Biochemical and Functional Characterization of the Interaction between Liprin-α1 and GIT1: Implications for the Regulation of Cell Motility

We have previously identified the scaffold protein liprin-alpha 1 as an important regulator of integrin-mediated cell motility and tumor cell invasion. Liprin-alpha 1 may interact with different proteins, and the functional significance of these interactions in the regulation of cell motility is poorly known. Here we have addressed the involvement of the liprin-alpha 1 partner GIT1 in liprin-alpha 1-mediated effects on cell spreading and migration. GIT1 depletion inhibited spreading by affecting the lamellipodia, and prevented liprin-alpha 1-enhanced spreading. Conversely inhibition of the formation of the liprin-alpha 1-GIT complex by expression of liprin-Delta CC3 could still enhance spreading, although to a lesser extent compared to full length liprin-alpha 1. No cumulative effects were observed after depletion of both liprin-alpha 1 and GIT1, suggesting that the two proteins belong to the same signaling network in the regulation of cell spreading. Our data suggest that liprin-alpha 1 may compete with paxillin for binding to GIT1, while binding of beta PIX to GIT1 was unaffected by the presence of liprin-alpha 1. Interestingly, GIT and liprin-alpha 1 reciprocally regulated their subcellular localization, since liprin-alpha 1 overexpression, but not the GIT binding-defective liprin-Delta CC3 mutant, affected the localization of endogenous GIT at peripheral and mature central focal adhesions, while the expression of a truncated, active form of GIT1 enhanced the localization of endogenous liprin-alpha 1 at the edge of spreading cells. Moreover, GIT1 was required for liprin-alpha 1-enhanced haptotatic migration, although the direct interaction between liprin-alpha 1 and GIT1 was not needed. Our findings show that the functional interaction between liprin-alpha 1 and GIT1 cooperate in the regulation of integrin-dependent cell spreading and motility on extracellular matrix. These findings and the possible competition of liprin-alpha 1 with paxillin for binding to GIT1 suggest that alternative binding of GIT1 to either liprin-alpha 1 or paxillin plays distinct roles in different phases of the protrusive activity in the cell.