Biochemistry of the Rap‐Specific Guanine Nucleotide Exchange Factors PDZ‐GEF1 and ‐2

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.