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-α1 as an important regulator of integrin-mediated cell motility and tumor cell invasion. Liprin-α1 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-α1 partner GIT1 in liprin-α1-mediated effects on cell spreading and migration.

Liprin-alpha1 promotes cell spreading on the extracellular matrix by affecting the distribution of activated integrins.

Integrin activation is needed to link the extracellular matrix with the actin cytoskeleton during cell motility. Protrusion requires coordination of actin dynamics with focal-adhesion turnover. We report that the adaptor protein liprin-alpha1 is stably associated with the cell membrane.

Identification of an intramolecular interaction important for the regulation of GIT1 functions.

G-protein coupled receptor kinase-interacting protein (GIT) proteins include an N-terminal Arf GTPase-activating protein domain, and a C terminus that binds proteins regulating adhesion and motility. Given their ability to form large molecular assemblies, the GIT1 protein must be tightly regulated. However, the mechanisms regulating GIT1 functions are poorly characterized.

betaPIX controls cell motility and neurite extension by regulating the distribution of GIT1.

Cell motility entails the reorganization of the cytoskeleton and membrane trafficking for effective protrusion. GIT1/p95-APP1 is a member of a family of GTPase-activating proteins for ARF GTPases that affect endocytosis, adhesion and migration. GIT1 associates with paxillin and a complex including the Rac/Cdc42 exchanging factors PIX/Cool and the kinase PAK.

Assay and properties of the GIT1/p95-APP1 ARFGAP.

GIT1/p95-APP1 is an adaptor protein with an aminoterminal ARFGAP domain involved in the regulation of ARF6 function. GIT1/p95-APP1 forms stable complexes with a number of proteins including downstream effectors and exchanging factors for members of the Rho family of small GTPases. This protein can also interact with other adaptor proteins implicated in the regulation of cell adhesion and synapse formation.

Characterization of the endogenous GIT1-betaPIX complex, and identification of its association to membranes.

G protein-coupled receptor kinase interactors (GITs) are adaptor proteins with ADP-ribosylating factor--GTPase-activating protein (ARF-GAP) activity that form complexes with the p21-activated kinase-interacting exchange factor (PIX) guanine nucleotide exchanging factors for Rac and Cdc42. In this study we have characterized the endogenous GIT1/p95-APP1/Cat1 (GIT1)- PIX complexes in neuronal and non-neuronal cells. In COS7 cells, immunocytochemical analysis shows the localization of endogenous GIT1 in the perinuclear region of the cell, as well as at the cell periphery, where GIT1 co-localizes with filamentous actin.

Generation and characterization of Rac3 knockout mice.

Rac proteins are members of the Rho family of GTPases involved in the regulation of actin dynamics. The three highly homologous Rac proteins in mammals are the ubiquitous Rac1, the hematopoiesis-specific Rac2, and the least-characterized Rac3. We show here that Rac3 mRNA is widely and specifically expressed in the developing nervous system, with highest concentration at embryonic day 13 in the dorsal root ganglia and ventral spinal cord.

ADP-ribosylation factor 6 and a functional PIX/p95-APP1 complex are required for Rac1B-mediated neurite outgrowth.

The mechanisms coordinating adhesion, actin organization, and membrane traffic during growth cone migration are poorly understood. Neuritogenesis and branching from retinal neurons are regulated by the Rac1B/Rac3 GTPase. We have identified a functional connection between ADP-ribosylation factor (Arf) 6 and p95-APP1 during the regulation of Rac1B-mediated neuritogenesis.

Leucine-zipper-mediated homo- and hetero-dimerization of GIT family p95-ARF GTPase-activating protein, PIX-, paxillin-interacting proteins 1 and 2.

ADP-ribosylation factor GTPase-activating proteins (ARFGAPs) of the G-protein-coupled receptor kinase interactor 1/p95 paxillin kinase linker/p95-ARFGAP Pak-interacting exchange factor paxillin-binding protein (APP)-1 family are multidomain proteins, which interact functionally with both ARF and Rac GTPases. These proteins are involved in the dynamic reorganization of adhesion and the cytoskeleton during cell motility. Our previous work [Di Cesare, Paris, Albertinazzi, Dariozzi, Andersen, Mann, Longhi and de Curtis (2000) Nat.

Analysis of the subcellular distribution of avian p95-APP2, an ARF-GAP orthologous to mammalian paxillin kinase linker.

We describe here the identification and characterization of avian p95-APP2, a multi-domain protein of a recently identified family of ADP-ribosylation factor (ARF)-GTPase-activating proteins (GAPs) including mammalian G protein-coupled receptor kinases (GRK)-interactor 1 (GIT1), paxillin kinase linker (PKL), and GIT2, as well as avian p95-APP1. The p95-APP2 is eluted from Rac-GTP-gamma-S, but not from Rac-GDP-beta-S columns. As other members of the family, p95-APP2 has binding regions for the focal adhesion protein paxillin, and for the Rac exchanging factor PIX.