title: Membrane-proximal {alpha}/{beta} stalk interactions differentially regulate integrin activation.
abstract: The affinity of integrin-ligand interaction is regulated extracellularly by divalent cations and intracellularly by inside-out signaling. We report here that the extracellular, membrane-proximal alpha/beta stalk interactions not only regulate cation-induced integrin activation but also play critical roles in propagating inside-out signaling. Two closely related integrins, alphaIIbbeta3 and alphaVbeta3, share high structural homology and bind to similar ligands in an RGD-dependent manner. Despite these structural and functional similarities, they exhibit distinct responses to Mn(2+). Although alphaVbeta3 showed robust ligand binding in the presence of Mn(2+), alphaIIbbeta3 showed a limited increase but failed to achieve full activation. Swapping alpha stalk regions between alphaIIb and alphaV revealed that the alpha stalk, but not the ligand-binding head region, was responsible for the difference. A series of alphaIIb/alphaV domain-swapping chimeras were constructed to identify the responsible domain. Surprisingly, the minimum component required to render alphaIIbbeta3 susceptible to Mn(2+) activation was the alphaV calf-2 domain, which does not contain any divalent cation-binding sites. The calf-2 domain makes interface with beta epidermal growth factor 4 and beta tail domain in three-dimensional structure. The effect of calf-2 domain swapping was partially reproduced by mutating the specific amino acid residues in the calf-2/epidermal growth factor 4-beta tail domain interface. When this interface was constrained by an artificially introduced disulfide bridge, the Mn(2+)-induced alphaVbeta3-fibrinogen interaction was significantly impaired. Notably, a similar disulfide bridge completely abrogated fibrinogen binding to alphaIIbbeta3 when alphaIIbbeta3 was activated by cytoplasmic tail truncation to mimic inside-out signaling. Thus, disruption/formation of the membrane-proximal alpha/beta stalk interface may act as an on/off switch that triggers integrin-mediated bidirectional signaling.
authors.affiliation: Departments of Anatomy, Transfusion Medicine and Cell Therapy, and Internal Medicine, Keio University School of Medicine, Tokyo 160-8582, Japan. kamata@sc.itc.keio.ac.jp
authors.forename: Tetsuji
authors.initials: T
authors.lastname: Kamata
authors.forename: Makoto
authors.initials: M
authors.lastname: Handa
authors.forename: Yukiko
authors.initials: Y
authors.lastname: Sato
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Ikeda
authors.forename: Sadakazu
authors.initials: S
authors.lastname: Aiso
mesh_terms: D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D002412:Cations; D006224:Cricetinae; D018076:DNA, Complementary / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry; D005340:Fibrinogen / Q000737:chemistry; D005434:Flow Cytometry; D039302:Integrin alphaVbeta3 / Q000737:chemistry* / Q000378:metabolism; D008024:Ligands; D008345:Manganese / Q000737:chemistry; D008954:Models, Biological; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000737:chemistry* / Q000378:metabolism; D010980:Platelet Membrane Glycoproteins / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D015398:Signal Transduction; D014162:Transfection
chemical_list: D002412:Cations; D018076:DNA, Complementary; D004220:Disulfides; D039302:Integrin alphaVbeta3; D008024:Ligands; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D010980:Platelet Membrane Glycoproteins; D008345:Manganese; D005340:Fibrinogen