title: Integrin cytoplasmic domains mediate inside-out signal transduction.
abstract: We analyzed the binding of fibronectin to integrin alpha 5 beta 1 in various cells; in some cells fibronectin bound with low affinity (e.g., K562 cells) whereas in others (e.g., CHO), it bound with high affinity (Kd approximately 100 nM) in an energy-dependent manner. We constructed chimeras of the extracellular and transmembrane domains of alpha IIb beta 3 joined to the cytoplasmic domains of alpha 5 beta 1. The affinity state of these chimeras was assessed by binding of fibrinogen or the monoclonal antibody, PAC1. The cytoplasmic domains of alpha 5 beta 1 conferred an energy-dependent high affinity state on alpha IIb beta 3 in CHO but not K562 cells. Three additional alpha cytoplasmic domains (alpha 2, alpha 6A, alpha 6B) conferred PAC1 binding in CHO cells, while three others (alpha M, alpha L, alpha v) did not. In the high affinity alpha chimeras, cotransfection with a truncated (beta 3 delta 724) or mutated (beta 3(S752-->P)) beta 3 subunit abolished high affinity binding. Thus, both cytoplasmic domains are required for energy-dependent, cell type-specific affinity modulation. In addition, mutations that disrupted a highly conserved alpha subunit GFFKR motif, resulted in high affinity binding of ligands to alpha IIb beta 3. In contrast to the chimeras, the high affinity state of these mutants was independent of cellular metabolism, cell type, and the bulk of the beta subunit cytoplasmic domain. Thus, integrin cytoplasmic domains mediate inside-out signaling. Furthermore, the highly conserved GFFKR motif of the alpha subunit cytoplasmic domain maintains the default low affinity state.
authors.affiliation: Department of Vascular Biology, Scripps Research Institute, La Jolla, California 92037.
authors.forename: T E
authors.initials: TE
authors.lastname: O'Toole
authors.forename: Y
authors.initials: Y
authors.lastname: Katagiri
authors.forename: R J
authors.initials: RJ
authors.lastname: Faull
authors.forename: K
authors.initials: K
authors.lastname: Peter
authors.forename: R
authors.initials: R
authors.lastname: Tamura
authors.forename: V
authors.initials: V
authors.lastname: Quaranta
authors.forename: J C
authors.initials: JC
authors.lastname: Loftus
authors.forename: S J
authors.initials: SJ
authors.lastname: Shattil
authors.forename: M H
authors.initials: MH
authors.lastname: Ginsberg
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002460:Cell Line; D017124:Conserved Sequence; D006224:Cricetinae; D003593:Cytoplasm / Q000737:chemistry; D004734:Energy Metabolism; D005353:Fibronectins / Q000378:metabolism*; D006801:Humans; D016023:Integrins / Q000737:chemistry / Q000378:metabolism*; D008024:Ligands; D008969:Molecular Sequence Data; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D017469:Receptors, Fibronectin; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction; D014162:Transfection
chemical_list: D005353:Fibronectins; D016023:Integrins; D008024:Ligands; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D017469:Receptors, Fibronectin; D011993:Recombinant Fusion Proteins