The GIT proteins, GIT1 and GIT2, are GTPase-activating proteins for the ADP-ribosylation factor family of small GTP binding proteins, but also serve as adaptors to link signaling proteins to distinct cellular locations. One role for GIT proteins is to link the PIX family of Rho guanine nucleotide exchange factors and their binding partners, the p21-activated protein kinases, to remodeling focal adhesions by interacting with the focal adhesion adaptor protein paxillin. We here identified the Cterminal domain of GIT1 responsible for paxillin binding. Combining structural and mutational analysis, we show that this region folds into an antiparallel four-helix domain highly reminiscent to the focal adhesion targeting (FAT) domain of focal adhesion kinase (FAK). Our results suggest that the GIT1 FAT-homology (FAH) domain and FAT bind the paxillin LD4 motif quite similarly. Since only a small fraction of GIT1 is bound to paxillin under normal conditions, regulation of paxillin binding was explored. Although paxillin binding to the FAT domain of FAK is regulated by tyrosine phosphorylation within this domain, we find that tyrosine phosphorylation of the FAH domain GIT1 is not involved in regulating binding to paxillin. Instead, we find that mutations within the FAH domain may alter binding to paxillin that has been phosphorylated within the LD4 motif. Thus, despite apparent structural similarity in their FAT domains, GIT1 and FAK binding to paxillin is differentially regulated. KeywordsGIT1 protein; paxillin; focal adhesion targeting domain; PIX protein; Arf GTPase-activating protein; guanine nucleotide exchange factor The GIT and PIX proteins tightly associate to form an oligomeric complex that functions as a recruitable scaffold for various signaling proteins [1]. The GIT proteins, GIT1 and GIT2 (also known as p95-APP1/2, p95-PKL, and Cat-2 proteins), are GTPase-activating proteins for the Arf family of small GTP-binding proteins [2][3][4]. The PIX proteins, α-PIX and β-PIX (also known as Cool-1/2, p85-SPR and ARHGEF6/7 proteins), are guanine nucleotide exchange 4To whom correspondence should be addressed: richard.premont@duke.edu.. 3 These authors contributed equally to this work.Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access Author ManuscriptCell Signal. Author manuscript; available in PMC 2008 August 1. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript factors for the Rho family of small GTP-binding proteins [5]. In addition to functioning as regulators of small GTP-binding proteins, GIT/PIX complexes have been reported to ...

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