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RGK Signaling and Biological Activity
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[39] Analyses of Rem/RGK Signaling and Biological Activity By DOUGLAS A. ANDRES, SHAWN M. CRUMP, ROBERT N. CORRELL, JONATHAN SATIN , and BRIAN S. FINLIN Abstract
Rem (Rad and Gem related) is a member of the RGK family of Ras‐ related GTPases that also includes Rad, Rem2, and Gem/Kir. All RGK proteins share structural features that are distinct from other Ras‐related proteins, including several nonconservative amino acid substitutions within regions known to participate in nucleotide binding and hydrolysis and a C‐terminal extension that contains regulatory sites that seem to control both subcellular location and function. Rem is known to modulate two distinct signal transduction pathways, regulating both cytoskeletal reorganization and voltage‐gated Ca2þ channel activity. In this chapter, we summarize the experimental approaches used to characterize the interaction of Rem with 14‐3‐3 proteins and Ca2þ channel ‐subunits and describe electrophysiological analyses for characterizing Rem‐mediated regulation of L‐type Ca2þ channel activity.
Introduction
The Ras subfamily of GTPases consists of approximately 35 members that share a high degree of amino acid conservation with H‐, K‐, and N‐ Ras, particularly within the their GTP‐binding/GTPase core (Colicelli, 2004). However, it is now clear that members of the family possess distinct biochemical and biological activities and express both overlapping and unique functions (Colicelli, 2004; Reuther and Der, 2000). Rem (Rad and Gem/Kir‐related) was originally identified as the product of polymerase chain reaction amplification (PCR) using oligonucleotide primers derived from conserved regions of the Rad and Gem/Kir GTPases (Finlin and Andres, 1997) and, together with Rem2 (Finlin et al., 2000), serves as the newest members of the RGK (Rem, Rem2, Rad, Gem/Kir) family of Ras‐ related GTP binding proteins (Cohen et al., 1994; Finlin and Andres, 1997; Finlin et al., 2000; Maguire et al., 1994; Reynet and Kahn, 1993). Despite the similarities between Rem and Ras, RGK proteins share several unique characteristics. These include nonconservative amino acid substitutions within regions known to be involved in guanine nucleotide binding and METHODS IN ENZYMOLOGY, VOL. 407 Copyright 2006, Elsevier Inc. All rights reserved.
0076-6879/06 $35.00 DOI: 10.1016/S0076-6879(05)07039-4
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hydrolysis, particularly a RGK signature DXWE G3 domain that replaces the DXXG motif (involved in nucleotide hydrolysis) found among most Ras superfamily proteins. Rem also contains a unique effector domain when compared with the larger Ras superfamily (Colicelli, 2004; Finlin and Andres, 1997). Surprisingly, Rem also differs from other RGK proteins within this putative effector domain, suggesting that each RGK protein may interact with distinct regulatory and effector proteins (Finlin et al., 2000). To date, no GTP‐dependent ‘‘classical’’ effectors for Rem have been identified, although several Rem binding proteins have been characterized (see the following). RGK proteins contain large N‐terminal and C‐terminal extensions relative to other Ras family proteins. These extensions contain multiple phosphorylation sites (Finlin and Andres, 1999; Maguire et al., 1994; Moyers et al., 1997, 1998; Ward et al., 2004), a C‐terminal calmodulin (CaM) binding domain in Rad and Gem (Beguin et al., 2005; Fischer et al., 1996; Moyers et al., 1997; Ward et al., 2004), and 14‐3‐3 binding sites (Beguin et al., 2005; Finlin and Andres, 1999; Ward et al., 2004). Protein phosphorylation and CaM/14‐3‐3 association have been proposed to play a role in regulating RGK function. RGK proteins do not have traditional lipid modification motifs at the C‐terminus, which are important for membrane anchorage of other Ras‐related GTPases. However, all RGK proteins contain a conserved 10 amino acid domain that includes a cysteine residue at position ‐7 from their C‐terminus that may serve as a unique modification site. Finally, RGK proteins are subject to transcriptional regulation and exhibit tissue‐ restricted expression patterns. Rem is expressed prominently in cardiac muscle but also in the lung, skeletal muscle, and kidney (Finlin and Andres, 1997). The administration of lipopolysaccharide results in a general repression of Rem mRNA levels, making Rem the first Ras family GTPase to be regulated by repression (Finlin and Andres, 1997). At present, questions remain concerning the physiological role of Rem. However, the recent identification of RGK binding proteins has begun to provide insight into the role of this GTPase subfamily. These include studies demonstrating RGK‐mediated regulation of cytoskeletal reorganization in various cell types, including human Rem (also termed Ges) promotion of endothelial cell sprouting (Pan et al., 2000). Although the mechanism of Rem‐mediated morphology change remains to be defined, the identification of Rho kinase (Ward et al., 2002), the kinesin‐like protein KIF‐9 (Piddini et al., 2001), and Gmip, a RhoGAP (Aresta et al., 2002), as Gem/Rad‐binding partners suggests mechanisms of regulating cytoskeletal morphology. However, many of these proteins associate with only a subset of the RGK proteins, and their roles in Rem signaling remain to be established. To date, the only common RGK family binding partners
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are voltage‐gated calcium channel subunits and 14‐3‐3 proteins. Moreover, expression of all members of the RGK GTPase family down regulates voltage‐gated calcium channel activity (Beguin et al., 2001; Finlin et al., 2003, 2005; Ward et al., 2004), leading to the notion that these G proteins provide a novel mechanism for regulating electrical signaling pathways. This chapter summarizes experimental approaches for evaluating Rem function. The first part of this chapter discusses a variety of binding analyses for characterizing the interaction of Rem with 14‐3‐3 proteins, Ca2þ channel ‐subunits, and for the isolation of novel interacting proteins. The second section describes electrophysiological analyses for characterizing Rem‐mediated regulation of L‐type Ca2þ channel activity. Mammalian Expression Vectors for Wild‐Type and Mutant Rem Proteins
The original full‐length Rem sequence encoding wild‐type (GenBank accession numbers U91601 [mouse] and AF084465 [human]) and a series of truncation and point mutants of mouse Rem were introduced into the mammalian vector pCDNA3 3.1zeo (Invitrogen) as described previously (Finlin and Andres, 1997). Expression of Rem in this vector is under control of the strong cytomegalovirus immediate‐early (CMV) promoter. These vectors also encode a zeocin resistance gene, under the control of the simian virus 40 (SV40) promoter, to allow selection in growth medium containing zeocin. pKH3 (Mattingly et al., 1994) and pEGFP‐C1 (Clontech) mammalian expression vectors encoding wild‐type Rem and a series of Rem deletion mutants were also generated (Finlin and Andres, 1999; Finlin et al., 2003). This results in the addition of N‐terminal sequences encoding a hemagglutinin (HA) protein epitope tag or fusion of the green fluorescent protein (GFP) onto the N‐terminus of the Rem protein. Fusions to the N‐terminus of Rem do not affect its biological activity (Finlin et al., 2003). Expression of HA‐Rem or GFP‐Rem proteins in transfected mammalian cells can be detected by Western blot analyses using anti‐HA monoclonal antibody (12CA5) or by use of a fluorescence microscope. We have used these expression vectors for both stable and transient expression analyses. Recombinant adenovirus coexpressing Rem and GFP or a series of Rem deletion mutants with GFP have also been used for Rem expression in difficult to transfect cell lines (Finlin et al., 2003). Rem adenoviral vectors are generated through homologous recombination using the AdEasy system as described previously (He et al., 1998). High‐titer viral stocks are generated by CsCl banding, and infectious titers are determined
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by limiting dilution plaque assay on HEK‐293 cells and used at m.o.i. values of 100–200 (Finlin et al., 2003). In Vitro Biochemical Assays: Identification of Rem Binding Proteins
Generation of Recombinant GST‐Rem We have used GST fusions to Rem for a variety of in vitro biochemical analyses (Finlin and Andres, 1997, 1999). Wild‐type Rem cDNA is introduced into the pGEX‐KG bacterial expression vector (Guan and Dixon, 1991), and bacterially expressed GST‐Rem proteins are isolated by conventional methods for the expression and purification of GST fusion proteins. Escherichia coli strain BL21‐DE3(LysE) (Novagen, Madison, WI) transformed with the various pGEX‐Rem plasmids are grown overnight at 37 to saturation in 2XYT broth supplemented with carbenicillin (50 g/ml) to maintain selection for the plasmid and then diluted 1:100 and grown to the start of log phase (4 h). After reaching an OD600 of 0.6, protein production is induced with 0.5 mM isopropylthio‐‐D‐ galactopyranoside (IPTG) for an additional 4 h. Bacteria are then collected by centrifugation and resuspended in 20 ml GST‐B buffer (10 mM Tris‐HCl [pH 7.4], 1 mM dithiothreitol [DTT], 10 M GDP, 1 mM MgCl2, and 1 mM PMSF) containing lysozyme (0.5 mg/ml), incubated for 15 min on ice, and broken using a French pressure cell. The lysate is diluted with 25 ml of GST‐C buffer (20 mM HEPES (pH 7.6), 100 mM KCl, 20% [v/v] glycerol, 1 mM DTT, 10 M GDP, 1 mM MgCl2, and 1 mM PMSF), cleared by centrifugation (30,000g for 20 min at 4 ), and tumbled at 4 with 1.0 ml of preswollen glutathione‐agarose bead resin (Sigma) for 30 min. The beads are washed three times with 20 ml of ice‐cold GST‐C buffer, the resin transferred to a small gravity flow column (BioRad), and eluted using release buffer (GST‐C buffer containing 25 mM glutathione, pH 7.4). The released protein is dialyzed against four changes (1 l) of TCB buffer (50 mM Tris‐HCl [pH 7.5], 10% [v/v] glycerol, 150 mM NaCl, 1 mM MgCl2, 1 mM DTT) and stored in 100‐g aliquots at 80 until needed. We have used recombinant GST‐Rem proteins for in vitro protein interaction studies (see later). In addition, recombinant Rem has been released from GST by thrombin cleavage and used to determine in vitro nucleotide binding and GTP hydrolysis (Finlin and Andres, 1997). Recombinant GST‐Rem protein has also been generated containing an N‐terminal heart muscle kinase (HMK) phosphorylatable RRASV sequence to allow expression of Rem fusion proteins that can be radiolabeled with 32P for use as a molecular probe to screen an expression library to identify Rem interacting proteins (Finlin and Andres, 1999). Rem cDNA
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sequences are introduced into the pGEX‐KG‐HMK plasmid (a pGEX‐KG derivative containing an HMK recognition site 50 to the BamHI site) to express GST‐HMK‐Rem (Finlin and Andres, 1999). Recombinant GST‐ HMK‐Rem is expressed in BL21DE3 bacteria and purified by glutathione– agarose affinity chromatography and cleaved with thrombin as described previously. This is followed by a kinase reaction in which HMK‐Rem (10 g) is incubated with 2.0 mCi [32P]ATP (6000 Ci/mmol, NEN) and 100 U HMK (Sigma, St. Louis, MO) in 100 l of 20 mM Tris, pH 7.5, 100 mM NaCl, and 12 mM MgCl2 for 30 min on ice. The kinase reaction is stopped by addition of 400 l of stop buffer (10 mM phosphate, 10 mM sodium pyrophosphate, 1 mg/ml bovine serum albumin [BSA]). The probe is then dialyzed against four changes (50 ml each) of dialysis buffer (20 mM Tris, pH 7.5, 100 mM NaCl, 12 mM MgCl2, 10 M GDP) to remove unincorporated label, counted in a scintillation counter, and stored in multiple aliquots at 80 . Radiolabeled Rem proteins have a 2‐week half‐life, so protein is generated as needed. ‘‘Far Western’’ Interaction Cloning to Identify Rem Binding Proteins To identify Rem‐interacting proteins, we have screened a 14‐day‐old mouse embryo lEXlox cDNA library (Novagen, Madison, WI) (Finlin and Andres, 1999). The library is plated at 40,000 plaques/plate (150 mm) by infection of BL21(DE3) bacteria. Once plaques reach 0.5–1 mm in size, the infected bacterial plates are overlaid with nitrocellulose filters and incubated overnight at 4 . The plates are then placed at 37 and incubated for another 4 h, and the primary filters are removed immediately and placed in Hyb75 (20 mM HEPES, pH 7.6, 75 mM KCl, 0.1 mM EDTA, 2.5 mM MgCl2, 1 mM DTT, 0.05% NP‐40) (Finlin and Andres, 1999). Secondary filters are generated by overlaying the plates with a second set of nitrocellulose filters and incubating for 4 h at 37 . These filters are immediately combined with the primary membranes in Hyb75. The filters are then blocked in Hyb75 with 1% nonfat milk for 4 h at 4 . Bacterial extract containing recombinant HMK‐GST is prepared from BL21DE3 cells transformed with pGEX‐KG‐HMK as follows. The bacteria are grown at 37 in LB medium to an A600 ¼ 0.6. Protein production is induced with 0.5 mM IPTG for 4 h. The bacteria are pelleted, resuspended in Hyb75, and broken using a French pressure cell, and the 100,000g cleared supernatant is used as a supplemental blocking agent. Library filters are incubated with Hyb75 containing 250 mM KCl, 1% nonfat milk, 400 g/ml HMK‐GST bacterial extract, 10 M GDP, and 200,000 cpm/ml [32P]HMK‐Rem probe for 16 h at 4 with shaking. Filters are washed four times at 4 with washing buffer (Hyb75 supplemented with 10 M GDP) and exposed to film for 4 h at room
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FIG. 1. Identification of 14‐3‐3 interaction with Rem by ‘‘Far‐Western’’ interaction cloning. A 14‐day‐old mouse embryo lEXlox cDNA expression library was plated at 4 104 plaques/filter and subjected to [32P]HMK‐Rem interaction screening. After extensive washing, filters were subjected to autoradiography (Kodak X‐OMAT film for 4 h at room temperature) and positive plaques selected and rescreened to allow the isolation of a single purified plaque. (A) Representative filter from the primary screen. (B) A representative filter after two rounds of plaque purification.
temperature. Positive clones are amplified and purified to tertiary clones (Fig. 1). Rem was found to interact strongly with a series of 14–3‐3 proteins, including the ", , , and 14‐3‐3 proteins (Finlin and Andres, 1999). Interaction with Rem Binding Proteins
We have used a variety of in vitro Rem‐binding assays to characterize the association of Rem with 14‐3‐3 proteins and the accessory ‐subunits of voltage‐gated calcium channels. We describe two standard methods of analysis: in vitro pull‐down assays to examine the interaction of bacterially expressed Rem with recombinant 14‐3‐3 or ‐subunits and coimmunoprecipitation analysis. In Vitro Binding Reactions Radiolabeled full‐length or truncation mutants of CaV 2a subunit are prepared by in vitro transcription and translation in the presence of [35S] methionine and examined for their ability to associate in a nucleotide‐ dependent fashion with GST‐Rem (Fig. 2). All manipulations are carried out at 4 . Glutathione‐sepharose beads (Pharmacia) (10 l) are washed with 500 l EDTA buffer (50 mM Tris, pH 7.5, 100 mM NaCl, 0.05% Tween‐20, 0.1 mM DTT, 1 mM EDTA) and resuspended in 1 ml EDTA buffer containing either GST (10 g) or GST‐Rem (10 g). The beads are incubated for 5 min with end‐over‐end rotation at 4 to allow GST fusion protein binding and then washed with 1 ml EDTA buffer to remove
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FIG. 2. In vitro association of Rem with CaV2a. Association of in vitro‐translated [35S]‐ 1‐355 (a short C‐terminal deletion mutant of CaV2a) with full‐length Rem but not labeled 2a GST alone, using an in vitro pull‐down assay. Rem‐mediated CaV2a binding to glutathione‐ sepharose resin was analyzed by resolving the bound fraction to 10% SDS‐PAGE and the dried gel subjected to autoradiography for 16 h.
unbound GST proteins and then incubated with either 1 ml GDP buffer (50 mM Tris, pH 7.5, 100 mM NaCl, 0.05% Tween‐20, 0.1 mM DTT, 10 mM MgCl2, 20 M GDP), 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 M GTPS) to facilitate nucleotide exchange or with EDTA buffer (to generate nucleotide‐free Rem). The GST‐Rem fusion proteins are stored in the presence of MgCl2 (see preceding), so EDTA washing is required to ensure that all Mg2þ is chelated. To examine nucleotide‐dependent ‐subunit binding, 76 l of either EDTA or GDP or GTP buffer is added to the washed pellet, and binding is initiated by the addition of 4 l [35S]2a subunit and incubated for 3 h with end‐over‐end rotation at 4 . The beads are then washed three times with the appropriate binding buffer, and bound [35S]2a is eluted from the beads with two 20‐l washes of assay buffer containing 25 mM glutathione. The eluted proteins are resolved on 10% SDS‐PAGE gels that is dried and exposed to film for 16–72 h. Coimmunoprecipitation Analysis HEK293 Cell Culture. HEK 293 cells are maintained in Dulbecco’s modified Eagle’s medium containing 5% (v/v) fetal bovine serum and 55 g/ml gentamicin and maintained at 37 in a humidified 5% CO2 atmosphere. Cell stocks are generated by plating 106 cells/100‐mm‐diameter tissue culture dish. Cells are fed every 48 h for 4–5 days (or until cells appear 70% confluent). Cells are then trypsinized as follows: growth medium is removed, the dish washed with 10 ml of phosphate‐buffered saline (PBS), and 1 ml of 0.05% (w/v) trypsin‐0.53 mM EDTA is added for 2–4 min or until cells can be readily dislodged. Cells are then pooled, pelleted at 800 rpm in a tabletop centrifuge for 5 min at room temperature,
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and then resuspended and frozen in 1 ml of ice‐cold freezing medium (95% [v/v] fetal bovine serum, and 5% dimethyl sulfoxide [DMSO]) per dish. Vials are placed in a polystyrene box and incubated overnight at 80 for gentle freezing before being transferred to liquid nitrogen for long‐term storage. Cells stocks are recovered by rapid thawing in a 37 water bath, diluted in warm culture medium, and then divided onto 3 100‐mm dishes. Once cells reach 70–80% confluence (3–5 days depending on the initial cell density), they are passaged by reseeding 2 106 cells/10‐mm dish. For expression studies, 100‐mm dishes are coated with polylysine (4 ml of 25 g/ml in PBS per 100‐mm plate). Cells are then plated at 107 cells/100‐mm dish and used the following day for transfection. HEK293 Transfection Protocol. REAGENTS FOR TRANSIENT TRANSFECTION HBS (2): Add the following to 900 ml of deionized water: 10 g of tissue culture‐tested HEPES and 16 g of NaCl. Adjust to pH 7.10 0.05 with 1 M NaOH, adjust the volume to 1 l, and sterilize by filtration. The correct pH is critical for good DNA precipitation. Phosphate (50): Combine 70 mM Na2HPO4 with 70 mM NaH2PO4 (1:1) and sterilize by autoclaving. Calcium chloride: Prepare 100 ml of 2 M CaCl2 and filter sterilize. Plasmid DNA: Plasmid DNA can be prepared by the standard alkaline lysis procedure and purified by banding twice in CsCl density gradients or by using a variety of commercial ion‐exchange column methods. DNA purity is a crucial factor in maintaining high transfection efficiency. Monolayers of HEK 293 cells are transiently transfected with 20 g of mammalian expression plasmid (pCDNA 3.1zeo) encoding hemagglutinin (HA) epitope–tagged Rem DNA/100‐mm dish using the calcium phosphate technique as described previously (Andres et al., 1997). After 48 h of recovery, stable cell lines may be generated by placing the cells under drug selection (250 mg/ml Zeocin) (Invitrogen) (Finlin and Andres, 1999). However, the Rem 14‐3‐3 interaction may also be demonstrated by transient transfection in this cell line. Rem is phosphorylated in vivo when expressed in HEK293 cells, which eliminates the necessity of kinase treatment (Finlin and Andres, 1999). Two days after transfection, the cells are harvested, resuspended in IPA buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 1% NP‐40, 1 mM PMSF), lysed by sonication, and a 100,000g supernatant (S100) is prepared. For analysis of 14–3‐3 coimmunoprecipitation, 1 mg of each S100 lysate is incubated with 20 l of a 50% slurry of protein G covalently crosslinked to antihemagglutinin (HA) monoclonal antibody (12CA5) in a 300‐l reaction for 2 h at 4 with gentle rotation. Immune
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complexes are pelleted, washed 3 1 ml IPA, and resolved by SDS‐PAGE as described (see preceding). 14‐3‐3 proteins are detected by immunoblot analysis using pan‐reactive rabbit anti‐14‐3‐3 antibody (S.C. 629; Santa Cruz). The in vitro assay may also be used to examine the interaction of bacterially expressed recombinant His6‐14‐3‐3 with HA‐Rem or Rem variant proteins (Finlin and Andres, 1999). Transiently transfected HEK 293 cell lysate (40 g) is incubated with 2 g of the His6‐tagged 14‐3‐3 proteins and HA‐Rem binding assessed by immunoblotting. Specific binding may be demonstrated by competition experiments in which 1–10 g of recombinant 14‐3‐3 (lacking the His6 epitope) is preincubated with the cell lysate for 1 h on ice before addition of His6‐14‐3‐3. REM‐CAV SUBUNIT INTERACTIONS. Hemagglutinin (HA)‐Rem and either Flag‐CaV or empty pFlag vector control are cotransfected into HEK293 cells by the calcium phosphate method (see preceding). Forty‐ eight hours after transfection, the cells are washed with PBS, placed into 1 ml of Verseen (GIBCO), harvested, pelleted, and then suspended in ice‐ cold immunoprecipitation (IP) buffer (20 mM Tris, pH 7.5, 250 mM NaCl, 1% TX‐100, 0.5 mM DTT, 1 protease inhibitor mixture [Calbiochem], 10 mM MgCl2, 10 M GTPS). The cells are lysed, subjected to centrifugation, and 1 mg of the supernatant incubated in a 500‐l reaction containing 10 l of packed Protein G Sepharose (Pharmacia) and 4 g of anti‐Flag M2 monoclonal antibody (Sigma) for 3 h with gentle rotation at 4 . The beads are pelleted, and 5 l of the supernatant is saved for analysis. The beads are then washed three times with 1 ml of IP buffer. The supernatant and bound fractions are resolved on SDS‐PAGE, transferred to nitrocellulose, and subjected to immunoblot analysis. HA‐Rem is detected by immunoblotting as described (Finlin, 2003), except that the biotinylated HA antibody is used at 1 g/ml, and bound protein was detected with streptavidin–horseradish peroxidase (Pierce) (1:40,000 dilution). The blot is subsequently probed for Flag‐CaV2a using anti‐Flag M2 monoclonal antibody (1 g/ml) to confirm the efficiency of immunoprecipitation. Analysis of L‐Type Calcium Channel Function
Voltage‐gated Ca2þ channels play crucial roles in the regulation of intracellular calcium concentrations in a diversity of cell types, including muscle cells, neuroendocrine cells, and neurons. The calcium that enters the cell through these channels serves as a second messenger to regulate a variety of processes including cardiac muscle excitation‐contraction coupling and neurotransmitter release (Catterall, 2000). Recent studies
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indicate that all members of the RGK GTPase family, by means of direct interaction with ‐subunits, serve as regulators of voltage‐gated Ca2þ channels (Beguin et al., 2001; Finlin et al., 2003, 2005; Ward et al., 2004). However, questions remain concerning the mechanism of RGK‐mediated control and how RGK regulation of voltage‐sensitive ion channels may affect excitation‐contraction coupling and calcium‐signaling pathways. The next section details assays for measuring Rem‐mediated regulation of Ca2þ channel function. Examination of RGK Effects on Voltage‐Gated Calcium Channel Activity A number of primary cells and cultured cell lines have been used to analyze voltage‐gated Ca2þ channel function. These have included the examination of native tissues that contain a complex array of ion channel signaling proteins, heterologous expression of channels in a cell lacking endogenous Ca2þ channels (e.g., Xenopus oocytes or human embryonic kidney [HEK293] cells) or cell lines that natively express functional Ca2þ channels (such as rat pheochromocytoma [PC12] cells). Two model systems have been used in our laboratory to analyze Rem‐ dependent calcium channel regulation by means of the whole‐cell configuration of the patch clamp technique. The first examines current through a minimal L‐type calcium channel complex consisting of the pore‐forming CaV subunit and an accessory CaV subunit in the presence of the RGK proteins Rem, Rem2, Rad, or mutants thereof transiently coexpressed in HEK293 cells by means of calcium‐phosphate transfection (Finlin et al., 2003, 2005). HEK293 cells are an excellent model system for examination of RGK effects because they lack endogenous calcium channel complexes, transfect with high efficiency with multiple plasmids, and are easily cultured. The second system used to examine Rem function examines current through voltage‐gated channel complexes endogenous to the Syrian golden hamster pancreatic beta cell line HIT‐T15 in the presence of Rem overexpressing adenovirus. The presence of native channel complexes makes HIT‐T15 cells well suited for examining the effects of RGK proteins on endogenous Ca2þ channel function (Finlin et al., 2005). Electrophysiological Recordings in HEK293 Cells The human embryonic kidney cell line HEK293 does not exhibit measurable endogenous calcium currents by means of whole‐cell patch clamp recording. Introduction of calcium channel components by transfection allows the detailed examination of the effect of Rem protein expression on calcium currents expressed from a variety of pore‐forming and accessory channel subunit combinations in a null‐background system.
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HEK293 cells are purchased from the American Type Culture Collection (Manassas, VA; www.ATCC.org) and cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented with 5% (v/v) fetal calf serum (HyClone). Cell Culture, Plating, and Transfection Procedures. Approximately 12–48 h before patch‐clamp analysis, HEK293 cells plated on poly‐L‐ lysine–coated glass coverslips at a density of 10,000 cells/slip are cotransfected in a 24‐well plate with a minimal calcium channel complex consisting of the pore‐forming CaV1.2, CaV1.3, or CaV3.2, along with the accessory CaV subunit 2A, 1B, or 4A (expressed in the single N‐terminal Flag vector pCMVT7/F2 (Sigma), and either GFP‐Rem (expressed in the N‐terminal enhanced green fluorescent protein [GFP] fusion vector pEGFP‐C1 [BD Biosciences]), or unfused GFP by the calcium phosphate transfection method. Typically, equal concentrations of each plasmid are used for transfection, to a total amount of 1 g of DNA per well/ transfection. It is also possible to use commercial liposomal reagents to transfect HEK293 cells. However, theses reagents are expensive and are not necessary for these assays. HEK293 cells cotransfected with CaV1 and CaV subunits and either GFP‐Rem or empty pEGFP‐C1 plasmid are evaluated for transfection efficiency and transfected cells visualized by fluorescence microscopy. With the exception of Rem cotransfection, all GFP‐positive cells cotransfected with plasmids encoding Ca2þ channel subunits express current (Fig. 3). Solutions for Electrophysiology. For recording barium currents through calcium channels, the external bath solution contains (in mM) 140 NaCl or CsCl, 2.5 (or 40) BaCl2 (or CaCl2), 1 MgCl2, 5 glucose, and 5 HEPES (pH 7.4). The internal recording solution contains (in mM) 110 K‐gluconate, 40 CsCl, 1 MgCl2, 5 Mg‐ATP, 10 EGTA (or Bapta), and 10 HEPES (pH 7.35). The choice and concentration of charge carrier (Ca2þ versus Ba2þ) is dictated by the specific experimental question to be addressed. Single channel conductance is higher and kinetics is slower with Ba2þ, whereas Ca2þ induces Ca2þ‐dependent inactivation. The use of 40 mM charge carrier increases the signal but sometimes leads to unstable whole‐cell recordings. Electrophysiology. Whole‐cell recordings are performed as previously described (Finlin et al., 2003). We select spherical cells with few processes to minimize series resistance and space clamp artifacts. Cell capacitance is typically about 15–25 pF. Quantification of macroscopic Ca2þ currents is complicated by the sporadic phenomenon known as run‐down. Five minutes after obtaining whole‐cell access (determined by the increase of capacitance) is typically sufficient time for equilibration of pipette solution with the cytosolic space. Whole‐cell Ca2þ current (ICa) is then tested for an additional 20 min to evaluate run‐down.
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FIG. 3. Rem or Rem2 prevents or attenuates de novo expression of CaV1.2 ionic current. (A and B) Representative Ba2þ current elicited by a þ5‐mV voltage step from 80 mV for cells expressing CaV1.2 þ 2a þ GFP. (C and D) HEK293 cells co‐transfected with CaV1.2 þ 2a þ GFP‐tagged wild‐type Rem2 or Rem. (E) Current voltage relationships for HEK293 cells transfected with CaV1.2 þ 2a þ GFP (filled squares), CaV1.2 þ 2a þ GFP‐Rem2 (open circles), or CaV1.2 þ 2a þ GFP‐Rem (open triangles). Rem completely inhibits CaV1.2 current expression, and Rem2 expression potently inhibits CaV1.2 current expression.
We use standard whole‐cell patch‐clamp methods to record calcium channel currents from individual transfected HEK293 cells under voltage clamp conditions. Coverslips containing transfected cells are placed in a chamber of 600 l volume. The chamber is then transferred to a stage of an inverted microscope (Warner Instruments, a subsidiary of Harvard Apparatus, Holliston, MA) outfitted with Hoffman modulation contrast and fluorescence optics. Cells are visualized for recording at 400 and assessed for EGFP expression using fluorescence imaging. Injection pipettes are pulled from glass capillary tubes (Warner Instruments, Hamden, CT) to resistances of 1–2 M on a model P‐97 Flaming/ Brown micropipette puller (Sutter Instrument Co., Novato, CA). Tips are flame‐polished using an MF‐830 Microfuge (Narishige, Japan), and Sylgard (Dow‐Corning) is applied to reduce pipette capacitance.
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All recordings are performed at room temperature (20–22 ). Stimulation protocols are generated with CLAMPEX 9.2 (Axon) and data acquired and amplified with an Axopatch 200B patch clamp in combination with a 333kHz A/D system (Axon Instruments, Union City, CA). The data obtained are analyzed with CLAMPFIT 9.2 (Axon) and ORIGIN statistical software (OriginLab, Northampton, MA). To measure Ca2þ (or Ba2þ) currents through voltage‐gated calcium channels, voltage steps of 800 msec duration from a 5‐sec holding potential of 80 mV to voltages between 90 mV and 80 mV at 5‐mV intervals are applied to assess the voltage dependence of channel gating and peak currents (Fig. 3). All currents are expressed as current density (peak inward current divided by whole‐cell capacitance). Electrophysiological Recordings in HIT‐T15 Cells The hamster pancreatic cell line HIT‐T15 is capable of insulin secretion and as such contains CaV1.2 and CaV1.3 channels, along with their associated CaV and CaV2 accessory subunits. Endogenous expression of the voltage‐gated calcium channel complex obviates the need for transient overexpression and provides a useful model system in which to examine the effects of RGK proteins on native calcium currents. HIT‐T15 cells are purchased from the American Type Culture Collection (Manassas, VA; www.ATCC.org) and cultured in F‐12K nutrient mixture (Gibco) supplemented with 10% (v/v) horse serum (Gibco) that has been dialyzed extensively against 0.15 M NaCl; 2.5% FBS (HyClone) and 55 mg/ml gentamicin (Gibco). Cell Culture, Plating, and Transfection Procedures. In preparation for whole‐cell recordings, HIT‐T15 cells are plated on poly‐L‐lysine–coated glass slips in a 24‐well plate at a density of 20,000 cells per slip. The next day, cells are infected with cesium‐purified adenovirus expressing GFP and Rem or Rem2 at a final concentration of 1 107 adenovirus particles/ml. Twenty‐two hours after infection, RGK effects on calcium current expression may be observed. Infected cells may be identified by expression of GFP. Alternately, HIT‐T15 cells may be transfected with commercial liposomal reagents. Although this cell line is resistant to many transient transfection methods, we have found that Lipofectamine (Invitrogen) works well at a DNA/lipid ratio of 2:5. Rem or Rem2 cloned into the N‐terminal GFP‐expressing vector pEGFP‐C1 may be expressed in this manner, and effects on calcium currents may be observed 48 h after transfection. Solutions for Electrophysiology. For whole‐cell patch clamp recordings, patch pipettes (Harvard Apparatus Ltd., Kent, UK) are pulled as previously to resistances of 1–3 M and contain a solution (in mM) of 110
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K‐gluconate, 40 CsCl, 3 EGTA, 1 MgCl2, 5 Mg‐ATP, and 5 HEPES (pH 7.36). Bath solution consists of (in mM) 102.5 (or 140) CsCl, 40 (or 2.5) BaCl2 or CaCl2, 1 MgCl2, 3 4‐AP, 10 TEA‐Cl, and 5 HEPES (pH 7.4). In contrast to HEK293 cells, overlapping Kþ and Naþ currents necessitate the need for K‐channel blockade by internal Csþ and external TEA, 4‐AP, and Csþ. Electrophysiology. All recordings are performed at room temperature (20–22 ). Signals are amplified with an Axopatch 200B amplifier and 333 kHz A/D system (Axon Instruments, Union City, CA). The data obtained are analyzed with CLAMPFIT 9.2 (Axon) and ORIGIN statistical software (OriginLab, Northampton, MA). The protocol used is identical to that used for examination of Ca2þ and Ba2þ currents from transiently transfected HEK293 cells (as described in detail previously). For cells containing overlapping Na channel current, a 1‐sec conditioning step to 50 mV steady‐state inactivates INa, allowing unimpeded ICa measurement. Acknowledgments We acknowledge W. Peavler for his contributions to these studies. This work was supported in part by United States Public Health Service Grants HL‐072936 (D. A. A.) and HL‐074091 (J. S.), National Institutes of Health Grant P20RR0171 from the COBRE program of the NCRR (to D. A. A.), and by a Predoctoral Fellowship from the American Heart Association, Ohio Valley Affiliate (to R. N. C). J. S. is an Established Investigator of the American Heart Association.
References Andres, D. A., Shao, H., Crick, D. C., and Finlin, B. S. (1997). Expression cloning of a novel farnesylated protein, RDJ2, encoding a DnaJ protein homologue. Arch. Biochem. Biophys. 346, 113–124. Aresta, S., de Tand‐Heim, M. F., Beranger, F., and de Gunzburg, J. (2002). A novel Rho GTPase‐activating‐protein interacts with Gem, a member of the Ras superfamily of GTPases. Biochem. J. 367, 57–65. Beguin, P., Mahalakshmi, R. N., Nagashima, K., Cher, D. H., Takahashi, A., Yamada, Y., Seino, Y., and Hunziker, W. (2005). 14‐3‐3 and calmodulin control subcellular distribution of Kir/Gem and its regulation of cell shape and calcium channel activity. J. Cell Sci. 118, 1923–1934. Beguin, P., Nagashima, K., Gonoi, T., Shibasaki, T., Takahashi, K., Kashima, Y., Ozaki, N., Geering, K., Iwanaga, T., and Seino, S. (2001). Regulation of Ca2þ channel expression at the cell surface by the small G‐protein kir/Gem. Nature 411, 701–706. Catterall, W. A. (2000). Structure and regulation of voltage‐gated Ca2þ channels. Annu. Rev. Cell Dev. Biol. 16, 521–555. Cohen, L., Mohr, R., Chen, Y. Y., Huang, M., Kato, R., Dorin, D., Tamanoi, F., Goga, A., Afar, D., Rosenberg, N., and Witte, O. (1994). Transcriptional activation of a ras‐like gene (kir) by oncogenic tyrosine kinases. Proc. Natl. Acad. Sci. USA 91, 12448–12452. Colicelli, J. (2004). Human RAS superfamily proteins and related GTPases. Sci. STKE 2004 RE13.
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[39]
Finlin, B. S., and Andres, D. A. (1997). Rem is a new member of the Rad‐ and Gem/Kir Ras‐ related GTP‐binding protein family repressed by lipopolysaccharide stimulation. J. Biol. Chem. 272, 21982–21988. Finlin, B. S., and Andres, D. A. (1999). Phosphorylation‐dependent association of the Ras‐ related GTP‐binding protein Rem with 14‐3‐3 proteins. Arch. Biochem. Biophys. 368, 401–412. Finlin, B. S., Crump, S. M., Satin, J., and Andres, D. A. (2003). Regulation of voltage‐gated calcium channel activity by the Rem and Rad GTPases. Proc. Natl. Acad. Sci. USA 100, 14469–14474. Finlin, B. S., Mosley, A. L., Crump, S. M., Correll, R. N., Ozcan, S., Satin, J., and Andres, D. A. (2005). Regulation of L‐type Ca2þ channel activity and insulin secretion by the Rem2 GTPase. J. Biol. Chem. Feb.22 [Epub ahead of print]. Finlin, B. S., Shao, H., Kadono‐Okuda, K., Guo, N., and Andres, D. A. (2000). Rem2, a new member of the Rem/Rad/Gem/Kir family of Ras‐related GTPases. Biochem. J. 347(Pt. 1), 223–231. Fischer, R., Wei, Y., Anagli, J., and Berchtold, M. W. (1996). Calmodulin binds to and inhibits GTP binding of the ras‐like GTPase Kir/Gem. J. Biol. Chem. 271, 25067–25070. Guan, K. L., and Dixon, J. E. (1991). Eukaryotic proteins expressed in Escherichia coli: An improved thrombin cleavage and purification procedure of fusion proteins with glutathione S‐transferase. Anal. Biochem. 192, 262–267. He, T. C., Zhou, S., da Costa, L. T., Yu, J., Kinzler, K. W., and Vogelstein, B. (1998). A simplified system for generating recombinant adenoviruses. Proc. Natl. Acad. Sci. USA 95, 2509–2514. Maguire, J., Santoro, T., Jensen, P., Siebenlist, U., Yewdell, J., and Kelly, K. (1994). Gem: An induced, immediate early protein belonging to the Ras family. Science 265, 241–244. Mattingly, R. R., Sorisky, A., Brann, M. R., and Macara, I. G. (1994). Muscarinic receptors transform NIH 3T3 cells through a Ras‐dependent signalling pathway inhibited by the Ras‐GTPase‐activating protein SH3 domain. Mol. Cell. Biol. 14, 7943–7952. Moyers, J. S., Bilan, P. J., Zhu, J., and Kahn, C. R. (1997). Rad and Rad‐related GTPases interact with calmodulin and calmodulin‐dependent protein kinase II. J. Biol. Chem. 272, 11832–11839. Moyers, J. S., Zhu, J., and Kahn, C. R. (1998). Effects of phosphorylation on function of the Rad GTPase. Biochem. J. 333, 609–614. Pan, J. Y., Fieles, W. E., White, A. M., Egerton, M. M., and Silberstein, D. S. (2000). Ges, A human GTPase of the Rad/Gem/Kir family, promotes endothelial cell sprouting and cytoskeleton reorganization [published erratum appears in J. Cell Biol. 2000 Jul 24; 150(2): following 401]. J. Cell Biol. 149, 1107–1116. Piddini, E., Schmid, J. A., de Martin, R., and Dotti, C. G. (2001). The Ras‐like GTPase Gem is involved in cell shape remodelling and interacts with the novel kinesin‐like protein KIF9. EMBO J. 20, 4076–4087. Reuther, G. W., and Der, C. J. (2000). The Ras branch of small GTPases: Ras family members don’t fall far from the tree. Curr. Opin. Cell Biol. 12, 157–165. Reynet, C., and Kahn, C. R. (1993). Rad: A member of the Ras family overexpressed in muscle of type II diabetic humans. Science 262, 1441–1444. Ward, Y., Spinelli, B., Quon, M. J., Chen, H., Ikeda, S. R., and Kelly, K. (2004). Phosphorylation of critical serine residues in gem separates cytoskeletal reorganization from down‐regulation of calcium channel activity. Mol. Cell. Biol. 24, 651–661. Ward, Y., Yap, S. F., Ravichandran, V., Matsumura, F., Ito, M., Spinelli, B., and Kelly, K. (2002). The GTP binding proteins Gem and Rad are negative regulators of the Rho‐Rho kinase pathway. J. Cell Biol. 157, 291–302.
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[39] Analyses of Rem/RGK Signaling and Biological Activity By DOUGLAS A. ANDRES, SHAWN M. CRUMP, ROBERT N. CORRELL, JONATHAN SATIN , and BRIAN S. FINLIN Abstract
Rem (Rad and Gem related) is a member of the RGK family of Ras‐ related GTPases that also includes Rad, Rem2, and Gem/Kir. All RGK proteins share structural features that are distinct from other Ras‐related proteins, including several nonconservative amino acid substitutions within regions known to participate in nucleotide binding and hydrolysis and a C‐terminal extension that contains regulatory sites that seem to control both subcellular location and function. Rem is known to modulate two distinct signal transduction pathways, regulating both cytoskeletal reorganization and voltage‐gated Ca2þ channel activity. In this chapter, we summarize the experimental approaches used to characterize the interaction of Rem with 14‐3‐3 proteins and Ca2þ channel ‐subunits and describe electrophysiological analyses for characterizing Rem‐mediated regulation of L‐type Ca2þ channel activity.
Introduction
The Ras subfamily of GTPases consists of approximately 35 members that share a high degree of amino acid conservation with H‐, K‐, and N‐ Ras, particularly within the their GTP‐binding/GTPase core (Colicelli, 2004). However, it is now clear that members of the family possess distinct biochemical and biological activities and express both overlapping and unique functions (Colicelli, 2004; Reuther and Der, 2000). Rem (Rad and Gem/Kir‐related) was originally identified as the product of polymerase chain reaction amplification (PCR) using oligonucleotide primers derived from conserved regions of the Rad and Gem/Kir GTPases (Finlin and Andres, 1997) and, together with Rem2 (Finlin et al., 2000), serves as the newest members of the RGK (Rem, Rem2, Rad, Gem/Kir) family of Ras‐ related GTP binding proteins (Cohen et al., 1994; Finlin and Andres, 1997; Finlin et al., 2000; Maguire et al., 1994; Reynet and Kahn, 1993). Despite the similarities between Rem and Ras, RGK proteins share several unique characteristics. These include nonconservative amino acid substitutions within regions known to be involved in guanine nucleotide binding and METHODS IN ENZYMOLOGY, VOL. 407 Copyright 2006, Elsevier Inc. All rights reserved.
0076-6879/06 $35.00 DOI: 10.1016/S0076-6879(05)07039-4
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hydrolysis, particularly a RGK signature DXWE G3 domain that replaces the DXXG motif (involved in nucleotide hydrolysis) found among most Ras superfamily proteins. Rem also contains a unique effector domain when compared with the larger Ras superfamily (Colicelli, 2004; Finlin and Andres, 1997). Surprisingly, Rem also differs from other RGK proteins within this putative effector domain, suggesting that each RGK protein may interact with distinct regulatory and effector proteins (Finlin et al., 2000). To date, no GTP‐dependent ‘‘classical’’ effectors for Rem have been identified, although several Rem binding proteins have been characterized (see the following). RGK proteins contain large N‐terminal and C‐terminal extensions relative to other Ras family proteins. These extensions contain multiple phosphorylation sites (Finlin and Andres, 1999; Maguire et al., 1994; Moyers et al., 1997, 1998; Ward et al., 2004), a C‐terminal calmodulin (CaM) binding domain in Rad and Gem (Beguin et al., 2005; Fischer et al., 1996; Moyers et al., 1997; Ward et al., 2004), and 14‐3‐3 binding sites (Beguin et al., 2005; Finlin and Andres, 1999; Ward et al., 2004). Protein phosphorylation and CaM/14‐3‐3 association have been proposed to play a role in regulating RGK function. RGK proteins do not have traditional lipid modification motifs at the C‐terminus, which are important for membrane anchorage of other Ras‐related GTPases. However, all RGK proteins contain a conserved 10 amino acid domain that includes a cysteine residue at position ‐7 from their C‐terminus that may serve as a unique modification site. Finally, RGK proteins are subject to transcriptional regulation and exhibit tissue‐ restricted expression patterns. Rem is expressed prominently in cardiac muscle but also in the lung, skeletal muscle, and kidney (Finlin and Andres, 1997). The administration of lipopolysaccharide results in a general repression of Rem mRNA levels, making Rem the first Ras family GTPase to be regulated by repression (Finlin and Andres, 1997). At present, questions remain concerning the physiological role of Rem. However, the recent identification of RGK binding proteins has begun to provide insight into the role of this GTPase subfamily. These include studies demonstrating RGK‐mediated regulation of cytoskeletal reorganization in various cell types, including human Rem (also termed Ges) promotion of endothelial cell sprouting (Pan et al., 2000). Although the mechanism of Rem‐mediated morphology change remains to be defined, the identification of Rho kinase (Ward et al., 2002), the kinesin‐like protein KIF‐9 (Piddini et al., 2001), and Gmip, a RhoGAP (Aresta et al., 2002), as Gem/Rad‐binding partners suggests mechanisms of regulating cytoskeletal morphology. However, many of these proteins associate with only a subset of the RGK proteins, and their roles in Rem signaling remain to be established. To date, the only common RGK family binding partners
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are voltage‐gated calcium channel subunits and 14‐3‐3 proteins. Moreover, expression of all members of the RGK GTPase family down regulates voltage‐gated calcium channel activity (Beguin et al., 2001; Finlin et al., 2003, 2005; Ward et al., 2004), leading to the notion that these G proteins provide a novel mechanism for regulating electrical signaling pathways. This chapter summarizes experimental approaches for evaluating Rem function. The first part of this chapter discusses a variety of binding analyses for characterizing the interaction of Rem with 14‐3‐3 proteins, Ca2þ channel ‐subunits, and for the isolation of novel interacting proteins. The second section describes electrophysiological analyses for characterizing Rem‐mediated regulation of L‐type Ca2þ channel activity. Mammalian Expression Vectors for Wild‐Type and Mutant Rem Proteins
The original full‐length Rem sequence encoding wild‐type (GenBank accession numbers U91601 [mouse] and AF084465 [human]) and a series of truncation and point mutants of mouse Rem were introduced into the mammalian vector pCDNA3 3.1zeo (Invitrogen) as described previously (Finlin and Andres, 1997). Expression of Rem in this vector is under control of the strong cytomegalovirus immediate‐early (CMV) promoter. These vectors also encode a zeocin resistance gene, under the control of the simian virus 40 (SV40) promoter, to allow selection in growth medium containing zeocin. pKH3 (Mattingly et al., 1994) and pEGFP‐C1 (Clontech) mammalian expression vectors encoding wild‐type Rem and a series of Rem deletion mutants were also generated (Finlin and Andres, 1999; Finlin et al., 2003). This results in the addition of N‐terminal sequences encoding a hemagglutinin (HA) protein epitope tag or fusion of the green fluorescent protein (GFP) onto the N‐terminus of the Rem protein. Fusions to the N‐terminus of Rem do not affect its biological activity (Finlin et al., 2003). Expression of HA‐Rem or GFP‐Rem proteins in transfected mammalian cells can be detected by Western blot analyses using anti‐HA monoclonal antibody (12CA5) or by use of a fluorescence microscope. We have used these expression vectors for both stable and transient expression analyses. Recombinant adenovirus coexpressing Rem and GFP or a series of Rem deletion mutants with GFP have also been used for Rem expression in difficult to transfect cell lines (Finlin et al., 2003). Rem adenoviral vectors are generated through homologous recombination using the AdEasy system as described previously (He et al., 1998). High‐titer viral stocks are generated by CsCl banding, and infectious titers are determined
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by limiting dilution plaque assay on HEK‐293 cells and used at m.o.i. values of 100–200 (Finlin et al., 2003). In Vitro Biochemical Assays: Identification of Rem Binding Proteins
Generation of Recombinant GST‐Rem We have used GST fusions to Rem for a variety of in vitro biochemical analyses (Finlin and Andres, 1997, 1999). Wild‐type Rem cDNA is introduced into the pGEX‐KG bacterial expression vector (Guan and Dixon, 1991), and bacterially expressed GST‐Rem proteins are isolated by conventional methods for the expression and purification of GST fusion proteins. Escherichia coli strain BL21‐DE3(LysE) (Novagen, Madison, WI) transformed with the various pGEX‐Rem plasmids are grown overnight at 37 to saturation in 2XYT broth supplemented with carbenicillin (50 g/ml) to maintain selection for the plasmid and then diluted 1:100 and grown to the start of log phase (4 h). After reaching an OD600 of 0.6, protein production is induced with 0.5 mM isopropylthio‐‐D‐ galactopyranoside (IPTG) for an additional 4 h. Bacteria are then collected by centrifugation and resuspended in 20 ml GST‐B buffer (10 mM Tris‐HCl [pH 7.4], 1 mM dithiothreitol [DTT], 10 M GDP, 1 mM MgCl2, and 1 mM PMSF) containing lysozyme (0.5 mg/ml), incubated for 15 min on ice, and broken using a French pressure cell. The lysate is diluted with 25 ml of GST‐C buffer (20 mM HEPES (pH 7.6), 100 mM KCl, 20% [v/v] glycerol, 1 mM DTT, 10 M GDP, 1 mM MgCl2, and 1 mM PMSF), cleared by centrifugation (30,000g for 20 min at 4 ), and tumbled at 4 with 1.0 ml of preswollen glutathione‐agarose bead resin (Sigma) for 30 min. The beads are washed three times with 20 ml of ice‐cold GST‐C buffer, the resin transferred to a small gravity flow column (BioRad), and eluted using release buffer (GST‐C buffer containing 25 mM glutathione, pH 7.4). The released protein is dialyzed against four changes (1 l) of TCB buffer (50 mM Tris‐HCl [pH 7.5], 10% [v/v] glycerol, 150 mM NaCl, 1 mM MgCl2, 1 mM DTT) and stored in 100‐g aliquots at 80 until needed. We have used recombinant GST‐Rem proteins for in vitro protein interaction studies (see later). In addition, recombinant Rem has been released from GST by thrombin cleavage and used to determine in vitro nucleotide binding and GTP hydrolysis (Finlin and Andres, 1997). Recombinant GST‐Rem protein has also been generated containing an N‐terminal heart muscle kinase (HMK) phosphorylatable RRASV sequence to allow expression of Rem fusion proteins that can be radiolabeled with 32P for use as a molecular probe to screen an expression library to identify Rem interacting proteins (Finlin and Andres, 1999). Rem cDNA
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sequences are introduced into the pGEX‐KG‐HMK plasmid (a pGEX‐KG derivative containing an HMK recognition site 50 to the BamHI site) to express GST‐HMK‐Rem (Finlin and Andres, 1999). Recombinant GST‐ HMK‐Rem is expressed in BL21DE3 bacteria and purified by glutathione– agarose affinity chromatography and cleaved with thrombin as described previously. This is followed by a kinase reaction in which HMK‐Rem (10 g) is incubated with 2.0 mCi [32P]ATP (6000 Ci/mmol, NEN) and 100 U HMK (Sigma, St. Louis, MO) in 100 l of 20 mM Tris, pH 7.5, 100 mM NaCl, and 12 mM MgCl2 for 30 min on ice. The kinase reaction is stopped by addition of 400 l of stop buffer (10 mM phosphate, 10 mM sodium pyrophosphate, 1 mg/ml bovine serum albumin [BSA]). The probe is then dialyzed against four changes (50 ml each) of dialysis buffer (20 mM Tris, pH 7.5, 100 mM NaCl, 12 mM MgCl2, 10 M GDP) to remove unincorporated label, counted in a scintillation counter, and stored in multiple aliquots at 80 . Radiolabeled Rem proteins have a 2‐week half‐life, so protein is generated as needed. ‘‘Far Western’’ Interaction Cloning to Identify Rem Binding Proteins To identify Rem‐interacting proteins, we have screened a 14‐day‐old mouse embryo lEXlox cDNA library (Novagen, Madison, WI) (Finlin and Andres, 1999). The library is plated at 40,000 plaques/plate (150 mm) by infection of BL21(DE3) bacteria. Once plaques reach 0.5–1 mm in size, the infected bacterial plates are overlaid with nitrocellulose filters and incubated overnight at 4 . The plates are then placed at 37 and incubated for another 4 h, and the primary filters are removed immediately and placed in Hyb75 (20 mM HEPES, pH 7.6, 75 mM KCl, 0.1 mM EDTA, 2.5 mM MgCl2, 1 mM DTT, 0.05% NP‐40) (Finlin and Andres, 1999). Secondary filters are generated by overlaying the plates with a second set of nitrocellulose filters and incubating for 4 h at 37 . These filters are immediately combined with the primary membranes in Hyb75. The filters are then blocked in Hyb75 with 1% nonfat milk for 4 h at 4 . Bacterial extract containing recombinant HMK‐GST is prepared from BL21DE3 cells transformed with pGEX‐KG‐HMK as follows. The bacteria are grown at 37 in LB medium to an A600 ¼ 0.6. Protein production is induced with 0.5 mM IPTG for 4 h. The bacteria are pelleted, resuspended in Hyb75, and broken using a French pressure cell, and the 100,000g cleared supernatant is used as a supplemental blocking agent. Library filters are incubated with Hyb75 containing 250 mM KCl, 1% nonfat milk, 400 g/ml HMK‐GST bacterial extract, 10 M GDP, and 200,000 cpm/ml [32P]HMK‐Rem probe for 16 h at 4 with shaking. Filters are washed four times at 4 with washing buffer (Hyb75 supplemented with 10 M GDP) and exposed to film for 4 h at room
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FIG. 1. Identification of 14‐3‐3 interaction with Rem by ‘‘Far‐Western’’ interaction cloning. A 14‐day‐old mouse embryo lEXlox cDNA expression library was plated at 4 104 plaques/filter and subjected to [32P]HMK‐Rem interaction screening. After extensive washing, filters were subjected to autoradiography (Kodak X‐OMAT film for 4 h at room temperature) and positive plaques selected and rescreened to allow the isolation of a single purified plaque. (A) Representative filter from the primary screen. (B) A representative filter after two rounds of plaque purification.
temperature. Positive clones are amplified and purified to tertiary clones (Fig. 1). Rem was found to interact strongly with a series of 14–3‐3 proteins, including the ", , , and 14‐3‐3 proteins (Finlin and Andres, 1999). Interaction with Rem Binding Proteins
We have used a variety of in vitro Rem‐binding assays to characterize the association of Rem with 14‐3‐3 proteins and the accessory ‐subunits of voltage‐gated calcium channels. We describe two standard methods of analysis: in vitro pull‐down assays to examine the interaction of bacterially expressed Rem with recombinant 14‐3‐3 or ‐subunits and coimmunoprecipitation analysis. In Vitro Binding Reactions Radiolabeled full‐length or truncation mutants of CaV 2a subunit are prepared by in vitro transcription and translation in the presence of [35S] methionine and examined for their ability to associate in a nucleotide‐ dependent fashion with GST‐Rem (Fig. 2). All manipulations are carried out at 4 . Glutathione‐sepharose beads (Pharmacia) (10 l) are washed with 500 l EDTA buffer (50 mM Tris, pH 7.5, 100 mM NaCl, 0.05% Tween‐20, 0.1 mM DTT, 1 mM EDTA) and resuspended in 1 ml EDTA buffer containing either GST (10 g) or GST‐Rem (10 g). The beads are incubated for 5 min with end‐over‐end rotation at 4 to allow GST fusion protein binding and then washed with 1 ml EDTA buffer to remove
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FIG. 2. In vitro association of Rem with CaV2a. Association of in vitro‐translated [35S]‐ 1‐355 (a short C‐terminal deletion mutant of CaV2a) with full‐length Rem but not labeled 2a GST alone, using an in vitro pull‐down assay. Rem‐mediated CaV2a binding to glutathione‐ sepharose resin was analyzed by resolving the bound fraction to 10% SDS‐PAGE and the dried gel subjected to autoradiography for 16 h.
unbound GST proteins and then incubated with either 1 ml GDP buffer (50 mM Tris, pH 7.5, 100 mM NaCl, 0.05% Tween‐20, 0.1 mM DTT, 10 mM MgCl2, 20 M GDP), 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 M GTPS) to facilitate nucleotide exchange or with EDTA buffer (to generate nucleotide‐free Rem). The GST‐Rem fusion proteins are stored in the presence of MgCl2 (see preceding), so EDTA washing is required to ensure that all Mg2þ is chelated. To examine nucleotide‐dependent ‐subunit binding, 76 l of either EDTA or GDP or GTP buffer is added to the washed pellet, and binding is initiated by the addition of 4 l [35S]2a subunit and incubated for 3 h with end‐over‐end rotation at 4 . The beads are then washed three times with the appropriate binding buffer, and bound [35S]2a is eluted from the beads with two 20‐l washes of assay buffer containing 25 mM glutathione. The eluted proteins are resolved on 10% SDS‐PAGE gels that is dried and exposed to film for 16–72 h. Coimmunoprecipitation Analysis HEK293 Cell Culture. HEK 293 cells are maintained in Dulbecco’s modified Eagle’s medium containing 5% (v/v) fetal bovine serum and 55 g/ml gentamicin and maintained at 37 in a humidified 5% CO2 atmosphere. Cell stocks are generated by plating 106 cells/100‐mm‐diameter tissue culture dish. Cells are fed every 48 h for 4–5 days (or until cells appear 70% confluent). Cells are then trypsinized as follows: growth medium is removed, the dish washed with 10 ml of phosphate‐buffered saline (PBS), and 1 ml of 0.05% (w/v) trypsin‐0.53 mM EDTA is added for 2–4 min or until cells can be readily dislodged. Cells are then pooled, pelleted at 800 rpm in a tabletop centrifuge for 5 min at room temperature,
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and then resuspended and frozen in 1 ml of ice‐cold freezing medium (95% [v/v] fetal bovine serum, and 5% dimethyl sulfoxide [DMSO]) per dish. Vials are placed in a polystyrene box and incubated overnight at 80 for gentle freezing before being transferred to liquid nitrogen for long‐term storage. Cells stocks are recovered by rapid thawing in a 37 water bath, diluted in warm culture medium, and then divided onto 3 100‐mm dishes. Once cells reach 70–80% confluence (3–5 days depending on the initial cell density), they are passaged by reseeding 2 106 cells/10‐mm dish. For expression studies, 100‐mm dishes are coated with polylysine (4 ml of 25 g/ml in PBS per 100‐mm plate). Cells are then plated at 107 cells/100‐mm dish and used the following day for transfection. HEK293 Transfection Protocol. REAGENTS FOR TRANSIENT TRANSFECTION HBS (2): Add the following to 900 ml of deionized water: 10 g of tissue culture‐tested HEPES and 16 g of NaCl. Adjust to pH 7.10 0.05 with 1 M NaOH, adjust the volume to 1 l, and sterilize by filtration. The correct pH is critical for good DNA precipitation. Phosphate (50): Combine 70 mM Na2HPO4 with 70 mM NaH2PO4 (1:1) and sterilize by autoclaving. Calcium chloride: Prepare 100 ml of 2 M CaCl2 and filter sterilize. Plasmid DNA: Plasmid DNA can be prepared by the standard alkaline lysis procedure and purified by banding twice in CsCl density gradients or by using a variety of commercial ion‐exchange column methods. DNA purity is a crucial factor in maintaining high transfection efficiency. Monolayers of HEK 293 cells are transiently transfected with 20 g of mammalian expression plasmid (pCDNA 3.1zeo) encoding hemagglutinin (HA) epitope–tagged Rem DNA/100‐mm dish using the calcium phosphate technique as described previously (Andres et al., 1997). After 48 h of recovery, stable cell lines may be generated by placing the cells under drug selection (250 mg/ml Zeocin) (Invitrogen) (Finlin and Andres, 1999). However, the Rem 14‐3‐3 interaction may also be demonstrated by transient transfection in this cell line. Rem is phosphorylated in vivo when expressed in HEK293 cells, which eliminates the necessity of kinase treatment (Finlin and Andres, 1999). Two days after transfection, the cells are harvested, resuspended in IPA buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 1% NP‐40, 1 mM PMSF), lysed by sonication, and a 100,000g supernatant (S100) is prepared. For analysis of 14–3‐3 coimmunoprecipitation, 1 mg of each S100 lysate is incubated with 20 l of a 50% slurry of protein G covalently crosslinked to antihemagglutinin (HA) monoclonal antibody (12CA5) in a 300‐l reaction for 2 h at 4 with gentle rotation. Immune
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complexes are pelleted, washed 3 1 ml IPA, and resolved by SDS‐PAGE as described (see preceding). 14‐3‐3 proteins are detected by immunoblot analysis using pan‐reactive rabbit anti‐14‐3‐3 antibody (S.C. 629; Santa Cruz). The in vitro assay may also be used to examine the interaction of bacterially expressed recombinant His6‐14‐3‐3 with HA‐Rem or Rem variant proteins (Finlin and Andres, 1999). Transiently transfected HEK 293 cell lysate (40 g) is incubated with 2 g of the His6‐tagged 14‐3‐3 proteins and HA‐Rem binding assessed by immunoblotting. Specific binding may be demonstrated by competition experiments in which 1–10 g of recombinant 14‐3‐3 (lacking the His6 epitope) is preincubated with the cell lysate for 1 h on ice before addition of His6‐14‐3‐3. REM‐CAV SUBUNIT INTERACTIONS. Hemagglutinin (HA)‐Rem and either Flag‐CaV or empty pFlag vector control are cotransfected into HEK293 cells by the calcium phosphate method (see preceding). Forty‐ eight hours after transfection, the cells are washed with PBS, placed into 1 ml of Verseen (GIBCO), harvested, pelleted, and then suspended in ice‐ cold immunoprecipitation (IP) buffer (20 mM Tris, pH 7.5, 250 mM NaCl, 1% TX‐100, 0.5 mM DTT, 1 protease inhibitor mixture [Calbiochem], 10 mM MgCl2, 10 M GTPS). The cells are lysed, subjected to centrifugation, and 1 mg of the supernatant incubated in a 500‐l reaction containing 10 l of packed Protein G Sepharose (Pharmacia) and 4 g of anti‐Flag M2 monoclonal antibody (Sigma) for 3 h with gentle rotation at 4 . The beads are pelleted, and 5 l of the supernatant is saved for analysis. The beads are then washed three times with 1 ml of IP buffer. The supernatant and bound fractions are resolved on SDS‐PAGE, transferred to nitrocellulose, and subjected to immunoblot analysis. HA‐Rem is detected by immunoblotting as described (Finlin, 2003), except that the biotinylated HA antibody is used at 1 g/ml, and bound protein was detected with streptavidin–horseradish peroxidase (Pierce) (1:40,000 dilution). The blot is subsequently probed for Flag‐CaV2a using anti‐Flag M2 monoclonal antibody (1 g/ml) to confirm the efficiency of immunoprecipitation. Analysis of L‐Type Calcium Channel Function
Voltage‐gated Ca2þ channels play crucial roles in the regulation of intracellular calcium concentrations in a diversity of cell types, including muscle cells, neuroendocrine cells, and neurons. The calcium that enters the cell through these channels serves as a second messenger to regulate a variety of processes including cardiac muscle excitation‐contraction coupling and neurotransmitter release (Catterall, 2000). Recent studies
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indicate that all members of the RGK GTPase family, by means of direct interaction with ‐subunits, serve as regulators of voltage‐gated Ca2þ channels (Beguin et al., 2001; Finlin et al., 2003, 2005; Ward et al., 2004). However, questions remain concerning the mechanism of RGK‐mediated control and how RGK regulation of voltage‐sensitive ion channels may affect excitation‐contraction coupling and calcium‐signaling pathways. The next section details assays for measuring Rem‐mediated regulation of Ca2þ channel function. Examination of RGK Effects on Voltage‐Gated Calcium Channel Activity A number of primary cells and cultured cell lines have been used to analyze voltage‐gated Ca2þ channel function. These have included the examination of native tissues that contain a complex array of ion channel signaling proteins, heterologous expression of channels in a cell lacking endogenous Ca2þ channels (e.g., Xenopus oocytes or human embryonic kidney [HEK293] cells) or cell lines that natively express functional Ca2þ channels (such as rat pheochromocytoma [PC12] cells). Two model systems have been used in our laboratory to analyze Rem‐ dependent calcium channel regulation by means of the whole‐cell configuration of the patch clamp technique. The first examines current through a minimal L‐type calcium channel complex consisting of the pore‐forming CaV subunit and an accessory CaV subunit in the presence of the RGK proteins Rem, Rem2, Rad, or mutants thereof transiently coexpressed in HEK293 cells by means of calcium‐phosphate transfection (Finlin et al., 2003, 2005). HEK293 cells are an excellent model system for examination of RGK effects because they lack endogenous calcium channel complexes, transfect with high efficiency with multiple plasmids, and are easily cultured. The second system used to examine Rem function examines current through voltage‐gated channel complexes endogenous to the Syrian golden hamster pancreatic beta cell line HIT‐T15 in the presence of Rem overexpressing adenovirus. The presence of native channel complexes makes HIT‐T15 cells well suited for examining the effects of RGK proteins on endogenous Ca2þ channel function (Finlin et al., 2005). Electrophysiological Recordings in HEK293 Cells The human embryonic kidney cell line HEK293 does not exhibit measurable endogenous calcium currents by means of whole‐cell patch clamp recording. Introduction of calcium channel components by transfection allows the detailed examination of the effect of Rem protein expression on calcium currents expressed from a variety of pore‐forming and accessory channel subunit combinations in a null‐background system.
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HEK293 cells are purchased from the American Type Culture Collection (Manassas, VA; www.ATCC.org) and cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented with 5% (v/v) fetal calf serum (HyClone). Cell Culture, Plating, and Transfection Procedures. Approximately 12–48 h before patch‐clamp analysis, HEK293 cells plated on poly‐L‐ lysine–coated glass coverslips at a density of 10,000 cells/slip are cotransfected in a 24‐well plate with a minimal calcium channel complex consisting of the pore‐forming CaV1.2, CaV1.3, or CaV3.2, along with the accessory CaV subunit 2A, 1B, or 4A (expressed in the single N‐terminal Flag vector pCMVT7/F2 (Sigma), and either GFP‐Rem (expressed in the N‐terminal enhanced green fluorescent protein [GFP] fusion vector pEGFP‐C1 [BD Biosciences]), or unfused GFP by the calcium phosphate transfection method. Typically, equal concentrations of each plasmid are used for transfection, to a total amount of 1 g of DNA per well/ transfection. It is also possible to use commercial liposomal reagents to transfect HEK293 cells. However, theses reagents are expensive and are not necessary for these assays. HEK293 cells cotransfected with CaV1 and CaV subunits and either GFP‐Rem or empty pEGFP‐C1 plasmid are evaluated for transfection efficiency and transfected cells visualized by fluorescence microscopy. With the exception of Rem cotransfection, all GFP‐positive cells cotransfected with plasmids encoding Ca2þ channel subunits express current (Fig. 3). Solutions for Electrophysiology. For recording barium currents through calcium channels, the external bath solution contains (in mM) 140 NaCl or CsCl, 2.5 (or 40) BaCl2 (or CaCl2), 1 MgCl2, 5 glucose, and 5 HEPES (pH 7.4). The internal recording solution contains (in mM) 110 K‐gluconate, 40 CsCl, 1 MgCl2, 5 Mg‐ATP, 10 EGTA (or Bapta), and 10 HEPES (pH 7.35). The choice and concentration of charge carrier (Ca2þ versus Ba2þ) is dictated by the specific experimental question to be addressed. Single channel conductance is higher and kinetics is slower with Ba2þ, whereas Ca2þ induces Ca2þ‐dependent inactivation. The use of 40 mM charge carrier increases the signal but sometimes leads to unstable whole‐cell recordings. Electrophysiology. Whole‐cell recordings are performed as previously described (Finlin et al., 2003). We select spherical cells with few processes to minimize series resistance and space clamp artifacts. Cell capacitance is typically about 15–25 pF. Quantification of macroscopic Ca2þ currents is complicated by the sporadic phenomenon known as run‐down. Five minutes after obtaining whole‐cell access (determined by the increase of capacitance) is typically sufficient time for equilibration of pipette solution with the cytosolic space. Whole‐cell Ca2þ current (ICa) is then tested for an additional 20 min to evaluate run‐down.
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FIG. 3. Rem or Rem2 prevents or attenuates de novo expression of CaV1.2 ionic current. (A and B) Representative Ba2þ current elicited by a þ5‐mV voltage step from 80 mV for cells expressing CaV1.2 þ 2a þ GFP. (C and D) HEK293 cells co‐transfected with CaV1.2 þ 2a þ GFP‐tagged wild‐type Rem2 or Rem. (E) Current voltage relationships for HEK293 cells transfected with CaV1.2 þ 2a þ GFP (filled squares), CaV1.2 þ 2a þ GFP‐Rem2 (open circles), or CaV1.2 þ 2a þ GFP‐Rem (open triangles). Rem completely inhibits CaV1.2 current expression, and Rem2 expression potently inhibits CaV1.2 current expression.
We use standard whole‐cell patch‐clamp methods to record calcium channel currents from individual transfected HEK293 cells under voltage clamp conditions. Coverslips containing transfected cells are placed in a chamber of 600 l volume. The chamber is then transferred to a stage of an inverted microscope (Warner Instruments, a subsidiary of Harvard Apparatus, Holliston, MA) outfitted with Hoffman modulation contrast and fluorescence optics. Cells are visualized for recording at 400 and assessed for EGFP expression using fluorescence imaging. Injection pipettes are pulled from glass capillary tubes (Warner Instruments, Hamden, CT) to resistances of 1–2 M on a model P‐97 Flaming/ Brown micropipette puller (Sutter Instrument Co., Novato, CA). Tips are flame‐polished using an MF‐830 Microfuge (Narishige, Japan), and Sylgard (Dow‐Corning) is applied to reduce pipette capacitance.
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All recordings are performed at room temperature (20–22 ). Stimulation protocols are generated with CLAMPEX 9.2 (Axon) and data acquired and amplified with an Axopatch 200B patch clamp in combination with a 333kHz A/D system (Axon Instruments, Union City, CA). The data obtained are analyzed with CLAMPFIT 9.2 (Axon) and ORIGIN statistical software (OriginLab, Northampton, MA). To measure Ca2þ (or Ba2þ) currents through voltage‐gated calcium channels, voltage steps of 800 msec duration from a 5‐sec holding potential of 80 mV to voltages between 90 mV and 80 mV at 5‐mV intervals are applied to assess the voltage dependence of channel gating and peak currents (Fig. 3). All currents are expressed as current density (peak inward current divided by whole‐cell capacitance). Electrophysiological Recordings in HIT‐T15 Cells The hamster pancreatic cell line HIT‐T15 is capable of insulin secretion and as such contains CaV1.2 and CaV1.3 channels, along with their associated CaV and CaV2 accessory subunits. Endogenous expression of the voltage‐gated calcium channel complex obviates the need for transient overexpression and provides a useful model system in which to examine the effects of RGK proteins on native calcium currents. HIT‐T15 cells are purchased from the American Type Culture Collection (Manassas, VA; www.ATCC.org) and cultured in F‐12K nutrient mixture (Gibco) supplemented with 10% (v/v) horse serum (Gibco) that has been dialyzed extensively against 0.15 M NaCl; 2.5% FBS (HyClone) and 55 mg/ml gentamicin (Gibco). Cell Culture, Plating, and Transfection Procedures. In preparation for whole‐cell recordings, HIT‐T15 cells are plated on poly‐L‐lysine–coated glass slips in a 24‐well plate at a density of 20,000 cells per slip. The next day, cells are infected with cesium‐purified adenovirus expressing GFP and Rem or Rem2 at a final concentration of 1 107 adenovirus particles/ml. Twenty‐two hours after infection, RGK effects on calcium current expression may be observed. Infected cells may be identified by expression of GFP. Alternately, HIT‐T15 cells may be transfected with commercial liposomal reagents. Although this cell line is resistant to many transient transfection methods, we have found that Lipofectamine (Invitrogen) works well at a DNA/lipid ratio of 2:5. Rem or Rem2 cloned into the N‐terminal GFP‐expressing vector pEGFP‐C1 may be expressed in this manner, and effects on calcium currents may be observed 48 h after transfection. Solutions for Electrophysiology. For whole‐cell patch clamp recordings, patch pipettes (Harvard Apparatus Ltd., Kent, UK) are pulled as previously to resistances of 1–3 M and contain a solution (in mM) of 110
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K‐gluconate, 40 CsCl, 3 EGTA, 1 MgCl2, 5 Mg‐ATP, and 5 HEPES (pH 7.36). Bath solution consists of (in mM) 102.5 (or 140) CsCl, 40 (or 2.5) BaCl2 or CaCl2, 1 MgCl2, 3 4‐AP, 10 TEA‐Cl, and 5 HEPES (pH 7.4). In contrast to HEK293 cells, overlapping Kþ and Naþ currents necessitate the need for K‐channel blockade by internal Csþ and external TEA, 4‐AP, and Csþ. Electrophysiology. All recordings are performed at room temperature (20–22 ). Signals are amplified with an Axopatch 200B amplifier and 333 kHz A/D system (Axon Instruments, Union City, CA). The data obtained are analyzed with CLAMPFIT 9.2 (Axon) and ORIGIN statistical software (OriginLab, Northampton, MA). The protocol used is identical to that used for examination of Ca2þ and Ba2þ currents from transiently transfected HEK293 cells (as described in detail previously). For cells containing overlapping Na channel current, a 1‐sec conditioning step to 50 mV steady‐state inactivates INa, allowing unimpeded ICa measurement. Acknowledgments We acknowledge W. Peavler for his contributions to these studies. This work was supported in part by United States Public Health Service Grants HL‐072936 (D. A. A.) and HL‐074091 (J. S.), National Institutes of Health Grant P20RR0171 from the COBRE program of the NCRR (to D. A. A.), and by a Predoctoral Fellowship from the American Heart Association, Ohio Valley Affiliate (to R. N. C). J. S. is an Established Investigator of the American Heart Association.
References Andres, D. A., Shao, H., Crick, D. C., and Finlin, B. S. (1997). Expression cloning of a novel farnesylated protein, RDJ2, encoding a DnaJ protein homologue. Arch. Biochem. Biophys. 346, 113–124. Aresta, S., de Tand‐Heim, M. F., Beranger, F., and de Gunzburg, J. (2002). A novel Rho GTPase‐activating‐protein interacts with Gem, a member of the Ras superfamily of GTPases. Biochem. J. 367, 57–65. Beguin, P., Mahalakshmi, R. N., Nagashima, K., Cher, D. H., Takahashi, A., Yamada, Y., Seino, Y., and Hunziker, W. (2005). 14‐3‐3 and calmodulin control subcellular distribution of Kir/Gem and its regulation of cell shape and calcium channel activity. J. Cell Sci. 118, 1923–1934. Beguin, P., Nagashima, K., Gonoi, T., Shibasaki, T., Takahashi, K., Kashima, Y., Ozaki, N., Geering, K., Iwanaga, T., and Seino, S. (2001). Regulation of Ca2þ channel expression at the cell surface by the small G‐protein kir/Gem. Nature 411, 701–706. Catterall, W. A. (2000). Structure and regulation of voltage‐gated Ca2þ channels. Annu. Rev. Cell Dev. Biol. 16, 521–555. Cohen, L., Mohr, R., Chen, Y. Y., Huang, M., Kato, R., Dorin, D., Tamanoi, F., Goga, A., Afar, D., Rosenberg, N., and Witte, O. (1994). Transcriptional activation of a ras‐like gene (kir) by oncogenic tyrosine kinases. Proc. Natl. Acad. Sci. USA 91, 12448–12452. Colicelli, J. (2004). Human RAS superfamily proteins and related GTPases. Sci. STKE 2004 RE13.
498
regulators and effectors of small GTPases: Ras family
[39]
Finlin, B. S., and Andres, D. A. (1997). Rem is a new member of the Rad‐ and Gem/Kir Ras‐ related GTP‐binding protein family repressed by lipopolysaccharide stimulation. J. Biol. Chem. 272, 21982–21988. Finlin, B. S., and Andres, D. A. (1999). Phosphorylation‐dependent association of the Ras‐ related GTP‐binding protein Rem with 14‐3‐3 proteins. Arch. Biochem. Biophys. 368, 401–412. Finlin, B. S., Crump, S. M., Satin, J., and Andres, D. A. (2003). Regulation of voltage‐gated calcium channel activity by the Rem and Rad GTPases. Proc. Natl. Acad. Sci. USA 100, 14469–14474. Finlin, B. S., Mosley, A. L., Crump, S. M., Correll, R. N., Ozcan, S., Satin, J., and Andres, D. A. (2005). Regulation of L‐type Ca2þ channel activity and insulin secretion by the Rem2 GTPase. J. Biol. Chem. Feb.22 [Epub ahead of print]. Finlin, B. S., Shao, H., Kadono‐Okuda, K., Guo, N., and Andres, D. A. (2000). Rem2, a new member of the Rem/Rad/Gem/Kir family of Ras‐related GTPases. Biochem. J. 347(Pt. 1), 223–231. Fischer, R., Wei, Y., Anagli, J., and Berchtold, M. W. (1996). Calmodulin binds to and inhibits GTP binding of the ras‐like GTPase Kir/Gem. J. Biol. Chem. 271, 25067–25070. Guan, K. L., and Dixon, J. E. (1991). Eukaryotic proteins expressed in Escherichia coli: An improved thrombin cleavage and purification procedure of fusion proteins with glutathione S‐transferase. Anal. Biochem. 192, 262–267. He, T. C., Zhou, S., da Costa, L. T., Yu, J., Kinzler, K. W., and Vogelstein, B. (1998). A simplified system for generating recombinant adenoviruses. Proc. Natl. Acad. Sci. USA 95, 2509–2514. Maguire, J., Santoro, T., Jensen, P., Siebenlist, U., Yewdell, J., and Kelly, K. (1994). Gem: An induced, immediate early protein belonging to the Ras family. Science 265, 241–244. Mattingly, R. R., Sorisky, A., Brann, M. R., and Macara, I. G. (1994). Muscarinic receptors transform NIH 3T3 cells through a Ras‐dependent signalling pathway inhibited by the Ras‐GTPase‐activating protein SH3 domain. Mol. Cell. Biol. 14, 7943–7952. Moyers, J. S., Bilan, P. J., Zhu, J., and Kahn, C. R. (1997). Rad and Rad‐related GTPases interact with calmodulin and calmodulin‐dependent protein kinase II. J. Biol. Chem. 272, 11832–11839. Moyers, J. S., Zhu, J., and Kahn, C. R. (1998). Effects of phosphorylation on function of the Rad GTPase. Biochem. J. 333, 609–614. Pan, J. Y., Fieles, W. E., White, A. M., Egerton, M. M., and Silberstein, D. S. (2000). Ges, A human GTPase of the Rad/Gem/Kir family, promotes endothelial cell sprouting and cytoskeleton reorganization [published erratum appears in J. Cell Biol. 2000 Jul 24; 150(2): following 401]. J. Cell Biol. 149, 1107–1116. Piddini, E., Schmid, J. A., de Martin, R., and Dotti, C. G. (2001). The Ras‐like GTPase Gem is involved in cell shape remodelling and interacts with the novel kinesin‐like protein KIF9. EMBO J. 20, 4076–4087. Reuther, G. W., and Der, C. J. (2000). The Ras branch of small GTPases: Ras family members don’t fall far from the tree. Curr. Opin. Cell Biol. 12, 157–165. Reynet, C., and Kahn, C. R. (1993). Rad: A member of the Ras family overexpressed in muscle of type II diabetic humans. Science 262, 1441–1444. Ward, Y., Spinelli, B., Quon, M. J., Chen, H., Ikeda, S. R., and Kelly, K. (2004). Phosphorylation of critical serine residues in gem separates cytoskeletal reorganization from down‐regulation of calcium channel activity. Mol. Cell. Biol. 24, 651–661. Ward, Y., Yap, S. F., Ravichandran, V., Matsumura, F., Ito, M., Spinelli, B., and Kelly, K. (2002). The GTP binding proteins Gem and Rad are negative regulators of the Rho‐Rho kinase pathway. J. Cell Biol. 157, 291–302.