Characterization of the Weak Interaction of C-Src with the β3 Cytoplasmic Tail
Authors: Moore Davi d T. , Ganjiwale Anjali , Cheng Hong , Roder Heinrich , DeGrado William F. , Bennett Joel S. 
Published: April 2020

Abstract
Integrins are ubiquitously expressed α/β heterodimers that mediate cell-cell and cellextracellular matrix interactions. Integrins reside on the plasma membrane in a highly regulated dynamic equilibrium between an inactive resting state and an active ligand binding conformation. An essential feature of this equilibrium is the association and dissociation of integrin transmembrane (TM) and cytoplasmic domains. Thus, when integrins are inactive, the TM and cytoplasmic domains of their α and β subunits are in proximity; the domains separate when integrins assume their active conformations. Subsequent to ligand binding, integrins cluster to generate “outside-in” signals, believed to be initiated by the activation of integrin-associated c-Src and related kinases. The prototypic example of regulated integrin activation and outside-in signaling is the major platelet integrin αIIbβ3. We have employed exogenous αIIb TM binding peptides to stabilize the separated conformation of αIIbβ3 and better understand the link between integrin conformation and downstream signal transduction. These peptides activate αIIbβ3 independent of “inside-out” signal transduction and induce αIIbβ3-mediated platelet aggregation despite the presence of the inhibitory prostaglandin PGE1 or the metabolic inhibitors 2-deoxyglucose and NaN3. When platelets are stimulated by natural agonists such as ADP or thrombin, fibrinogen binds to αIIbβ3 after which the c-Src associated with the β3 cytoplasmic tail is activated. This results in a characteristic phosphotyrosine signal that can be detected by immunoblotting platelet lysates using phosphotyrosine-specific antibodies and can be inhibited by the αIIbβ3 antagonists abciximab and RGDS that inhibit fibrinogen binding and clustering of αIIbβ3 in the membrane. By contrast, exogenous αIIb TM binding peptides activate phosphotyrosine signaling in platelets in the presence of abciximab and RGDS. This clustering-independent signal transduction is both integrin- and Src-dependent as it is absent in β3 knockout mice and can be blocked by the Src inhibitors PP2 and dasatinib. To better understand how β3-associated Src kinases are activated, we investigated the interaction of c-Src with αIIbβ3 by NMR spectroscopy. It has been reported that the SH3 domain of c-Src interacts with the C-terminal three amino acids of β3, Arg-Gly-Thr. We synthesized short fragments of the β3 cytoplasmic domain containing this sequence and observed perturbations in the 15N-1H HSQC NMR spectrum of the c-Src SH3 domain. There were significant shifts in the backbone amide spectra for residues Arg19, Asp23, Leu24, Glu21, Thr20, Tyr55, and Asn36, as well as shifts in the spectra for the side chains of Trp42, Trp43, Asn37, and Asn59. Titration experiments revealed a dissociation constant of approximately 4 mM for β3 peptide binding to the SH3 domain. It is noteworthy that the perturbed residues are localized to one region of the SH3 domain and are located outside of the canonical polyproline binding groove, implying a new mode of SH3 interaction. Moreover, the weak interaction of c-Src with the β3 cytoplasmic domain is consistent with the high local concentrations of β3 and Src kinases at the platelet membrane and the dynamic nature of integrin protein-protein interactions. These results suggest that the cytoplasmic domain separation that accompanies αIIbβ3 activation may be sufficient to activate the Src kinases weakly associated with β3 cytoplasmic domain. Subsequent αIIbβ3 clustering and/or c-Src dissociation may then amplify the tyrosine phosphorylation signal to complete outside-in signaling.