title: Reconstructing and deconstructing agonist-induced activation of integrin alphaIIbbeta3.
abstract: BACKGROUND
  Integrin receptors, composed of transmembrane alpha and beta subunits, are essential for the development and functioning of multicellular animals. Agonist stimulation leads cells to regulate integrin affinity ("activation"), thus controlling cell adhesion and migration, controlling extracellular-matrix assembly, and contributing to angiogenesis, tumor cell metastasis, inflammation, the immune response, and hemostasis. A final step in integrin activation is the binding of talin, a cytoskeletal protein, to integrin beta cytoplasmic domains. Many different signaling molecules that regulate integrin affinity have been described, but a pathway that connects agonist stimulation to talin binding and activation has not been mapped.
  
  
  RESULTS
  We used forward, reverse, and synthetic genetics to engineer and order an integrin activation pathway in cells expressing a prototype activatable integrin, platelet alphaIIbbeta3. Phorbol myristate acetate (PMA) activated alphaIIbbeta3 only after the increased expression of both recombinant protein kinase Calpha (PKCalpha) and talin to levels approximating those in platelets. Inhibition of Rap1 GTPase reduced alphaIIbbeta3 activation, whereas activated Rap1A(G12V) bypassed the requirement for PKC, establishing that Rap1 is downstream of PKC. Talin binding to integrins mediates Rap1-induced activation because Rap1A(G12V) failed to activate alphaIIbbeta3 in cells expressing integrin binding-defective talin (W359A). Rap1 activated integrins by forming an integrin-associated complex containing talin in combination with the Rap effector, RIAM. Furthermore, siRNA-mediated knockdown of RIAM blocked integrin activation.
  
  
  CONCLUSIONS
  We have, for the first time, ordered a pathway from agonist stimulation to integrin activation and established the Rap1-induced formation of an "integrin activation complex," containing RIAM and talin, that binds to and activates the integrin.
authors.affiliation: Department of Medicine, University of California, San Diego, La Jolla, California 92093, USA.
authors.forename: Jaewon
authors.initials: J
authors.lastname: Han
authors.forename: Chinten James
authors.initials: CJ
authors.lastname: Lim
authors.forename: Naohide
authors.initials: N
authors.lastname: Watanabe
authors.forename: Alessandra
authors.initials: A
authors.lastname: Soriani
authors.forename: Boris
authors.initials: B
authors.lastname: Ratnikov
authors.forename: David A
authors.initials: DA
authors.lastname: Calderwood
authors.forename: Wilma
authors.initials: W
authors.lastname: Puzon-McLaughlin
authors.forename: Esther M
authors.initials: EM
authors.lastname: Lafuente
authors.forename: Vassiliki A
authors.initials: VA
authors.lastname: Boussiotis
authors.forename: Sanford J
authors.initials: SJ
authors.lastname: Shattil
authors.forename: Mark H
authors.initials: MH
authors.lastname: Ginsberg
mesh_terms: D000818:Animals; D001665:Binding Sites; D001792:Blood Platelets / Q000378:metabolism; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D049452:Green Fluorescent Proteins / Q000032:analysis; D006801:Humans; D008954:Models, Biological; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000819:agonists* / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000032:analysis; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D016608:Talin / Q000378:metabolism / Q000502:physiology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D020827:rap1 GTP-Binding Proteins / Q000378:metabolism / Q000502:physiology
chemical_list: D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D011993:Recombinant Fusion Proteins; D016608:Talin; D049452:Green Fluorescent Proteins; D011493:Protein Kinase C; D020827:rap1 GTP-Binding Proteins; D013755:Tetradecanoylphorbol Acetate