Mechanism of regulation of the Epac family of cAMP-dependent RapGEFs.
Epac1 (cAMP-GEFI) and Epac2 (cAMP-GEFII) are closely related guanine nucleotide exchange factors (GEFs) for the small GTPase Rap1, which are directly regulated by cAMP. Here we show that both GEFs efficiently activate Rap2 as well. A third member of the family, Repac (GFR), which lacks the cAMP dependent regulatory sequences, is a constitutive activator of both Rap1 and Rap2. In contrast to Epac1, Epac2 contains a second cAMP binding domain at the N terminus, as does the Epac homologue from Caenorhabditis elegans. Affinity measurements show that this distal cAMP binding domain (the A-site) binds cAMP with much lower affinity than the cAMP binding domain proximal to the catalytic domain (the B-site), which is present in both Epac1 and Epac2. Deletion mutant analysis shows that the high affinity cAMP binding domains are sufficient to regulate the GEFs in vitro. Interestingly, isolated fragments containing the B-sites of either Epac1 or Epac2, but not the A-site from Epac2, inhibit the catalytic domains in trans. This inhibition is relieved by the addition of cAMP. In addition to the cAMP binding domains, both Epac1 and Epac2 have a DEP domain. Deletion of this domain does not affect regulation of Epac1 activity but affects membrane localization. From these results, we conclude that all three members of the Epac family regulate both Rap1 and Rap2. Furthermore, we conclude that the catalytic activity of Epac1 is constrained by a direct interaction between GEF and high affinity cAMP binding domains in the absence of cAMP. Epac1 becomes activated by a release of this inhibition when cAMP is bound.
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