Direct Inhibition of the Interaction between (cid:1) -Interaction Domain and (cid:2) -Interaction Domain of Voltage-dependent Ca 2 (cid:3) Channels by Gem*

TheRas-relatedsmallG-proteinGemregulatesvoltage-dependent Ca 2 (cid:3) channels (VDCCs) through interaction with the (cid:2) -subunit of the VDCC. This action of Gem is mediated by regulated (cid:1) 1 -subunit expression at the plasma membrane. In the present study, we examined the mechanism of the inhibition of VDCC activity by Gem. The (cid:2) -interaction domain (BID) of the (cid:2) -subunit, which specifically interacts with the (cid:1) -interaction domain (AID) of the (cid:1) 1 -subunit, is shown to be essential for the interaction between Gem and (cid:2) -subunits. In addition, the AID peptide inhibited interaction between Gem and (cid:2) -subunits in a dose-dependent manner. GemS88N mutant, which has low binding affinity for guanine nucleotide, did not interact with (cid:2) -subunits, allowing (cid:1) 1 subunit expression at the plasma membrane. This inhibitory effect of wild-type Gem on VDCC activity was reduced in cells expressing GemS88N. The overexpression of wild-type Gem in pancreatic (cid:2) -cell line MIN6 cells suppressed Ca 2 (cid:3) -triggered secretion, whereas overexpression of GemS88N induced Ca 2 (cid:3) -triggered secretion to control level. These results suggest that GTPase activity of Gem is required for the binding of Gem to BID that regulates VDCC activity through interaction with AID.

Coexpression of a ␤-subunit with an ␣ 1 -subunit results in an increase of peak current amplitude and an increase in the number of binding sites for drugs and toxins, acceleration of activation and inactivation kinetics, and an increase the number of ␣ 1 -subunits in plasma membrane (16 -19). The effects of ␤-subunits are suggested to be mediated by interaction with the intracellular I-II loop of ␣ 1 -subunits (20 -22). The I-II loop of the ␣ 1 -subunit includes the endoplasmic reticulum retention signal and inhibits trafficking of ␣ 1 -subunits to the plasma membrane. The interaction of the loop with the ␤-subunit masks the retention signal in the loop, leading to channel expression at the plasma membrane (23). ␤-Subunits function as chaperone-like molecules in the trafficking of ␣ 1 -subunits to plasma membrane (17). ␣ 1 -and ␤-subunits interact with each other at the ␣-interaction domain (AID) and the ␤-interaction domain (BID), respectively (see Fig. 1A) (20,24). AID comprises a minimal 18 amino acids in the I-II loop of the ␣-subunit. BID comprises the amino-terminal 30 amino acids of the second conserved domain of the ␤-subunit.
Gem was identified originally in mitogen-induced peripheral blood T cells (25) and oncogenic tyrosine kinase-transformed cells (26). Gem is a member of the RGK (Rem, Rad, and Gem/ kir) family consisting of a long amino-terminal region, a Rasrelated core domain, and a carboxyl-terminal region lacking a CAAX motif (27,28). Subcellular localization of Gem requires interaction of its carboxyl-terminal region with Ca 2ϩ /calmodulin. We previously reported that Gem inhibits the trafficking of the ␣ 1 -subunits of N, P/Q, and L-type VDCCs to the plasma membrane by interacting with the ␤-subunits (29), inhibiting channel activities. Inhibition of cell surface expression also is involved in the interaction of the Gem carboxyl terminus with Ca 2ϩ /calmodulin. Recently, it has been shown that Rad and Rem, members of the RGK family, also inhibit VDCC activity by binding to the ␤-subunit (30).
In the present study, we examined the mechanism of regu-lation of VDCC activity by Gem. We find that Gem inhibits VDCC activity through its interaction with BID and that the binding of guanine nucleotide to Gem is required for expression of ␣ 1 -subunits at plasma membrane, VDCC activity, and Ca 2ϩtriggered hormone secretion. Because the RGK proteins Rad, Rem, and Gem all share conserved structural features (25-28), a common mechanism may exist in the regulation of VDCC activities by small G-proteins.
Recombinant Proteins-Mouse Gem was expressed as glutathione S-transferase (GST) fusion protein and purified by affinity chromatography with glutathione-resin (Amersham Biosciences Corp.). AID peptides (amino acid residues 458 -475) in the I-II loop of rabbit ␣ 1 1.2subunit were synthesized and purified using reverse-phase chromatography.
Electrophysiology-The whole-cell VDCC currents in PC12 cells were recorded as described (29). Briefly, Ba 2ϩ was used as a charged carrier for measurement of VDCC currents. The extracellular solution contained 40 mM Ba(OH) 2 , 20 mM 4-aminopyridine, 110 mM tetraethylammonium hydroxide, 10 mM tetraethylammonium chloride, 140 mM methanesulfonate, and 10 mM MOPS, pH 7.4. The pipette solution contained 10 mM cesium chloride, 130 mM cesium aspartate, 10 mM EGTA, 10 mM MOPS, and 5 mM Mg-ATP, pH 7.2. Cells were maintained at a holding potential of Ϫ60 mV. For recording VDCC currents, square pulses of 400-ms duration at potentials between Ϫ40 and ϩ60 mV in steps of 10 mV were applied every 4 s. Currents were normalized by dividing by the membrane capacitance for each cell (31). Recordings were performed using the Axopatch200B amplifier (Axon Instruments, Foster City, CA).
Measurement of Growth Hormone (GH) Secretion-MIN6 cells were cotransfected with pXGH5-human (h)GH (Nichols Institute, San Juan Capistrano, CA) and pSR␣-wild-type Gem or pSR␣-GemS88N. As a control, the cells were cotransfected with pXGH5 and pSR␣-␤-galactosidase. One day after transfection, MIN6 cells were harvested and split to a 12-well plate at a density of 5.0 ϫ 10 5 cells/well. Two days after transfection, MIN6 cells were washed three times with KRB G1.0 (140 mM NaCl, 4.7 mM KCl, 1.2 mM KH 2 PO 4 , 1.2 mM MgSO 4 , 5.0 mM NaHCO 3 , 2.5 mM CaCl 2 , 10 mM HEPES, pH7.4, 0.1% bovine serum albumin, and 1.0 mM glucose) and then preincubated at 37°C for 30 min. After washing with KRB G1.0, the cells were incubated at 37°C for 10 min with a high K ϩ solution (KRB G1.0 containing 60 mM KCl and 85 mM NaCl) or a low K ϩ solution (KRB G1.0 containing 4.7 mM KCl and 140 mM NaCl). Amounts of hGH released into medium were measured using enzyme-linked immunosorbent assay kit (Roche Applied Science). Secretion was expressed as percent of the amount of hGH released into medium relative to the total cellular GH amounts (32).
Immunofluorescence Staining and Confocal Microscopy-The tsA201 cells were transfected with FLAG-tagged ␣ 1 1.2-subunit, ␤ 3 -subunit, and wild-type Gem or GemS88N. The transfected cells were fixed with 4% paraformaldehyde, followed by permeabilization with 0.2% Triton X-100, and incubated with 10% normal goat serum in phosphate-buffered saline. The cells were incubated at room temperature for 60 min with anti-FLAG M2 antibody. As the secondary antibody, Cy3-conjugated goat anti-mouse IgG antibody (Chemicon International, Temecula, CA) was used. The cells were mounted on cover slips using PermaFluor (Immunon Pittsburgh, CA) and observed using LSM510-Ver3.0 laser scanning microscope (Carl Zeiss Co., Ltd.).

RESULTS
Binding of Gem to ␤ 3 -Subunit of VDCC through BID in ␤ 3 -Subunit-It has been shown that the BID of ␤-subunits participates in modulation of VDCC activity through interaction with AID in ␣-subunits (24). We investigated the involvement of BID in the interaction between Gem and the ␤ 3 -subunit. FLAG-tagged wild-type ␤ 3 -subunit, FLAG-tagged ␤ 3subunit mutant lacking BID (␤ 3 ⌬BID), EGFP-tagged AID, and Myc-tagged Gem (Fig. 1A) were expressed in COS-1 cells. FLAG-tagged ␤ 3 ⌬BID did not bind to EGFP-tagged AID, as reported previously (Fig. 1B) (24). In addition, FLAG-tagged ␤ 3 ⌬BID did not bind to Myc-tagged Gem (Fig. 1C), suggesting that Gem specifically binds to BID in the ␤ 3 -subunit.
Because both AID and Gem bind to BID in the ␤ 3 -subunit, we investigated AID inhibition of the binding of Gem and BID. As shown in Fig. 1D, the binding of GST-tagged Gem and FLAGtagged ␤ 3 -subunit was inhibited in an AID peptide dose (0 -1 g)-dependent manner, suggesting that the inhibitory effect of Gem on the trafficking of ␣ 1 -subunits to the plasma membrane is because of its blocking of the interaction between BID and AID.
Requirement of Guanine Nucleotide Binding of Gem for Interaction between Gem and ␤ 3 -Subunit-Activation of small G-proteins leads to stimulation of the downstream pathway through effector proteins. Because the RasS17N mutant, which has low binding affinity for the guanine nucleotide, cannot bind to Ras effector proteins and functions in a dominant negative manner, the mutant has been used to investigate the effect of GTP-bound form (active form) of Ras on its physiological functions (33). By analogy to RasS17N mutant, we constructed Gem mutant in which Ser at 88 was substituted by Asn (GemS88N), as described previously (34,35). The mutant has been shown to have reduced guanine nucleotide-binding affinity (35). We investigated to find out whether the guanine nucleotide-binding of Gem is required for binding Gem and the ␤ 3 -subunit using GemS88N. GST-tagged GemS88N and FLAG-tagged ␤ 3 -subunit expressed in COS-1 cells was subjected to an in vitro binding assay. The binding of GST-tagged GemS88N to the FLAG-tagged ␤ 3 -subunit was significantly decreased compared with that of wild-type Gem to FLAG-tagged ␤ 3 -subunit ( Fig.  2A). In COS-1 cells coexpressing Myc-tagged GemS88N and FLAG-tagged ␤ 3 -subunit, Myc-tagged GemS88N did not bind to FLAG-tagged ␤ 3 -subunit (Fig. 2B). These results suggest that guanine nucleotide-binding of Gem is required for the interaction between Gem and the ␤ 3 -subunit.
Subcellular Localization of ␣ 1 1.2-Subunit in tsA201 Cells Expressing Wild-type Gem and GemS88N-We investigated to find out whether the guanine nucleotide-binding of Gem participates in inhibition of ␣ 1 -subunits at cell surface expression. Subcellular localization of the FLAG-tagged ␣ 1 1.2-subunit in tsA201 cells expressing GemS88N, FLAG-tagged ␣ 1 1.2-subunit, and ␤ 3 -subunit were analyzed by confocal microscopy. In cells expressing FLAG-tagged ␣ 1 1.2-subunit alone, the ␣ 1 1.2subunit was not localized to the plasma membrane (Fig. 3A). In cells expressing the FLAG-tagged ␣ 1 1.2-subunit and ␤ 3 -subunit, the ␣ 1 1.2-subunit was localized to plasma membrane (Fig.  3B). When wild-type Gem was expressed together with the FLAG-tagged ␣ 1 1.2-subunit and ␤ 3 -subunit, the ␣ 1 1.2-subunit was not localized to plasma membrane (Fig. 3C), as described previously (29). However, when GemS88N was expressed together with the FLAG-tagged ␣ 1 1.2-subunit and ␤ 3 -subunit, the ␣ 1 1.2-subunit was mainly localized to plasma membrane (Fig. 3D). No effects of GemS88N on the subcellular localization of the ␣ 1 1.2-subunit were found in any of the transfected tsA201 cells.
Effect of GemS88N on VDCC Activity and Hormone Secretion-To elucidate the effect of guanine nucleotide-binding of Gem on inhibition of VDCC activity, we examined the effects of Gem in PC12 cells, in which Ca 2ϩ -induced exocytosis is triggered through the activation of L-type VDCC (36). In PC12 cells expressing wild-type Gem or GemS88N, the I-V relationship of VDCC was measured (Fig. 4). The overexpression of wild-type Gem in PC12 cells resulted in a significant inhibition of endogenous Ca 2ϩ channel currents, whereas overexpression of the GemS88N had only a small inhibitory effect (Fig. 4).
Overexpression of wild-type Gem in PC12 cells and MIN6 cells prevents Ca 2ϩ -triggered exocytosis through inhibition of VDCC activity (29). We examined the effect of GemS88N on Ca 2ϩ -triggered exocytosis in MIN6 cells. For this purpose, we utilized MIN6 cells transfected with hGH (32) and monitored secretion by measuring the hGH release from MIN6 cells transfected with GemS88N under high K ϩ stimulation. The overexpression of wild-type Gem in MIN6 cells inhibited hGH secretion from 10.6 Ϯ 0.6% of total (control, MIN6 cells overexpressing ␤-galactosidase) to 7.9 Ϯ 0.8% of total. In contrast, in MIN6 cells overexpressing GemS88N, the hGH secretion (11.5 Ϯ 0.9% of total) was comparable with the control level (Fig. 5). These results suggest that inhibitory effects of Gem on both VDCC activity and hormone secretion are involved in its guanine nucleotide-binding.

DISCUSSION
The ␤-subunits of VDCCs are critical in determining the properties of the channels including modification of kinetics, amplitude, and trafficking of ␣ 1 -subunits to the plasma membrane (16 -19). We previously reported that interaction of ␤-subunits with Gem prevents the trafficking of ␣ 1 -subunits to the plasma membrane in the regulation of VDCC activity (29). It also has been found that infection of guinea pig heart with adenovirus carrying Gem markedly decreased L-type VDCC density at the sarcolemma of isolated ventricular myocytes (37). However, the mechanism of the inhibition of VDCC activity by Gem was not known. BID in ␤-subunits has been shown to interact with AID in the I-II loop of ␣-subunits in the modulation of VDCC activity (24). We found that BID also is required for interaction with Gem and that the AID peptide inhibits the binding of Gem to the ␤-subunits. These findings suggest a competitive interaction between Gem and the ␣ 1subunits at the BID site. In addition to BID, the ␤ 3 -subunit contains Src homology 3 (SH3) and guanylate kinase domains (38). The tandem arrangement of these domains is characterized in membrane-associated guanylate kinase, which serves as the scaffold proteins that organize the signaling pathway (39). These motifs are required in the modulation of VDCC activity (40). The finding that the mutant ␤ 3 -subunit (␤ 3 ⌬BID) lacking BID but containing both SH3 and guanylate kinase domains cannot bind to Gem indicates that neither the SH3 nor the guanylate kinase domain is required for the inhibitory effect of Gem on VDCC activity.
The GTP-bound form (active form) of small G-proteins binds with high affinity to a set of effector proteins that initiate distinct intracellular signaling cascades. The active form of Gem specifically interacts with the ␤-subunits in vitro (29). We found that mutant GemS88N, which has reduced guanine nucleotide-binding affinity, did not bind to the ␤ 3 -subunit and had no inhibitory effect on the trafficking of ␣ 1 -subunits to the plasma membrane. These findings suggest that the GTP form of Gem is required for its inhibitory effect on interaction of the ␣ 1 -and ␤-subunits. Indeed, overexpression of wild-type Gem in PC12 cells inhibited endogenous VDCC currents, whereas overexpression of mutant GemS88N reduced them only slightly.
We also examined the effect of mutant GemS88N in MIN6 cells, in which insulin granule exocytosis is triggered by Ca 2ϩ influx through activation of L-type VDCCs (41), and found that Ca 2ϩ -triggered hormone secretion was inhibited in MIN6 cells expressing wild-type Gem, whereas secretion in MIN6 cells expressing GemS88N was not inhibited. This suggests that cycling between the GTP-and GDP-form is important in the effect of Gem on Ca 2ϩ -triggered hormone secretion most likely through regulation of the activity of the VDCCs.
NM23, which was identified as GTPase-activating protein and guanine nucleotide exchange factor (GEF) for Rad, acts as Gem-GEF in vitro (42). It has been shown that NM23 inhibits Rad-induced growth and tumorigenicity of breast cancer in breast tissues (43). However, it is not known whether NM23 activates Gem in intact cells. The GTPase-activating protein and GEF for Gem, which have yet to be identified, may play important roles in Gem-mediated cellular functions. Gem interacts with the small G-protein Rho effector, Rho kinase, and negatively regulates cytoskeletal organization mediated by Rho/Rho kinase signaling (44). Rho/Rho kinase signaling has been shown to modulate the translocation of plasma membrane proteins including Na ϩ ,K ϩ -ATPase and water channel aquaporin-2 (45,46). Although the effect of Rho kinase on the interaction between Gem and the ␤-subunits is not clear, translocation to the plasma membrane of ␣ 1 -subunits regulated by Gem/␤-subunits may well be involved in Rho/Rho kinase signaling.
Recently, it has been shown that Rad and Rem, members of the RGK family, also regulate VDCC activity through interaction with the ␤-subunits of the VDCCs (30). As RGK proteins Rad, Rem, and Gem share conserved structural features (25)(26)(27)(28), a similar mechanism may well be involved in regulation of VDCC activities by these small G-proteins. were incubated in buffer (1 mM glucose ϩ 4.7 mM K ϩ ). After 30 min, the cells were stimulated with high K ϩ buffer (1 mM glucose ϩ 60 mM K ϩ ). GH secretion by high K ϩ stimulation in MIN6 cells was measured. Values are expressed as secreted hGH/total contents (%). Open column, stimulation with 1 mM glucose ϩ 4.7 mM K ϩ ; filled column, stimulation with 1 mM glucose ϩ 60 mM K ϩ . Values are mean Ϯ S.E. from five independent experiments (n ϭ 20). *, p Ͻ 0.05; NS, not significant.