Functional analyses of the human leukotriene C4 synthase gene promoter molecular haplotypes

Integrin receptors, composed of transmembrane α and β 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 β 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.We used forward, reverse, and synthetic genetics to engineer and order an integrin activation pathway in cells expressing a prototype activatable integrin, platelet αIIbβ3. Phorbol myristate acetate (PMA) activated αIIbβ3 only after the increased expression of both recombinant protein kinase Cα (PKCα) and talin to levels approximating those in platelets. Inhibition of Rap1 GTPase reduced αIIbβ3 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 αIIbβ3 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.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.