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.
Kuiperij
H Bea
HB
Department of Physiological Chemistry, Centre for Biomedical Genetics, University Medical Center Utrecht, Utrecht, The Netherlands.
Rehmann
Holger
H
Zwartkruis
Fried J T
FJ
D000818:Animals; D002151:Calorimetry; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000378:metabolism; D020662:Guanine Nucleotide Exchange Factors / Q000032:analysis / Q000378:metabolism*; D006801:Humans; D051379:Mice; D041681:NIH 3T3 Cells; D009419:Nerve Tissue Proteins / Q000032:analysis / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D051381:Rats; D018411:Spodoptera
D020662:Guanine Nucleotide Exchange Factors; D009419:Nerve Tissue Proteins; C403525:RAPGEF2 protein, human