCl, 0.05% Tween 20, 0.1 mm DTT, 1 mm EDTA). The beads were resuspended in 1 ml of EDTA buffer, and either GST (10 μg) or GST·Rem (10 μg) was added. The beads were incubated for 5 min with end-over-end rotation at 4 °C. The beads were washed 2 times with 1 ml of EDTA buffer and then with 1 ml of EDTA buffer or 1 ml of GDP buffer (50 mm Tris, pH 7.5, 100 mm NaCl, 0.05% Tween 20, 0.1 mm DTT, 10 mm MgCl2, 20 μm GDP) or 1 ml GTP buffer (50 mm Tris pH 7.5, 100 mm NaCl, 0.05% Tween 20, 0.1 mm DTT, 10 mm MgCl2, 20 μm GTPγS) as indicated to facilitate nucleotide exchange. The buffer was aspirated so that 20 μl of a 50% slurry was allowed to remain in the tube. EDTA, GDP, or GTP buffer (76 μl) was added as indicated, and binding was initiated by the addition of 35S-labeled β2a-subunit (4 μl). The reaction was incubated for 3 h with end-over-end rotation. The beads were then washed three times with the same buffer used in the assay. GST or GST Rem was eluted from the beads with two 20-μl washes of assay buffer supplemented with 25 mm glutathione. The eluted proteins were resolved on 10% SDS-PAGE gels, which were dried and exposed to film for 16–72 h. Competition Assays—The ability of Rem to compete with CaVα subunit intracellular loop I-II (AID) was assayed as follows. First, the minimal amount of GST·AID protein required to bind 35S-labeled β2a was determined by adding the indicated amount of GST·AID or unfused GST to 10 μl of pre-equilibrated glutathione-Sepharose beads (GE Healthcare). GTP buffer (76 μl) was then added, and binding was initiated by the addition of 4 μl of 35S-labeled β2a subunit. The reaction was incubated for 3 h with end-over-end rotation. The beads were then washed 3 times with 500 μl of GTP buffer. Bound protein was eluted from the beads with two 20-μl washes of assay buffer containing 25 mm glutathione. The eluted proteins were resolved on 10% SDS-PAGE gels, which were dried and exposed to film for 16–72 h to examine β2a/AID association. The ability of Rem to compete with this interaction was determined by adding the indicated amount of thrombin cleaved Rem to the reaction. As a positive control, I-II loop that had been thrombin-cleaved from GST was used. Also, a mock cleavage buffer treatment was used to confirm that thrombin had been completely removed from the cleaved proteins. In Vivo GST Pulldown Assays—HEK293 cells were transiently transfected with the indicated plasmids as described (35Andres D.A. Shao H. Crick D.C. Finlin B.S. Arch. Biochem. Biophys. 1997; 346: 113-124Crossref PubMed Scopus (32) Google Scholar). 48 h post-transfection cells were harvested and lysed in 20 mm Tris, pH 7.5, 250 mm NaCl, 1% Triton X-100, 0.5 mm DTT, 1× protease inhibitor mixture (Calbiochem), 10 mm MgCl2, and 10 μm GTPγS and centrifuged at 100,000 × g for 10 min. The supernatant was harvested, and protein concentration was determined using the Bradford assay. Cleared supernatant (1 mg) was incubated with GST (control) or GST·AID and 10 μl of glutathione-agarose (GE Healthcare) as indicated for 3 h at 4 °C with gentle end-over-end rotation. The beads were then isolated by centrifugation for 10 s at 14000 rpm in a microcentrifuge, and 5 μl of the supernatant was retained for analysis. The beads were washed 3 times with 1 ml of lysis buffer and then eluted with assay buffer containing 25 mm glutathione. The eluted fraction was then analyzed with the supernatant for the presence of FLAG-CaVβ2a as follows. The supernatant and pellet were boiled in SDS-PAGE buffer and resolved on 10% SDS-PAGE gels. The gels were transferred to nitrocellulose and immunoblotted with either anti-FLAG or anti-HA monoclonal antibodies as indicated. Surface Biotinylation Studies—HIT-T15 cells were obtained from ATCC and maintained in F12-K media (Invitrogen) supplemented with 50 μg/ml gentamicin, 2.5% fetal bovine serum, and 10% dialyzed horse serum (prepared by dialyzing horse serum in 14,000-kDa cutoff dialysis bags extensively versus 0.15 m NaCl at 4 °C). Ten-cm dishes were seeded with cells the day before infection. Cells were either cultured alone (uninfected control) or incubated for 24 h with CsCl-purified adenovirus-expressing GFP (control) or co-expressing GFP and Rem or GFP and Rem1–265 (107 plaque-forming units/ml) (22Finlin B.S. Crump S.M. Satin J. Andres D.A. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 14469-14474Crossref PubMed Scopus (178) Google Scholar). This resulted in near complete HIT-T15 cell infection based on the analysis of GFP-positive cells (24 h post-infection). Monolayers were washed three times with ice-cold phosphate-buffered saline (PBS), and surface proteins were biotinylated using 1 mg/ml membrane-impermeant sulfo-NHS-LC-biotin (Pierce) in PBS for 1 h at 4°C with gentle rocking. Cells were then washed three times with ice-cold phosphate-buffered saline, harvested on ice in 1 ml of Versene (Invitrogen), and pelleted by gentle centrifugation, and the Versene was aspirated. Radioimmune precipitation assay buffer (1% Triton X-100, 1% deoxycholic acid, 0.1% SDS, 50 mm Tris-HCl, pH 7.4, 1× protease inhibitor mixture (Calbiochem)) was added to the cell pellet, which was sonicated twice for 10 s (Kontes). The soluble fraction was isolated after centrifugation at 100,000 × g for 10 min. Protein concentrations were determined using the Bio-Rad assay kit with bovine serum albumin as a standard. Biotinylated proteins were isolated by adding cleared cell lysates (500 μg) to 100 μl (50% slurry) of streptavidin beads (Pierce) in a total volume of 1 ml of radioimmune precipitation assay buffer. The reaction was gently rotated end over end at 4 °C for 1.5 h, and resin was pelleted by centrifugation, washed once with radioimmune precipitation assay buffer (RIPA) containing 0.3 m NaCl (two times), twice with RIPA containing 0.15 m NaCl, and finally twice with wash buffer containing no detergent (150 mm NaCl, 50 mm Tris-HCl, pH 7.4, 2.5 mm EDTA). The beads were resuspended in 30 μl of 2× SDS loading buffer and boiled for 5 min. The released protein as well as 10 μg of the input was resolved using 6% SDS-PAGE gel electrophoresis, transferred to nitrocellulose, and subjected to immunoblot analysis with affinity-purified L-type calcium channel α-subunit polyclonal antibody at 2 μg/ml and horseradish peroxidate goat anti-rabbit (Zymed Laboratories Inc.) secondary antibody at a 1:20,000 dilution. Super signal (Pierce) was used as the enhanced chemiluminescent reagent. For inhibition studies, immunoblotting was performed as above, but the α-subunit polyclonal antibody was preincubated for 1 h at 20°C with 10 μg/ml GST-fused CaV1.2 II-III loop protein. To assure that the cells remained intact throughout the surface labeling, biotinylation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a cytosolic protein, was analyzed by immunoblotting with GAPDH monoclonal antibody (Ambion) at 1:2000 dilution. Electrophysiology—HEK293 cells were transiently transfected with plasmids 48 h before recordings using Effectene (Qiagen) according to the manufacturer's instructions. tsA201 cells were transiently transfected 48 h before recordings using the calcium phosphate method as described previously (35Andres D.A. Shao H. Crick D.C. Finlin B.S. Arch. Biochem. Biophys. 1997; 346: 113-124Crossref PubMed Scopus (32) Google Scholar). Transfected cells were identified by GFP expression. HIT-T15 cells were plated on polylysine-coated coverslips in 24-well tissue culture dishes at 20,000 cells/well. The next day the cells were infected with the indicated adenovirus at 1 × 107 adenovirus per ml. Adenovirus-infected cells were identified by GFP expression, and recordings were made 22 h post-infection (23Finlin B.S. Mosley A.L. Crump S.M. Correll R.N. Ozcan S. Satin J. Andres D.A. J. Biol. Chem. 2005; 280: 41864-41871Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). The whole-cell configuration of the patch clamp technique was used to measure ionic current. Patch electrodes with resistances of 2–4 megaohms contained 110 (or 0) mm potassium gluconate, 40 (or 150) mm CsCl, 1 mm MgCl2, 5 mm Mg-ATP, 3 mm EGTA, 5 mm Hepes, pH 7.36. The bath solution for HEK and tsA201 cells consisted of 112.5 mm CsCl, 30 mm BaCl2, 1 mm MgCl2, 10 mm tetraethylammonium chloride, 5 mm glucose, 5 mm Hepes, pH 7.4. The bath solution for adenoviral infected HIT-T15 cells consisted of 102.5 mm CsCl, 40 mm BaCl2, 1 mm MgCl2, 10 mm tetraethylammonium chloride, and 5 mm Hepes, pH 7.4. Signals were amplified with an Axopatch 200B amplifier and 333-kHz A/D system (Axon Instruments, Union City, CA). Data were analyzed with Clampfit 9 (Axon Instruments) and Origin statistical software (OriginLab Corp., Northampton, MA). All recordings were performed at room temperature (20–22 °C). Identification of the Rem Binding Domain in β2a—We previously reported that Rem binds to a variety of CaVβ subunits, including CaVβ2a (22Finlin B.S. Crump S.M. Satin J. Andres D.A. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 14469-14474Crossref PubMed Scopus (178) Google Scholar). Because RGK proteins are capable of associating with all four β-subunit gene products (18Finlin B.S. Shao H. Kadono-Okuda K. Guo N. Andres D.A. Biochem. J. 2000; 347: 223-231Crossref PubMed Scopus (90) Google Scholar, 21Beguin P. Nagashima K. Gonoi T. Shibasaki T. Takahashi K. Kashima Y. Ozaki N. Geering K. Iwanaga T. Seino S. Nature. 2001; 411: 701-706Crossref PubMed Scopus (254) Google Scholar, 22Finlin B.S. Crump S.M. Satin J. Andres D.A. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 14469-14474Crossref PubMed Scopus (178) Google Scholar) and CaVβ-subunit binding appears critical to the ability of RGK proteins to disrupt CaVα1 function (21Beguin P. Nagashima K. Gonoi T. Shibasaki T. Takahashi K. Kashima Y. Ozaki N. Geering K. Iwanaga T. Seino S. Nature. 2001; 411: 701-706Crossref PubMed Scopus (254) Google Scholar, 22Finlin B.S. Crump S.M. Satin J. Andres D.A. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 14469-14474Crossref PubMed Scopus (178) Google Scholar, 23Finlin B.S. Mosley A.L. Crump S.M. Correll R.N. Ozcan S. Satin J. Andres D.A. J. Biol. Chem. 2005; 280: 41864-41871Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 26Beguin P. Mahalakshmi R.N. Nagashima K. Cher D.H. Kuwamura N. Yamada Y. Seino Y. Hunziker W. Biochem. J. 2005; 390: 67-75Crossref PubMed Scopus (65) Google Scholar), we reasoned that RGK binding occurs via a conserved CaVβ-subunit sequence. Although previous work suggested that CaVβ binding to RGK GTPases may be GTP-dependent (21Beguin P. Nagashima K. Gonoi T. Shibasaki T. Takahashi K. Kashima Y. Ozaki N. Geering K. Iwanaga T. Seino S. Nature. 2001; 411: 701-706Crossref PubMed Scopus (254) Google Scholar), in vitro CaVβ2a binding to Rem was found to be nucleotide-independent, with both GDP-bound and GTPγS-bound Rem displaying equivalent CaVβ-subunit binding (Fig. 1B). To identify the structural domain of CaVβ2a responsible for Rem association, we used 35S-labeled, in vivo translated wild-type and truncated CaVβ2a-subunit probes to examine the interaction between the various structural domains of CaVβ2a and both wild-type Rem and the loop I-II (AID) domain of CaV1.2 (Fig. 1A). GST·Rem and GST·AID both associated with 35S-labeled in vitro translated wild-type CaVβ2a and, more importantly, with the CaVβ2a226-604 truncation mutant (Fig. 1C). This localized the Rem interaction site within the highly conserved GK domain (GK) found in all CaVβ subunit genes and known to harbor the ABP binding domain primarily responsible for CaVβ-subunit/AID binding (2Richards M.W. Butcher A.J. Dolphin A.C. Trends Pharmacol. Sci. 2004; 25: 626-632Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). No int
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