Spatial association of the Cav 1 . 2 calcium channel with 5 1-integrin

Chao J, Gui P, Zamponi GW, Davis GE, Davis MJ. Spatial association of the Cav1.2 calcium channel with 5 1-integrin. Am J Physiol Cell Physiol 300: C477–C489, 2011. First published December 22, 2010; doi:10.1152/ajpcell.00171.2010.—Engagement of 5 1-integrin by fibronectin (FN) acutely enhances Cav1.2 channel (CaL) current in rat arteriolar smooth muscle and human embryonic kidney cells (HEK293-T) expressing CaL. Using coimmunoprecipitation strategies, we show that coassociation of CaL with 5or 1-integrin in HEK293-T cells is specific and depends on cell adhesion to FN. In rat arteriolar smooth muscle, coassociations between CaL and 5 1-integrin and between CaL and phosphorylated c-Src are also revealed and enhanced by FN treatment. Using site-directed mutagenesis of CaL heterologously expressed in HEK293-T cells, we identified two regions of CaL required for these interactions: 1) COOH-terminal residues Ser and Tyr, known to be phosphorylated by protein kinase A (PKA) and c-Src, respectively; and 2) two proline-rich domains (PRDs) near the middle of the COOH terminus. Immunofluorescence confocal imaging revealed a moderate degree of wild-type CaL colocalization with 1-integrin on the plasma membrane. Collectively, our results strongly suggest that 1) upon ligation by FN, CaL associates with 5 1-integrin in a macromolecular complex including PKA, c-Src, and potentially other protein kinases; 2) phosphorylation of CaL at Y and/or S is required for association of CaL with 5 1-integrin; and 3) c-Src, via binding to PRDs that reside in the II–III linker region and/or the COOH terminus of CaL, mediates current potentiation following 5 1-integrin engagement. These findings provide new evidence for how interactions between 5 1-integrin and FN can modulate CaL entry and consequently alter the physiological function of multiple types of excitable cells.

suggesting that Ca L may be modulated by mechanical stress applied to the plasma membrane (22).A link between Ca L and integrins in transducing mechanical force has also been demonstrated in arterioles.The application of peptides containing an Arg-Gly-Asp (RGD) sequence, a cryptic binding motif in ECM that becomes exposed during vascular injury (7), inhibits pressure-dependent myogenic tone, through interactions with multiple VSM and endothelial cell integrins (8).Moreover, function-blocking antibodies against ␣ 5 -, ␤ 1 -, and ␤ 3 -integrin significantly reduce the degree of myogenic constriction to pressure elevation (29).The effects of integrin antibodies on myogenic tone are mediated in part by altered Ca 2ϩ entry through Ca L because electrophysiological studies reveal selective effects of integrin ligands on Ca L current; the engagement and clustering of ␣ 5 ␤ 1 -integrin by insoluble fibronectin (FN) or anti-␣ 5 -integrin antibody acutely potentiates Ca L current, whereas engagement of ␣ v ␤ 3 -integrin by vitronectin or soluble ligands inhibits Ca L current in isolated VSM cells (41).Using site-directed mutagenesis strategies, two phosphorylation sites within the COOH terminus of the ␣ 1C -subunit of Ca L (␣ 1C -Ca L ) were identified as targets of protein kinase A (PKA) and c-Src that become activated downstream from ␣ 5 ␤ 1 -integrin ligation (14).
A critical remaining issue is the extent to which ␣ 5 ␤ 1integrin, PKA, c-Src, and Ca L are spatially coupled within a cell.In the present study, we tested the hypothesis that ␣ 5 ␤ 1integrins spatially associate with Ca L and that this association is required to modulate Ca L function.Using human embryonic kidney cells (HEK293-T) overexpressing the neuronal isoform of Ca L and VSM cells from rat skeletal muscle arterioles as model systems, the interactions between endogenous ␣ 5 ␤ 1integrin and Ca L and between c-Src and Ca L were examined using immunoprecipitation (IP), immunoblotting (IB), immunofluorescence confocal microscopy, and patch-clamp methods.

METHODS
Cell culture and transient transfection.HEK293-T/(TSA201) cells were maintained in 10% FBS (Hyclone)-DMEM (high glucose) supplemented with 2 mM glutamine, 100 units of penicillin, and 100 g of streptomycin in 5% CO 2 at 37°C.When cells reached 70 -80% confluence, wild-type (WT) or mutant neuronal Cav1.2 calcium channel (Ca L) cDNAs composed of ␣1C-c (6 g), ␤1b (3 g), and ␣2␦ (2 g) subunits, in a total volume of 14 l, were transfected into HEK293-T cells grown in 60-mm tissue culture dishes containing 2.5 ml of DMEM with Lipofectamine 2000 (20 l) for 18 -24 h.Green fluorescent protein (GFP; 0.5-1 g) was cotransfected with Ca L to monitor transfection efficiency.Untransfected cells were used as a control.Cells were incubated for another 24 h posttransfection before passing and plating onto either FN (catalog no.354403, BD BioCoat, BD Biosciences), BSA (2%), or poly-L-lysine-treated plates for 1 h, followed by protein isolation or immunofluorescence imaging.To account for variation in the expression level of ␣ 1C-CaL due to variation in transfection efficiency among the different CaL mutants, the experiments for all CaL mutants were performed in parallel with WT-CaL transfection using the same passage of cells.
For electrophysiological experiments, HEK293-T cells were transfected using calcium phosphate, after which patch-clamp recordings were performed 48 -72 h posttransfection as previously described (14).Only single cells expressing GFP were used for electrophysiological protocols.
The various mutations in the ␣ 1C-COOH terminus were generated using site-directed mutagenesis as described previously (14) and as illustrated in Fig. 1.
Isolation of skeletal muscle arterioles.The isolation of first-and second-order arterioles from rat cremaster muscle was performed as described previously (21,40,41), except for addition of 1ϫ Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Scientific) into the dissection chamber immediately before dissection.All animal protocols were approved by the University of Missouri Animal Care and Use Committee and conformed to the Public Health Service Policy for the Humane Care and Use of Laboratory Animals (PHS Policy, 1996).
Electrophysiological recording.Patch-clamp recordings were made using an EPC9 amplifier under the control of Pulse software (HEKA Instruments) in the conventional whole cell mode, as previously described (14).Pipettes were filled with Cs ϩ pipette solution containing (in mM) 110 CsCl, 20 TEA chloride, 10 EGTA, 2 MgCl2, 10 HEPES, and 1 CaCl2 (pH 7.2 with CsOH).Ba 2ϩ was used as the charge carrier to increase the magnitude of the inward current and to minimize calcium-dependent current inactivation.Cs ϩ was used to block endogenous K ϩ current.
Immunoprecipitation and immunoblotting.Proteins from HEK293-T cells were isolated as described by Ling et al. (27) with minor modifications.The lysis buffer was composed of 1% Triton X-100 containing the following: 2.5 mM Tris, pH 7.4, 13.8 mM NaCl, 0.3 mM KCl, 2 mM EDTA, 1 mM EGTA, 10 mM NaF, 1 mM benzamidine, 1 mM Na 3VO4, 4 mM Pefabloc, 10 g/ml leupeptin, 10 g/ml aprotinin, 10 g/ml of calpain I and II, and 10 g/ml pepstatin A. Isolation of total proteins in arterioles was performed as described above except for using 0.25% Triton X-100, along with 2% n-octyl-␤-D-glucopyranoside to lyse arterioles.Arterioles (1st order and 2nd order) were snap-frozen in liquid nitrogen after dissection.Vessels were cut into smaller pieces and pulverized with a plastic pestle in 0.65-ml centrifuge tubes in the presence of an optimal amount of lysis buffer.Tubes containing arterioles were frequently dipped into liquid nitrogen and kept on dry ice during pulverization.Three hundred to four hundred micrograms of total protein (150 -250 g for arterioles) were precleared with protein A/G agarose beads (catalog no.SC-2003; Santa Cruz) in the presence of either rabbit or mouse IgG (catalog no.PP64 and PP54, respectively; Millipore), followed by overnight IP in lysis buffer at 4°C with one of the following antibodies: 1) either rabbit anti-Cav1.2polyclonal antibody (Ab) (3.2 g; catalog no.AB5156, Millipore) or pan Calcium Channel Ab (catalog no.Ab6298, Abcam), recognizing the ␣ 1C-subunit of CaL (␣1C-CaL); 2) mouse anti-human ␤ 1-integrin monoclonal Ab (5 g, clone B3B11, catalog no.MAB2251Z, Millipore); 3) purified mouse anti-human ␣ 5-integrin Ab (5 g, clone 1/CD49e, catalog no.610634, BD Biosciences); 4) mouse anti-pp60c-Src (4 g, clone GD11, catalog no.05-184, Millipore); or 5) anti-phosphorylated-Tyr Ab (5 g, clone 4G10, catalog no.05-321, Millipore).The immune complex was captured using either protein A (catalog no.SC-2001, Santa Cruz) or protein G agarose beads (catalog no.17088601, Amersham) prior to SDS-PAGE (6%), followed by overnight transfer to nitrocellulose membrane (0.45 nm) and IB.To confirm binding specificity, the whole cell lysate was also immunoprecipitated with 1) control IgG for the appropriate host species and captured by protein A or protein G beads, or 2) protein A or protein G beads alone.Equal loading of total protein was confirmed by Ponceau staining (Sigma) before IB.Ca L was detected using rabbit anti-CaL ␣1C-subunit Ab (catalog no.AB5156, 1:6,500 dilution) for HEK293-T cells or pan Calcium Channel Ab (1:2,500 dilution) for rat arterioles.Endogenous ␤ 1- integrin was detected using mouse anti-human ␤ 1-integrin Ab or rabbit anti-rat ␤1-integrin Ab (catalog no.MAB2251Z, 1:1,400 dilution and catalog no.AB1952P, 1:1,500 dilution, respectively; Millipore).c-Src or phosphorylated c-Src was detected using anti-pp60c-Src (catalog no.05-184, 1:1,200 dilution, Millipore) or rabbit anti-rat phospho-Src family (Tyr416) Ab (catalog no.2101S, 1:1,000 dilution, Cell Signaling Technology), respectively.The immune complexes were incubated with either goat anti-rabbit IgG-horseradish peroxidase (HRP)-conjugated antibody (H&L, 1:120,000 dilution, catalog no.AP132P, Millipore) or donkey anti-mouse-IgG-HRP-conjugated antibodies (H&L, 1:60,000 dilution, catalog no.715-035-150, Jackson Research Laboratory) followed by detection with Pierce SuperSignal extended Dura or Femto substrate (Thermo Scientific).After probing, the blot was stripped with Restore Western blot stripping buffer (Thermo Scientific) and reprobed with either anti-␣1C-CaL, anti-␤1integrin, or anti-c-Src Ab to assess the relative loading.For detection of the immune complex from arterioles, the blot was processed as described above except that SuperSignal Western Blot Enhancer (catalog no.46641, Thermo Scientific) was used according to the manufacturer's instruction before IB.The results were captured by exposure to Kodak Biomax MR, MS, or Light film (Sigma), and the band intensity for the protein of interest was quantified using Bio-Rad Quantity One.Quantification of co-IP was determined as described by Cherubini et al. (4).The ratio of immunoprecipitated ␣1C-CaL to ␤1-integrin in WT-␣1C-CaL-expressing cells was set as 1.The ratio of immunoprecipitated ␣1C-CaL to ␤1-integrin in mutant ␣1C-CaL-expressing cells was expressed relative to WT-␣1C-CaL.
Immunofluorescence images were taken using a laser-scanning confocal system (Leica TCS SP5 Microsystems) attached to an upright microscope (Leica DM 6000 CS) and were collected using a ϫ63 oil objective (numerical aperture 1.42).Excitation of the fluorophores was achieved using argon 488, He/Ne 543, or He/Ne 633 nm laser combinations with pinhole size set at 1 airy disk.Images were taken at 512 ϫ 512 pixels with a step size of 0.2 m and a zoom factor of 5.The image size was ϳ49.2 m 2 .
To prevent bleed-through of different wavelengths that might interfere with colocalization analysis, all the images were taken using a sequential acquisition procedure at wavelengths of 488/519 nm (excitation/emission) for the ␣ 1C-CaL-subunit; 543/569 nm (excitation/emission) for ␤1-integrin, and 633/656 nm (excitation/emission) for nuclear staining.
Quantification of ␣1C-CaL association with ␤1-integrin.The association of ␣1C-CaL with ␤1-integrin was analyzed with ImageJ software (version 1.39d, National Institutes of Health, Bethesda, MD).The degree of ␣1C-CaL association with ␤1-integrin was quantified using Pearson's coefficient, Mander's coefficient, and intensity correlation analysis (ICA) (24,33).The advantage of using ICA analysis is that it avoids taking the overlap of randomly distributed proteins into account for colocalization if the intensity of the two target proteins does not vary coincidently (24).ICA values are distributed between Ϫ0.5 and ϩ0.5.ICA values close to 0 indicate random staining, values Ϫ0.5 Յ ICA Ͻ 0 represent segregated staining, and values 0 Ͻ ICA Յ ϩ0.5 represent interdependent staining.The degree of ␣ 1C-CaL association with ␤1-integrin was normalized to the average ICA value for the coassociation of paxillin with vinculin (0.25), which was shown previously to be very strong (37).
Statistical analysis.Results are presented as means Ϯ SE from at least three independent experiments for IP or four independent experiments for immunofluorescence confocal imaging (IF).Statistical differences were analyzed using GraphPad InStat or Prism (version 3.06 and version 5.0, respectively; GraphPad, San Diego, CA) with either analysis of variance or unpaired t-tests.P Ͻ 0.05 was considered significant.

RESULTS
Ca L association with ␣ 5 ␤ 1 -integrin depends on adhesion to fibronectin.To determine whether ␣ 1C -Ca L associated with ␣ 5 ␤ 1 -integrin, IP of whole cell lysates from HEK293-T cells expressing the neuronal Ca L using either anti-␤ 1 -or anti-␣ 5integrin antibody was followed by IB to probe for the presence of ␣ 1C -Ca L .The neuronal Ca L isoform was used as a model system because ␣ 1C -Ca L mutants had been previously generated by site-directed mutagenesis strategies; neuronal and smooth muscle (SM) cell (SMC) Ca L isoform share 90% homology in amino acid sequence, and we previously demonstrated that heterologously expressed rat neuronal and SMC isoforms (Cav1.2cand Cav1.2b, respectively) showed a similar degree of current potentiation following ␣ 5 ␤ 1 -integrin ligation (14).We first determined the association between ␣ 1C -Ca L and ␤ 1 -integrin in cells plated on poly-L-lysine-treated dishes.As shown in Fig. 2A, no appreciable ␣ 1C -Ca L was observed after IP using anti-␤ 1 -integrin Ab in either control (untransfected cells, lane 2) or cells expressing WT ␣ 1C -Ca L (lane 7) when plated on poly-L-lysine-treated dishes.However, when cells expressing WT ␣ 1C -Ca L were plated on FN (Fig. 2B), ␣ 1C -Ca L was detected after IP of the lysate using either anti-␣ 5 -(lane 4) or anti-␤ 1 -integrin antibody (lane 3).No specific ␣ 1C -Ca L band was observed in cells expressing ␣ 1C -Ca L plated on BSAcoated dishes after IP using anti-␤ 1 -integrin or anti-c-Src Ab (Supplemental Fig. S1A, lanes 6 and 7, respectively) or after IP of the lysate using mouse IgG or protein-G (Fig. 2B, lanes 2, 5, and 9).These results indicate that the association of ␣ 1C -Ca L with ␣ 5 or ␤ 1 -integrin in HEK293-T cells is specific and depends on cell adhesion to FN.
To confirm that the effect of FN on ␣ 1C -Ca L association with ␤ 1 -integrin was not an artifact of Ca L overexpression in HEK293-T cells, we examined the same interaction in lysates from arteriolar SM.Arterioles were dissected and then incubated with exogenous FN abluminally (300 g/ml) for 1 h.When arteriolar SM lysates were immunoprecipitated with  anti-␣ 1C -Ca L , ␤ 1 -integrin was detected only in arterioles incubated with FN (Fig. 3A, lanes 1 and 2), compared with control (lanes 5 and 6).The same blot was also stripped and reprobed for the expression of ␣ 1C -Ca L .In addition, incubation with FN did not substantially change the level of ␣ 1C -Ca L expression in arterioles (Supplemental Fig. S1B).These results indicate that the association of ␣ 1C -Ca L with ␤ 1 -integrin in SMC depends on ligation of ␤ 1 -integrin by FN.
c-Src, a tyrosine kinase activated upon integrin ligation by ECM ligands (18), has been shown to modulate Ca L function in SMC (2,3,19,39).Thus, we also examined whether c-Src associated with ␣ 1C -Ca L in arteriolar SM and whether FN was important for this association.As shown in Fig. 3B, c-Src was detected in immunoprecipitates using two different anti-␣ 1C -Ca L antibodies (Fig. 3B, lanes 1 and 2).However, abluminal incubation with FN did not substantially change the degree of c-Src association with ␣ 1C -Ca L (Fig. 3B, lanes 1 and 2 vs. lanes 5 and 6).Next, we tested whether incubation with FN altered the phosphorylation status of c-Src in association with ␣ 1C -Ca L .A basal level of phosphorylated c-Src was observed in untreated control lysate (Fig. 3C, lane 3) and in lysate from arteriolar SM incubated with FN (lane 4).In contrast to Fig. 3B, phosphorylated c-Src coassociated with ␣ 1C -Ca L only in arteriolar SM incubated with FN, not in control arteriolar SM (Fig. 3C, lanes 1 and 2 vs. lanes 5 and 6, respectively).These results suggest that the association of ␣ 1C -Ca L with c-Src in arteriolar SM is independent of FN, whereas the association of ␣ 1C -Ca L with phosphorylated c-Src depends on integrin engagement by FN.
The COOH terminus of ␣ 1C -Ca L is essential for association with ␤ 1 -integrin.To determine whether the specific regions in the COOH terminus of ␣ 1C are required for the association of ␣ 1C -Ca L with ␣ 5 ␤ 1 -integrin, three mutant ␣ 1C -Ca L constructs were overexpressed in HEK293-T cells (Fig. 1).If any of the specific regions is required for association between the two proteins, we predicted to observe less ␣ 1C -Ca L pulled down by anti-␤ 1 -integrin Ab in cells expressing the mutant ␣ 1C -Ca L .As shown in Fig. 4A, truncated ␣ 1C -Ca L exhibited a reduced level of association with ␤ 1integrin (lane 8) and a reduced level of association with c-Src (lane 7), compared with their respective levels of association in WT ␣ 1C -Ca L (lanes 1 and 2, respectively).Figure 4B shows the same membrane after stripping and reprobing for ␤ 1 -integrin as a loading control.The expression of ␤ 1 -integrin, c-Src (Supplemental Fig. S3A), and the degrees of association between ␤ 1 -integrin and c-Src were not changed in cells expressing truncated ␣ 1C -Ca L , compared with WT ␣ 1C -Ca L (quantified data not shown).When normalized to the total amount of WT ␣ 1C -Ca L , the associations between truncated ␣ 1C -Ca L and ␤ 1 -integrin as well as between truncated ␣ 1C -Ca L and c-Src were reduced to 40 Ϯ 9% and 55 Ϯ 8% of WT ␣ 1C -Ca L , respectively (Fig. 4C).These results provide evidence for the requirement of the distal COOH terminus in the associations between ␣ 1C -Ca L and ␤ 1 -integrin and between ␣ 1C -Ca L and c-Src.
To confirm that the reduced associations between ␣ 1C -Ca L and ␤ 1 -integrin and between ␣ 1C -Ca L and c-Src were not due to decreased expression of truncated ␣ 1C -Ca L construct, we increased the amount of total protein for IP from cells expressing the truncated ␣ 1C -Ca L construct to twofold that of WT ␣ 1C -Ca L and reexamined the association of truncated ␣ 1C -Ca L with ␤ 1 -integrin or with c-Src.As shown in Supplemental Fig. S2, A and C, reduced degrees of association between ␣ 1C -Ca L and ␤ 1 -integrin (Fig. S2A, lane 3), ␣ 1C -Ca L and phosphorylated tyrosine (Tyr-Pi, Fig. S2A, lane 2), and ␣ 1C -Ca L and c-Src (Fig. S2A, lane 1) were still observed in cells expressing truncated ␣ 1C -Ca L , when compared with the degrees of their respective associations in cells expressing WT ␣ 1C -Ca L (70 Ϯ 7%, 51 Ϯ 4%, and 58 Ϯ 11%; % of WT, respectively).However, the expressions of ␤ 1 -integrin and c-Src, the degrees of associations between ␤ 1 -integrin and c-Src, or between ␤ 1 -integrin and proteins containing phosphorylated tyrosine were not changed in cells expressing truncated ␣ 1C -Ca L , compared with WT ␣ 1C -Ca L (Supplemental Figs.S2B and S3B; quantified data not shown).These results suggest a requirement for the distal COOH terminus in ␣ 1C -Ca L association with the ␤ 1 -integrin and c-Src.
The collaborative contribution of PKA and c-Src in the association of Ca L with ␤ 1 -integrin.We previously showed that ␣ 5 ␤ 1 -integrin engagement leads to potentiation of Ca L current via phosphorylation of ␣ 1C -Ca L serine (S 1901 ) and tyrosine (Y 2122 ) by PKA and c-Src, respectively (14).Thus, a next logical step was to determine whether phosphorylation of ␣ 1C -Ca L by PKA and c-Src is required for Ca L association with ␤ 1 -integrin or c-Src.As shown in Fig. 5A, reduced associations between ␣ 1C -Ca L and ␤ 1 -integrin (lane 3), ␣ 1C -Ca L and Tyr-Pi (lane 2), and ␣ 1C -Ca L and c-Src (lane 1) were observed in cells expressing S 1901 A/Y 2122 F-Ca L , compared with WT ␣ 1C -Ca L (Fig. 5C, 47 Ϯ 8%, 48 Ϯ 12%, and, 27 Ϯ 7%; % of WT, respectively).However, the expressions of ␤ 1 -integrin (Fig. 5B) and c-Src, the degrees of associations between ␤ 1 -integrin and c-Src, or between ␤ 1 -integrin and proteins containing phosphorylated tyrosine were unchanged in cells expressing S 1901 A/Y 2122 F-Ca L , compared with WT ␣ 1C -Ca L (Supplemental Fig. S4A, quantified data not shown).These results suggest that phosphorylation of ␣ 1C -Ca L by PKA and/or c-Src is required for the associations between ␣ 1C -Ca L and ␤ 1 -integrin and between ␣ 1C -Ca L and c-Src.
The proline-rich domains in the COOH terminus are required for the association of ␣ 1C -Ca L with ␤ 1 -integrin.Prolinerich domains (PRDs) are binding motifs for Src homology 3 domain (SH3)-containing proteins, such as c-Src (34).Two PRDs have been identified in the COOH terminus of ␣ 1C -Ca L and have been shown to mediate the association of Ca L with some proteins containing SH3 domains (12,13).Thus, we investigated the roles of the COOH-terminal PRDs in determining ␣ 1C -Ca L association with ␤ 1 -integrin or c-Src by expressing a ⌬P1/⌬P2-Ca L mutant with selective deletion of both PRDs in the COOH terminus of ␣ 1C .As shown in Fig. 6A, significantly less association between ␣ 1C -Ca L and ␤ 1 -integrin (lane 6) and between ␣ 1C -Ca L and c-Src (lane 8), was observed in cells expressing the ⌬P1/⌬P2 ␣ 1C -Ca L mutant, compared with WT ␣ 1C -Ca L (Fig. 6C, 54 Ϯ 9%, 54 Ϯ 6%; % of WT, respectively).However, the expression of ␤ 1integrin (Fig. 6B), c-Src, and the degree of association between ␤ 1 -integrin and c-Src was unchanged in cells expressing ⌬P1/⌬P2 ␣ 1C -Ca L (Supplemental Fig. S4B, quantified data not shown).These results suggest that the PRDs in the ␣ 1C -Ca L COOH terminus are partially required for associations between ␣ 1C -Ca L and ␤ 1 -integrin and between ␣ 1C -Ca L and c-Src.
Functional modulation of Ca L current by ␣ 5 ␤ 1 -integrin is independent of the PRDs in the COOH terminus.Given the results shown in Figs. 5 and 6, we also investigated the roles of the two PRDs in the modulation of Ca L current following ␣ 5 ␤ 1 -integrin engagement.As shown in Fig. 7A, the application of ␣ 5 ␤ 1 -integrin antibody to the bath solution potentiated whole cell Ca L current in HEK293-T cells expressing WT-Ca L , consistent with our previous findings (14).To our surprise, no significant electrophysiological difference was observed between WT-Ca L and ⌬P1/⌬P2-Ca L (82% of WT) in the degree of current potentiation by ␣ 5 ␤ 1 -integrin engagement, suggesting that the COOH-terminal PRDs are not required for potentiation of Ca L current by ␣ 5 ␤ 1 -integrin engagement.When the degree of Ca L current potentiation following ␣ 5 ␤ 1 -integrin ligation in the mutant constructs was normalized to that of WT, both the truncated ␣ 1C -Ca L and S 1901 A/Y 2122 F-Ca L mutants displayed significant impairment in the amount of current potentiation by integrin ligation (Fig. 7B, 9% and 1% potentiation of WT-Ca L , respectively; see also Ref. 14).The above results confirm that S 1901 and Y 2122 residues in the COOH terminus of ␣ 1C -Ca L are required for Ca 2ϩ current potentiation following ␣ 5 ␤ 1 -integrin ligation.However, selective deletion of the two PRDs in the COOH terminus of ␣ 1C -Ca L does not impair the current potentiation by ␣ 5 ␤ 1 -integrin engagement.Immunofluorescence imaging analysis demonstrates association of Ca L with ␤ 1 -integrin on the plasma membrane.The COOH terminus of ␣ 1C -Ca L has been shown to mediate membrane targeting of the channel (11), so we further investigated the effect of the abovementioned regions in the COOH terminus on membrane targeting of ␣ 1C -Ca L and its association with ␤ 1 -integrin.Immunofluorescence confocal microscopy (IF) was performed to determine the expression, distribution, and relative localization of ␣ 1C -Ca L and ␤ 1 -integrin on the plasma membrane.Images of cells without staining or cells stained with either anti-mouse IgG, anti-rabbit IgG, or the combination of both, exhibited only faint, diffuse, and nonspecific staining (Supplemental Fig. S5).As shown in Fig. 8, neither the membrane expression of ␣ 1C -Ca L nor ␤ 1 -integrin was detectably altered in S 1901 A/Y 2122 F-Ca L , truncated ␣ 1C -Ca L , or ⌬P1/ ⌬P2-Ca L , compared with WT-Ca L .
When we examined the association between WT ␣ 1C -Ca L and endogenous ␤ 1 -integrin on the plasma membrane, a moderate degree of colocalization was observed.To quantify the degree of colocalization between ␣ 1C -Ca L and ␤ 1 -integrin, we used the overlap of paxillin and vinculin staining as a normalization standard for the following analyses, since these two proteins highly associate at the focal adhesion site (ICA value 0.25) (37).The relatively high degree of colocalization between paxillin and vinculin was set as 100% for subsequent analysis of colocalization between ␣ 1C -Ca L and ␤ 1 -integrin.We used Pearson's coefficient (R), Mander's coefficient (R r ), and ICA to determine the degree of ␣ 1C -Ca L -␤ 1 -integrin colocalization, as listed in Table 1.The degrees of ␣ 1C -Ca L colocalization with ␤ 1 -integrin in WT-Ca L , truncated ␣ 1C -Ca L , S 1901 A/Y 2122 F-Ca L , or ⌬P1/⌬P2 ␣ 1C -Ca L were significantly less than the degree of colocalization between paxillin and vinculin (60 -88% of paxillin-vinculin colocalization).
Using ICA analysis (Fig. 9), modest, but not significant, decreases in the degrees of ␣ 1C -Ca L association with ␤ 1integrin were observed in the truncated ␣ 1C -Ca L , S 1901 A/ Y 2122 F ␣ 1C -Ca L , and ⌬P1/⌬P2 ␣ 1C -Ca L mutants, compared with WT-Ca L (99 Ϯ 5.9%, 99 Ϯ 6.2%, and 88 Ϯ 5.1% of WT ␣ 1C -Ca L , respectively).Deconvolution analysis was also used to eliminate the signals collected from out-of-focus emission.However, no substantial differences were noted in the same comparisons when deconvolution analysis was applied (data not shown).Collectively, confocal image analysis demonstrated that the association between ␣ 1C -Ca L and ␤ 1 -integrin on the cell membrane was moderate in cells expressing WT ␣ 1C -Ca L .No significant differences were observed in the degree of ␣ 1C -Ca L colocalization with ␤ 1 -integrin between the three mutant ␣ 1C -Ca L constructs, compared with WT ␣ 1C -Ca L .Taken together, the IF results suggest that ␣ 1C -Ca L and ␤ 1integrin colocalize to a moderate degree on the plasma mem- brane and that the modifications we made in the COOH terminus of ␣ 1C -Ca L did not significantly alter the membrane targeting of ␣ 1C -Ca L or ␤ 1 -integrin on the plasma membrane.Thus, the reduced associations between the mutant forms of ␣ 1C -Ca L and ␤ 1 -integrin observed in IP protocol are not due to deficient membrane targeting.

DISCUSSION
We present evidence that the pore-forming ␣ 1C -subunit of the L-type calcium channel (␣ 1C -Ca L ) associates with ␤ 1integrin and c-Src in arteriolar SM as well as when the channel is heterologously expressed in HEK293-T cells.These associations depend on interactions of the cells with the ECM protein FN, which is known from previous studies to potentiate Ca L current following ␣ 5 ␤ 1 -integrin engagement (40).Furthermore, our results show that the associations between ␣ 1C -Ca L and ␣ 5 ␤ 1 -integrin and between ␣ 1C -Ca L and c-Src require specific regions in the COOH terminus of ␣ 1C -Ca L .Two specific domains that are critical for these interactions are the PKA and c-Src phosphorylation sites that we previously identified as essential for modulation of Ca L function following ␣ 5 ␤ 1 -integrin engagement (14).In addition, we found that Ca L preferentially associates with the phosphorylated, active, form of c-Src in arteriolar SM and that this association depends on FN.The latter result provides evidence for a unique local functional regulation of Ca L by ␣ 5 ␤ 1 -integrin through phosphorylation of c-Src.Collectively, our data support the concept of a macromolecular complex composed of multiple focal adhesion molecules including PKA, c-Src, and some fraction of the Ca L population, which is assembled upon integrin ligation and required for potentiation of Ca L current following ␣ 5 ␤ 1 -integrin engagement.
In this study, three different approaches, IP, IF, and patchclamp recording of Ca L current, were used to assess structural and functional interactions between ␣ 1C -Ca L and ␣ 5 ␤ 1 -integrin.In some respects, the data obtained using these methods support each other, while in other respects the data conflict.How can we reconcile the conflicting results?What do the collective data tell us about the direct or indirect association of the channel with ␣ 5 ␤ 1 -integrin?What signaling components are required to mediate ␣ 1C -Ca L association with ␣ 5 ␤ 1 -integrin upon adhesion to FN? How does the spatial interaction between ␣ 1C -Ca L and ␣ 5 ␤ 1 -integrin contribute to the functional regulation of Ca L ?What is the physiological significance of ␣ 1C -Ca L association with ␣ 5 ␤ 1 -integrin?These issues will be addressed in the following sections.
Direct or indirect association of Ca L with ␣ 5 ␤ 1 -integrin.After demonstrating an association between ␣ 1C -Ca L and ␣ 5 ␤ 1 -integrin, using IP and IB methods, we sought to determine the nature of the interaction and the regions in ␣ 1C -Ca L that were required.Because the crystal structure of ␣ 1C -Ca L has not been resolved, interactions between these proteins are difficult to predict.If ␣ 1C -Ca L directly interacts with ␣ 5 ␤ 1integrin, a potential binding region would be the canonical Arg-Gly-Asp (RGD)-integrin-binding motif.In fact, a previous study by McPhee et al. (31) identified the functional significance of an RGD sequence located in the extracellular loop, between the first membrane-spanning region and the pore, of the G protein-activated inward rectifier K ϩ channel (GIRK).This RGD sequence mediates the direct interaction between GIRK and the ␤ 1 -integrin.The functional significance of the interaction appears to involve targeting of GIRK to the plasma membrane and/or regulation of its stability rather than acute regulation of channel function (31).However, in Cav1.2, the only conserved RGD sequence (aa 448 -450) is found in a conserved transmembrane region of all three Ca L isoforms, so that direct binding of ␣ 1C -Ca L to RGD-binding integrins is unlikely due to potential steric hindrance.Thus, an indirect association between ␣ 1C -Ca L and ␣ 5 ␤ 1 -integrin through cytoplasmic or cytoskeletal proteins seems more probable.
To determine regions in ␣ 1C -Ca L that provide binding motifs for other focal adhesion proteins that are known to associate with ␣ 5 ␤ 1 -integrin, we used a site-directed mutagenesis strategy.On the basis of differences in the amount of coimmuno- Representative confocal immunofluorescence images from the cells expressing WT-, truncated ␣1C-, S 1901 A/Y 2122 F-, or ⌬P1/⌬P2-CaL.␣1C and endogenous ␤1-integrin expression on the plasma membrane appear as green and red punctate staining in a, e, i, and m and in b, f, j, and n, respectively.The colocalization of ␣1C-CaL with ␤1-integrin on the membrane appears as yellow to orange punctate staining (c, g, k, and o).A noticeably higher degree of ␣1C-CaL colocalization with ␤1-integrin was seen in cells expressing WT-␣1C-CaL, compared with mutant ␣1C-CaL construct-expressing cells.Merged images of the previous three panels with nuclei counterstaining appear in blue (d, h, l, and p).Endogenous paxillin and vinculin staining in HEK293-T cells appears as green and red in q and r, respectively.The degree of colocalization between paxillin and vinculin (yellow to orange staining; s) appears to be much higher than that between ␣1C-CaL and ␤1-integrin in the WT-or mutant ␣1C-CaL-expressing cells.Merged image with nuclear counterstaining in blue appears in t.Magnification, ϫ63 oil; scale bar, 10 m.

C485
Cav1.2 ASSOCIATES WITH ␣5␤1-INTEGRIN precipitated ␣ 1C -Ca L with ␤ 1 -integrin in WT versus mutated ␣ 1C -Ca L , we identified several regions in the ␣ 1C -Ca L COOH terminus that are involved in the interaction of ␣ 1C -Ca L with ␣ 5 ␤ 1 -integrin: two phosphorylation sites for PKA and c-Src, along with the PRDs.These findings therefore implicate PKA, c-Src, and potentially other SH3 domain-containing proteins in mediating the interaction of ␣ 1C -Ca L with ␣ 5 ␤ 1 -integrin.
The roles of PKA in the functional regulation of Ca L and its association with ␣ 5 ␤ 1 -integrin.PKA is known to regulate all three Ca L isoforms.In neurons, PKA colocalizes with adenylate cyclase, ␤ 2 -adrenergic receptor (␤ 2 -AR), A-kinase anchoring protein (AKAP), and protein phosphatase (PP2A) in a macromolecular signaling complex; PKA mediates the increase in neuronal Ca L current following stimulation of ␤ 2 -AR (6).Our results suggest that PKA is also a component of another macromolecular complex containing various focal adhesion proteins recruited following ligation of ␣ 5 ␤ 1 -integrin and that signaling between proteins in this complex also can exert a significant degree of control over Ca L function.Evidence to support this conclusion derives from two aspects of our results.First, reduced association between ␣ 1C -Ca L and ␤ 1 -integrin was observed in an ␣ 1C -Ca L mutant with altered PKA and c-Src phosphorylation sites.Second, our previous electrophysiological studies demonstrated a substantial reduction in the amount of Ca L current that was potentiated following ligation of ␣ 5 ␤ 1 -integrin when ␣ 1C -Ca L mutants with altered PKA and/or c-Src phosphorylation were used (14).When S 1901 in ␣ 1C is mutated, it not only leads to reduction in the association between ␣ 1C -Ca L and ␤ 1 -integrin, but also to reduction in the phosphorylation of Ca L by PKA (14).Findings from other laboratories also support the notion that S 1901 in the COOH terminus of ␣ 1C -Ca L is a focal assembly point for PKA, AKAP, PP2A, and WAVE/WASP, which collectively modulate Ca L function (5).In support of this idea, the macromolecular complex composed of PKA, AKAP, PP2A, and WAVE/ WASP has been shown to provide a spatial link to the integrincytoskeleton network in modulation of other cellular functions (15) and engagement of ␤ 1 -integrin is known to facilitate PKA targeting to specific cellular locations (26,32).On the basis of all the above, faulty trafficking/targeting of PKA in the cytoplasm could contribute to reduced interaction of PKA with other focal adhesion components in the macromolecular complex, thereby disrupting the spatial link to the integrin-cytoskeleton network and reducing the extent to which Ca L function is influenced by ␣ 5 ␤ 1 -integrin signaling.
The roles of PRDs and c-Src in functional regulation of Ca L and its association with ␣ 5 ␤ 1 -integrin.c-Src is another kinase that is implicated in the signaling pathway between ␣ 5 ␤ 1integrin and Ca L .The involvement of c-Src is supported by several findings in the present study: 1) co-IP of ␣ 1C -Ca L with c-Src in arteriolar SM appears to be independent of FN; 2) co-IP of ␣ 1C -Ca L and phosphorylated c-Src is only observed in arteriolar SM incubated with FN; 3) co-IP of ␣ 1C -Ca L and c-Src is only observed upon adhesion of HEK293-T cells to FN; and 4) mutation of the phosphorylation site for c-Src in the COOH terminus in ␣ 1C -Ca L (Y 2122 ) leads to reduced co-IP of ␣ 1C -Ca L with ␤ 1 -integrin and of ␣ 1C -Ca L with c-Src.These results are consistent with previous findings that purified c-Src associates with the COOH terminus of ␣ 1C -Ca L (19,20) and that a basal level of Ca L current depends on c-Src activity in colonic smooth muscle (16).Our co-IP findings are also consistent with a previous functional study from our laboratory showing that the potentiation of Ca L current following ␣ 5 ␤ 1integrin ligation is significantly reduced in Y 2122 F ␣ 1C -Ca L constructs (14).Thus, preventing this tyrosine residue from being phosphorylated not only reduces the overall phosphorylation of Ca L by exogenous c-Src, but also reduces the spatial interaction between ␣ 1C -Ca L and ␤ 1 -integrin and between ␣ 1C -Ca L and endogenous c-Src, indicating that the interactions between ␣ 1C -Ca L and ␤ 1 -integrin and between ␣ 1C -Ca L and Values are means Ϯ SE, taken from at least eight cells in either wild-type (WT) or mutant L-type voltage-gated Cav1.2 calcium channel (CaL) per experiment.The degree of CaL association with ␤1-integrin was normalized using the association of paxillin (Pax) and vinculin (Vin) as a reference and set as 100% for Pearson's, Mander's, or intensity correlation analysis (ICA).The value of CaL association with ␤1-integrin in WT, truncated ␣1C-, S 1901 A/Y 2122 F, or ⌬P1/⌬P2-CaL is presented as the percentage Ϯ SE of paxillin-vinculin association.Rr, Pearson's coefficient; R, Mander's coefficient.Statistical analysis was performed on the basis of the average from at least four individual experiments.c-Src are required for current potentiation following ␣ 5 ␤ 1integrin ligation.
Our IP studies also identified a region in the ␣ 1C -Ca L COOH terminus containing two PRDs (P1/P2) as another potential binding domain for c-Src.Reduced associations between ␣ 1C -Ca L and ␤ 1 -integrin and between ␣ 1C -Ca L and c-Src were observed in the ⌬P1/⌬P2 ␣ 1C -Ca L mutant.Our results agree with a previous study demonstrating that fusion proteins containing ␣ 1C PRDs mediate interactions with c-Src in SY5Y cells expressing the neuronal Ca L isoform upon IGF stimulation (1).Taken together, the results suggest multiple roles for PRDs in association with the macromolecular complex containing ␤ 1 -integrin, c-Src, and ␣ 1C -Ca L .
FAK is another proline-rich tyrosine kinase known to provide a binding site for SH2 and SH3 domain-containing proteins such as paxillin, vinculin, talin, p130Cas, and Crk (42).Although we did not specifically examine whether ␣ 1C -Ca L associates with FAK, our previous studies in vascular SMC revealed that a significant amount of the Ca L current potentiation following ␣ 5 ␤ 1 -integrin engagement was attenuated by dialysis of the cells with an antibody against FAK (40).A precedent for an association between FAK and another ion channel has been established by studies of Cherubini et al. (4), who demonstrated the association between FAK and the human ether-a-go-go-related gene (hERG) channel.In addition, PYK2, a family member of the FAK tyrosine kinases, was demonstrated to associate with cardiac ␣ 1C -Ca L via binding to PRDs (9).On the basis of the above, the PRDs in the ␣ 1C -Ca L appear to play significant roles in the assembly of a macromolecular complex containing Ca L , ␣ 5 ␤ 1 -integrin, c-Src, and potentially other protein tyrosine kinases.
It should be noted that the roles of PRDs in the physical and functional association of ␣ 1C -Ca L with ␣ 5 ␤ 1 -integrin may be more complicated than at first suggested by the preceding discussion.When the ⌬P1/⌬P2 ␣ 1C -Ca L mutant is expressed, the IP results (Fig. 6, A and C) reveal a reduced association between ␣ 1C -Ca L and ␤ 1 -integrin and between ␣ 1C -Ca L and c-Src, compared with WT ␣ 1C -Ca L , whereas the electrophysiological data (Fig. 7, A and B) reveal no substantial impairment in the degree of Ca L current potentiation following ␣ 5 ␤ 1integrin ligation.The discrepancy may arise from two issues.First, the PRD regions that we deleted from ␣ 1C -Ca L lie within the distal COOH terminus inhibitory region (DCT).Two studies have shown that deletion of the homologous PRDs (aa 1966 -2004) in the cardiac Ca L isoform leads to enhancement of basal Ca L current (13,38).Deletion of the PRDs removes part of the DCT, which may partially relieve constitutive channel inhibition by the DCT segment, though this has not been specifically tested.However, the major differences between the neuronal, cardiac, and smooth muscle isoforms of the Ca L channel reside in the more distal regions of the respective COOH termini, and whether or not the DCT segment exerts strong inhibitory control of the neuronal Ca L has not been determined.
A second explanation for the discrepancy between the structural and functional PRD results is the possible contribution of another PRD that has been identified in the II-III linker region of ␣ 1C -Ca L (aa 857-861).A study by Dubuis et al. (9) suggests that the II-III linker region appears to play a role in extending the activation range of cardiac Ca L , which consequently alters Ca L function.Our IP results revealed that deletion of the COOH-terminal PRDs only reduced c-Src coassociation with ␣ 1C -Ca L by 42%, even when the amount of truncated COOHterminal ␣ 1C -Ca L mutants for IP was increased by twofold (Supplemental Fig. S2, A and C).Thus, deletion of only the PRDs in the COOH terminus of ␣ 1C -Ca L may not be sufficient to fully abolish the potentiation in Ca L current following ␣ 5 ␤ 1 -integrin engagement.Overall, our electrophysiological and IP results agree with the findings of Dubuis et al. and suggest the possibility of another important c-Src binding region in ␣ 1C -Ca L .
Reduced association between ␣ 1C -Ca L and ␤ 1 -integrin is not due to faulty membrane targeting of Ca L .The amino acid residues 1623-1733 in the COOH terminus of cardiac ␣ 1C -Ca L have been shown to mediate the membrane targeting of the channel (11).Using IF, we detected no significant difference in membrane targeting of ␣ 1C -Ca L or ␤ 1 -integrin in S 1901 A/ Y 2122 F-Ca L , truncated ␣ 1C -Ca L at amino acid residue 1862, or ⌬P1/⌬P2-Ca L , compared with WT-Ca L .Our results suggest that the reduced association between ␣ 1C -Ca L and ␤ 1 -integrin observed in the above mentioned ␣ 1C -Ca L mutants is not due to unsuccessful membrane targeting of ␣ 1C -Ca L .Our findings agree with those of Gao et al. (11) that the regions in the COOH terminus required for successful membrane targeting of cardiac Ca L are amino acid residues 1623-1733.Because those specific regions are not altered in our mutant ␣ 1C -Ca L constructs, the reduced association between ␣ 1C -Ca L and ␤ 1integrin in our mutant constructs are unlikely attributable to incorrect membrane targeting of Ca L .In addition, our IF results also suggest that the association between ␣ 1C -Ca L and ␤ 1integrin on the plasma membrane is independent of the PKA/ c-Src phosphorylation sites, the PRD domains, or the distal COOH terminus after amino acid residue 1862 in ␣ 1C -Ca L .These results suggest that ␣ 5 ␤ 1 -integrin modulates Ca L function independent of the interactions of the two proteins on the plasma membrane.The IF results agree with the IP and patch-clamp findings in that the formation of a macromolecular complex including PKA, c-Src, ␣ 5 ␤ 1 -integrin, and Ca L in the cytoplasm following ␣ 5 ␤ 1 -integrin engagement appears to play an important role in Ca L current potentiation by ␣ 5 ␤ 1 -integrin.
Physiological significance of Ca L association with integrin.Our findings of an association between Ca L and ␤ 1 -integrin and between Ca L and c-Src provide evidence for a macromolecular signaling complex composed of ␣ 5 ␤ 1 -integrin, Ca L , c-Src, PKA, and possibly other focal adhesion components.Formation of this complex appears to be important for regulation of Ca L function following integrin-ECM interaction.Our IP results are consistent with previous findings that c-Src is involved in the increase of Ca L current potentiation in response to PDGF (17) or IGF (1) and with the finding that a c-Src-FAK complex forms in colonic smooth muscle in response to PDGF stimulation (16).Interestingly, cross talk between integrins and growth factors has been demonstrated in several studies, with the interaction of FAK and c-Src as a point of convergence (10,28,35).Although it is not known whether these two signaling pathways converge to regulate an ion channel, an intriguing possibility is that PDGF or IGF act in conjunction with ␣ 5 ␤ 1integrin to produce additive or synergistic potentiation of Ca L function by integrin.
In summary, this is the first study to provide evidence for the association of ␣ 1C -Ca L with ␣ 5 ␤ 1 -integrin or ␣ 1C -Ca L with c-Src in arteriolar smooth muscle.This association depends on engagement of ␣ 5 ␤ 1 -integrin by FN and specific regions in the ␣ 1C -Ca L COOH terminus, including two phosphorylation sites for PKA and c-Src and proline-rich domains that provide a binding motif for SH3 domain-containing proteins, such as c-Src.Identification of these regions, along with our electrophysiological findings, suggests that a macromolecular complex is assembled upon engagement of ␣ 5 ␤ 1 -integrin and that the interactions of proteins within this complex contribute to modulation of Ca L function.Our study provides new insights into a Ca L macromolecular regulatory complex that is required for functional regulation of the channel following ␣ 5 ␤ 1 -integrin engagement.

Fig. 3 .
Fig. 3. ␣1C-CaL associates with ␤1-integrin in arteriolar smooth muscle incubated with FN.A: representative blot showing co-IP protocol on protein lysed from arteriolar smooth muscle (pulled down with anti-␣1C-CaL).␤1-Integrin and ␣1C-CaL coimmunoprecipitated when arterioles were incubated with FN for 1 h (lanes 1 and 2, respectively).Lanes 1 and 6 were immunoprecipitated with anti-␣1C-CaL from Chemicon, whereas lanes 2 and 5 were immunoprecipitated with anti-␣1C-CaL from Abcam.Lanes 3 and 4 were lysates alone isolated from control arterioles or arterioles incubated with FN, respectively.The absence of coimmunoprecipitated ␤1-integrin by rabbit IgG (lane 7) indicates binding specificity.B: representative blot showing co-IP protocol on protein lysed from arteriolar smooth muscle (pulled down with anti-␣1C-CaL).c-Src coimmunoprecipitated with ␣1C-CaL whether or not arterioles were incubated with FN (lanes 1 and 2 vs. lanes 5 and 6, respectively).IP was performed as described in A. Abluminal incubation of arterioles with exogenous FN did not change the degree of association between ␣1C-CaL and c-Src (lane 2, 90% and lane 5, 86% of arterioles alone, respectively).The absence of coimmunoprecipitated c-Src by rabbit IgG (lane 7) indicates binding specificity.C: representative blot showing co-IP protocol on protein lysed from arteriolar smooth muscle (pulled down with anti-␣1C-CaL).Phosphorylated c-Src was only observed in arterioles incubated with exogenous FN (lanes 1 and 2).IP was performed as described in A. The absence of phosphorylated c-Src by rabbit IgG (lane 7) indicates binding specificity.

Fig. 4 .
Fig. 4. Requirement of the ␣1C-CaL COOH terminus for association of ␣1C-CaL with c-Src or ␤1-integrin.A: representative blot comparing WT-and truncated ␣1C-CaL immunoprecipitated with either anti-c-Src, anti-␤1-integrin Ab, or mouse IgG, then probed for ␣1C.␣1C was only detected in lysates immunoprecipitated with anti-c-Src (lanes 2 and 7) or anti-␤1-integrin Ab (lanes 1 and 8) but not with mouse IgG (lanes 3 and 6).Relatively lower amounts of ␣1C-CaL were pulled down by anti-c-Src or anti-␤1-integrin Ab in truncated ␣1C-CaL (lanes 7 and 8, respectively), compared with WT.B: the same blot in A after stripping and reprobing for ␤1-integrin as a control for IP and immunoblotting.C: summary graph of the relative levels of immunoprecipitated ␣1C in cells expressing WT-or truncated ␣1C-CaL.Values were obtained as described in METHODS and are based on the average of at least three experiments.*P Ͻ 0.05 vs. WT ϩ anti-␤1-integrin Ab or WT ϩ anti-c-Src Ab.

Fig. 5 .
Fig. 5. S 1901 and Y 2122 in ␣1C-CaL are required for ␣1C-CaL association with ␤1-integrin or c-Src.A: representative blot comparing WT ␣1C-CaL and S 1901 A/Y 2122 F ␣1C-CaL immunoprecipitated with either anti-c-Src, anti-phosphorylated tyrosine (Tyr-Pi), or anti-␤1-integrin Ab, and probed for ␣1C.Relatively lower amounts of ␣1C-CaL appear to be pulled down by anti-c-Src, anti-Tyr-Pi, or anti-␤1-integrin Ab (lanes 1, 2, and 3, respectively) in S 1901 A/Y 2122 F ␣1C-CaL-expressing cells, compared with WT-␣1C-CaL.B: the same blot in A after stripping and reprobing for ␤1-integrin.C: summary graph showing the relative levels of immunoprecipitated ␣1C in WT-or S 1901 A/Y 2122 F-CaL.Values were obtained as described in METHODS and are based on the average of at least three experiments.*P Ͻ 0.05 vs. WT ϩ anti-␤1-integrin Ab, WT ϩ anti-Tyr-Pi Ab, or WT ϩ anti-c-Src Ab.

Fig. 8 .
Fig. 8. Confocal immunofluorescence image analysis to determine the distribution and association of ␣1C-CaL and ␤1-integrin on the plasma membrane.Representative confocal immunofluorescence images from the cells expressing WT-, truncated ␣1C-, S 1901 A/Y 2122 F-, or ⌬P1/⌬P2-CaL.␣1C and endogenous ␤1-integrin expression on the plasma membrane appear as green and red punctate staining in a, e, i, and m and in b, f, j, and n, respectively.The colocalization of ␣1C-CaL with ␤1-integrin on the membrane appears as yellow to orange punctate staining (c, g, k, and o).A noticeably higher degree of ␣1C-CaL colocalization with ␤1-integrin was seen in cells expressing WT-␣1C-CaL, compared with mutant ␣1C-CaL construct-expressing cells.Merged images of the previous three panels with nuclei counterstaining appear in blue (d, h, l, and p).Endogenous paxillin and vinculin staining in HEK293-T cells appears as green and red in q and r, respectively.The degree of colocalization between paxillin and vinculin (yellow to orange staining; s) appears to be much higher than that between ␣1C-CaL and ␤1-integrin in the WT-or mutant ␣1C-CaL-expressing cells.Merged image with nuclear counterstaining in blue appears in t.Magnification, ϫ63 oil; scale bar, 10 m.

Fig. 9 .
Fig. 9. Bar graph showing the quantified degree of ␣1C-CaL association with ␤1-integrin normalized to the degree of paxillin (Pax)-vinculin (Vin) association using intensity correlation analysis (ICA).The association of paxillin with vinculin was set as 100%.The ICA value of ␣1C-CaL-␤1-integrin association in WT ␣1C-CaL, truncated ␣1C-CaL, S 1901 A/Y 2122 F-␣1C-CaL, or ⌬P1/⌬P2 ␣1C-CaL is presented as the percentage of paxillin-vinculin association.Values were taken from at least 8 cells in either WT-or mutant CaL per experiment, and the values in the graph were calculated on the basis of the average of at least four experiments.Statistical analysis was performed on the basis of the average from at least four individual experiments.#P Ͻ 0.01 vs. the degree of colocalization between paxillin and vinculin.

Table 1 .
Semiquantitative analysis of Ca L association with ␤ 1 -integrin