enous paxillin coprecipitated with DCC-2036 FLAG-GIT1 that includes the full paxillin binding region . On the other hand, constructs including short deletions at the carboxy-terminus abolished the interaction with both liprin-a1 and paxillin. Altogether the results show that an extended region of the carboxy-terminal portion of GIT1 is required for efficient interaction with liprin-a1, and that the region of GIT1 required for the binding to liprin-a1 includes the paxillin-binding region. Based on these findings, we tested the hypothesis that liprin-a1 may interfere with the binding of paxillin to the carboxy-terminus of GIT1 in the cell. For this, we first immunoprecipitated endogenous paxillin from lysates of cells transfected either with HA-GIT1-C2 alone, or with both HA-GIT1-C2 and full length FLAG-liprin-a1. Under conditions in which endogenous paxillin was virtually immunodepleted from lysates, the interaction of paxillin with HA-GIT1-C2 was strongly reduced in the lysates 
from co-transfected cells. We then tested the hypothesis that the decrease of binding of paxillin to GIT1-C2 may be due to binding of the overexpressed liprin-a1 to GIT1-C2 itself. For this, the unbound fraction after immunoprecipitation with anti-paxillin from lysates of cells cotransfected with HA-GIT1-C2 and FLAG-liprin-a1, was used in a second round of immunoprecipitation with anti-liprin-a1 antibody. This immunoprecipitation showed a strong interaction of FLAG-liprin-a1 with HA-GIT1-C2. These data suggest that binding of overexpressed liprin-a1 to the carboxy-terminal portion of GIT1 interferes with the binding of paxillin to the same region of GIT1, and indicate that the formation of a trimeric liprin-a1/GIT1/paxillin complex in the cell is not likely. GIT1 and bPIX form stable hetero-complexes in COS7 cells. We thus tested if bPIX binding to the SHD domain of GIT1 interfered with the binding of liprin-a1 to the contiguous GIT1 carboxy-terminus. We used co-immunoprecipitation from transfected cell lysates to test for the possible interference between liprin-a1 and bPIX binding to GIT1. COS7 cells co-transfected with HA-GIT1-C2 and HA-bPIX, with HA-GIT1-C2 and FLAGLiprin-a1, or triple-transfected with HA-GIT1-C2, HA-bPIX and FLAG-Liprin-a1 were immunoprecipitated with anti-FLAG antibodies. Similar amounts of GIT1-C2 were co-immunoprecipitated with anti-liprin-a1 antibodies in the presence or absence of bPIX, indicating that binding of liprin-a1 to GIT1-C2 did not affect the interaction of GIT1-C2 with bPIX. These results indicate that GIT1 may be found in complex with both bPIX and liprin-a1 at the same time. On the other side, we found that immunoprecipitation of bPIX from co-transfected cells resulted in efficient co-precipitation of GIT1-C2 both in the presence and absence of liprin-a1. These results show that a trimeric bPIX/GIT1/Liprin-a1 complex may form in the cell. June 2011 | Volume 6 | Issue 6 | e20757 Liprin-a1 and GIT1 Regulate Migration 3 June 2011 | Volume 6 | Issue 6 | e20757 Liprin-a1 and GIT1 Regulate Migration FLAG-GIT1, or with FLAG-liprin-a1 and FLAG-GIT1 and HA-bPIX. 200 mg of each lysate were immunoprecipitated with anti-GIT1 antiserum. Lysates, unbound fractions and immunoprecipitates were blotted and incubated with antibodies specific for the indicated proteins. Overexpression of bPix did not increase the interaction of liprin-a1 with GIT1. Model for the regulated interaction of GIT1 with paxillin and liprina1. Either ligand binds poorly to fu
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