Abstract
Engagement of α(5)β(1)-integrin by fibronectin (FN) acutely enhances Cav1.2 channel (Ca(L)) current in rat arteriolar smooth muscle and human embryonic kidney cells (HEK293-T) expressing Ca(L). Using coimmunoprecipitation strategies, we show that coassociation of Ca(L) with α(5)- or β(1)-integrin in HEK293-T cells is specific and depends on cell adhesion to FN. In rat arteriolar smooth muscle, coassociations between Ca(L) and α(5)β(1)-integrin and between Ca(L) and phosphorylated c-Src are also revealed and enhanced by FN treatment. Using site-directed mutagenesis of Ca(L) heterologously expressed in HEK293-T cells, we identified two regions of Ca(L) required for these interactions: 1) COOH-terminal residues Ser(1901) and Tyr(2122), 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 Ca(L) colocalization with β(1)-integrin on the plasma membrane. Collectively, our results strongly suggest that 1) upon ligation by FN, Ca(L) associates with α(5)β(1)-integrin in a macromolecular complex including PKA, c-Src, and potentially other protein kinases; 2) phosphorylation of Ca(L) at Y(2122) and/or S(1901) is required for association of Ca(L) with α(5)β(1)-integrin; and 3) c-Src, via binding to PRDs that reside in the II-III linker region and/or the COOH terminus of Ca(L), mediates current potentiation following α(5)β(1)-integrin engagement. These findings provide new evidence for how interactions between α(5)β(1)-integrin and FN can modulate Ca(L) entry and consequently alter the physiological function of multiple types of excitable cells.
Highlights
Spatial association of the Cav1.2 calcium channel with αJu5n-βT1zu-iCnhtaeo,gPreiicnhun Gui, Gerald W
We present evidence that the pore-forming ␣1C-subunit of the L-type calcium channel (␣1C-CaL) associates with 1integrin and c-Src in arteriolar smooth muscle (SM) as well as when the channel is heterologously expressed in HEK293-T cells
These associations depend on interactions of the cells with the extracellular matrix proteins (ECM) protein FN, which is known from previous studies to potentiate CaL current following ␣51-integrin engagement [40]
Summary
Spatial association of the Cav1.2 calcium channel with αJu5n-βT1zu-iCnhtaeo,gPreiicnhun Gui, Gerald W. Ca2ϩ entry through the L-type voltage-gated Cav1.2 calcium channel (CaL) is required for stretch-induced contraction of vascular smooth muscle (VSM). 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]. The effects of integrin antibodies on myogenic tone are mediated in part by altered Ca2ϩ entry through CaL because electrophysiological studies reveal selective effects of integrin ligands on CaL current; the engagement and clustering of ␣51-integrin by insoluble fibronectin (FN) or anti-␣5-integrin antibody acutely potentiates CaL current, whereas engagement of ␣v3-integrin by vitronectin or soluble ligands inhibits CaL current in isolated VSM cells [41]. Using human embryonic kidney cells (HEK293-T) overexpressing the neuronal isoform of CaL and VSM cells from rat skeletal muscle arterioles as model systems, the interactions between endogenous ␣51integrin and CaL and between c-Src and CaL were examined using immunoprecipitation (IP), immunoblotting (IB), immunofluorescence confocal microscopy, and patch-clamp methods
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