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Methods Enzymol. 2006;407:174-86.

Biochemistry of the Rap-specific guanine nucleotide exchange factors PDZ-GEF1 and -2.

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Department of Physiological Chemistry, Centre for Biomedical Genetics, University Medical Center Utrecht, Utrecht, The Netherlands.


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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