title: Characterisation of PDZ-GEFs, a family of guanine nucleotide exchange factors specific for Rap1 and Rap2.
abstract: PDZ-GEF1 (RA-GEF/nRapGEP/CNrasGEF) is a guanine nucleotide exchange factor (GEF) characterised by the presence of a PSD-95/DlgA/ZO-1 (PDZ) domain, a Ras-association (RA) domain and a region related to a cyclic nucleotide binding domain (RCBD). These domains are in addition to a Ras exchange motif (REM) and GEF domain characteristic for GEFs for Ras-like small GTPases. PDZ-GEF1 efficiently exchanges nucleotides of both Rap1 and Rap2, but has also been implicated in mediating cAMP-induced Ras activation through binding of cAMP to the RCBD. Here we describe a new family member, PDZ-GEF2, of which we isolated two splice variants (PDZ-GEF2A and 2B). PDZ-GEF2 contains, in addition to the domains characteristic for PDZ-GEF1, a second, less conserved RCBD at the N-terminus. PDZ-GEF2 is also specific for both Rap1 and Rap2. We further investigated the possibility that PDZ-GEF2, like PDZ-GEF1, is a cAMP-responsive GEF for Ras. However, in contrast to previous results, we did not find any effect of either PDZ-GEF1 or PDZ-GEF2 on Ras in the absence or presence of cAMP. Moreover, affinity measurements by isothermic calorimetry showed that the RCBD of PDZ-GEF1 does not bind cAMP with a physiologically relevant affinity. We conclude that both PDZ-GEF1 and 2 are specific for Rap1 and Rap2 and unresponsive to cAMP and various other nucleotides.
authors.affiliation: Department of Physiological Chemistry and Centre for Biomedical Genetics, University Medical Centre Utrecht, Universiteitsweg 100, 3584 CG, Utrecht, The Netherlands.
authors.forename: H Bea
authors.initials: HB
authors.lastname: Kuiperij
authors.forename: Johan
authors.initials: J
authors.lastname: de Rooij
authors.forename: Holger
authors.initials: H
authors.lastname: Rehmann
authors.forename: Miranda
authors.initials: M
authors.lastname: van Triest
authors.forename: Alfred
authors.initials: A
authors.lastname: Wittinghofer
authors.forename: Johannes L
authors.initials: JL
authors.lastname: Bos
authors.forename: Fried J T
authors.initials: FJ
authors.lastname: Zwartkruis
mesh_terms: D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D000242:Cyclic AMP / Q000494:pharmacology; D020662:Guanine Nucleotide Exchange Factors / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins; D020668:rap GTP-Binding Proteins / Q000378:metabolism; D020827:rap1 GTP-Binding Proteins / Q000378:metabolism*
chemical_list: D020662:Guanine Nucleotide Exchange Factors; D009419:Nerve Tissue Proteins; C403525:RAPGEF2 protein, human; D000242:Cyclic AMP; C492137:Rap2a protein, mouse; D020668:rap GTP-Binding Proteins; D020827:rap1 GTP-Binding Proteins