Human Molecular Genetics, 2003, Vol. 12, No. 2 155-167
© 2003 Oxford University Press
Interaction of 
PIX (ARHGEF6) with ß-parvin (PARVB) suggests an involvement of PIX in integrin-mediated signaling
Institut für Humangenetik, Universitätsklinikum Hamburg-Eppendorf, Butenfeld 42, 22529 Hamburg, Germany
Received September 19, 2002; Accepted November 16, 2002
Members of the Rho GTPase family are key regulatory molecules that link surface receptors to the organization of the actin cytoskeleton. It is now well established that these small GTPases are also crucial for neuronal morphogenesis and connectivity. Moreover, mutations in ARHGEF6 (also known as 
PIX or Cool-2 ), encoding a Rac1/Cdc42-specific guanine nucleotide exchange factor, have been implicated in X-linked mental retardation. In an attempt to get insight into the biological function of ARHGEF6 and the upstream signaling cascades leading to its activation, we used the full-length coding region of ARHGEF6 as bait in yeast-two hybrid screens and identified PARVB (ß-parvin or affixin) as a novel binding partner. The interaction was confirmed by co-immunoprecipitation and GST pull-down. We showed by immunofluorescence that ARHGEF6 and PARVB co-localize at the cell periphery to lamellipodia and ruffles in well-spread and actively spreading cells adhered to fibronectin. In addition, interaction of ARHGEF6 to ARHGEF7 (ßPIX or Cool-1), a close homolog of ARHGEF6, was confirmed. In in vivo assays, two ARHGEF6 mutations identified previously in patients with X-linked non-specific mental retardation, ARHGEF6 
aa56–83 and aa396–776, abolished interaction of ARHGEF6 to PARVB. Binding between ARHGEF6 and ARHGEF7 was not affected by ARHGEF6 aa56–83 but did not occur with ARHGEF6 aa396–776. These data suggest that both the N-terminal calponin homology (CH) and C-terminal coiled-coil domains are necessary for the ARHGEF6-PARVB binding. In contrast, it seems that only the coiled-coil domain is required for the interaction and heterodimerization of ARHGEF6 and ARHGEF7. PARVB is known to interact with integrin-linked kinase (ILK) and is involved in the early stage of cell–substrate interaction through integrins. The identification of PARVB as an ARHGEF6 interacting partner together with the co-localization of ARHGEF6 and ILK in spreading cells suggest that ARHGEF6 is involved in integrin-mediated signaling leading to activation of the GTPases Rac1 and/or Cdc42.
* To whom correspondence should be addressed. Tel: +49 40428034597; Fax: +49 40428035138; Email: kkutsche{at}uke.uni-hamburg.de
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