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Affinity Precipitation of Active Rho-GEFs Using a GST-tagged Mutant Rho Protein (GST-RhoA(G17A)) from Epithelial Cell Lysates
Published on: March 31, 2012
Biochemistry of the Rap-specific guanine nucleotide exchange factors PDZ-GEF1 and -2
H Bea Kuiperij1, Holger Rehmann, Fried J T Zwartkruis
1Department of Physiological Chemistry, Centre for Biomedical Genetics, University Medical Center Utrecht, Utrecht, The Netherlands.
Abstract:
PDZ-GEFs represent one of four types of highly conserved Rap-specific guanine nucleotide exchange factors. They contain a number of well-known protein domains, including a "related to cyclic nucleotide binding domain" (RCBD), a PDZ-domain, a Ras-associating domain (RA), and, of course, a catalytic domain required for their exchange activity. Since their cloning more than 5 years ago, relatively little has been learned about their mode of regulation. Although their activity may in part depend on regulated membrane localization by means of the RA and/or PDZ domain, it seems highly likely that PDZ-GEFs can be modified by additional mechanisms as well. Based on analogy of the regulatory mechanisms of the cAMP-responsive GEF Epac, in the past we postulated a role for the RCBD domain in this. In this chapter, we give a detailed description of the methods that were used to unravel this mechanism in vitro and in vivo.
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