title: Spatial association of the Cav1.2 calcium channel with α5β1-integrin.
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.
authors.affiliation: Dept. of Medical Pharmacology and Physiology, M451 Med. Sci. Bldg., Univ. of Missouri, Columbia, 1 Hospital Dr., Columbia, MO 65211, USA.
authors.forename: Jun-Tzu
authors.initials: JT
authors.lastname: Chao
authors.forename: Peichun
authors.initials: P
authors.lastname: Gui
authors.forename: Gerald W
authors.initials: GW
authors.lastname: Zamponi
authors.forename: George E
authors.initials: GE
authors.lastname: Davis
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Davis
mesh_terms: D000818:Animals; D020746:Calcium Channels, L-Type / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020013:Calcium Signaling / Q000187:drug effects / Q000235:genetics*; D002448:Cell Adhesion / Q000235:genetics / Q000502:physiology; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D002478:Cells, Cultured; D004847:Epithelial Cells / Q000737:chemistry / Q000378:metabolism*; D005353:Fibronectins / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D057809:HEK293 Cells; D006801:Humans; D039081:Integrin alpha5beta1 / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D018613:Microscopy, Confocal; D009131:Muscle, Smooth, Vascular / Q000737:chemistry / Q000166:cytology / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation / Q000235:genetics / Q000502:physiology; D051381:Rats
chemical_list: D020746:Calcium Channels, L-Type; D005353:Fibronectins; D039081:Integrin alpha5beta1; C419385:L-type calcium channel alpha(1C)