Structural Basis for Talin-Mediated Integrin Activation

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Platelet receptor alpha-IIb-beta3 belongs to a class of cell adhesion receptors, the integrins. alpha-IIb-beta3 mediates platelet aggregation, which is essential for hemostasis but also can result in the formation of acute occlusive thrombi, leading to heart attacks and strokes. Central to the function of alpha-IIb-beta3 is its capacity to undergo activation, a transition from a low to a high affinity/avidity state for ligand recognition. The small cytoplasmic face of alpha-IIb-beta3 regulates such transition through a distinct process called "inside-out" signaling, i.e., upon platelet stimulation by agonists, such as thrombin or ADP, the cytoplasmic face of alpha-IIb-beta3 undergoes a conformational change that propagates to the extracellutar domain allowing it to bind soluble fibrinogen or von Willebrand Factor with high affinity. The exact signal(s) or protein(s) that directly initiates this conformational change has been under intensive investigation over the past decade. Talin provides a model for one such activating signal. Talin is a cytoskeletal protein that binds directly to the alpha-IIb-beta3 cytoplasmic face and activates the receptor. Deletion and biochemical analyses revealed that the N-terminal head domain of talin (talin-H) or its smaller fragment (talin-HS) specifically recognizes the beta3 cytoplasmic tail and is responsible for inducing the alpha-IIb-beta3 activation. Interestingly, intact talin has significantly lower affinity for the beta3 tail than the isolated talin-H, suggesting a conformation-based regulatory process for the talin-beta3 interaction. A molecular level understanding of the talin-beta3 interaction in regulating the alpha-IIb-beta3 activation remains unclear. We propose to address this issue by using highly integrated and systematic structural/biochemical approaches. We will determine NMR structure of the talin-HS domain (27 kDa) in complex with the beta3 cytoplasmic tail (5 kDa) and perform structure-based mutagenesis to evaluate the significance of the interaction in mediating the integrin activation. We will also examine other factors in regulating the integrin activation such as tyrosine-phosphorylation and integrin-skelemin interaction. Finally we will investigate how the talin-H/beta3 interaction undergoes the transition from a lower affinity state to a higher affinity state. These studies will significantly impact on understanding the mechanisms of alpha-IIb-beta3 activation, which will ultimately provide insight into a fundamental process in thrombosis.