Novel Rap1 dominant-negative mutants interfere selectively with C3G and Epac.
Rap1 is a Ras-related GTPase that is principally involved in integrin- and E-cadherin-mediated adhesion. Rap1 is transiently activated in response to many incoming signals via a large family of guanine nucleotide exchange factors (GEFs). The lack of potent Rap1 dominant-negative mutants has limited our ability to decipher Rap1-dependent pathways; we have therefore developed a procedure to generate such mutants consisting in the oligonucleotide-mediated mutagenesis of residues 14-19, selection of mutants presenting an enhanced interaction with Epac2 by yeast two-hybrid screening and counter-screening for mutants still interacting with Rap effectors. In detail analysis of their interaction capacity with various Rap-GEFs in the yeast two-hybrid system revealed that mutants of residues 15 and 16 interacted with Epacs, C3G and CalDAG-GEFI, whereas mutants of position 17 had selectively lost their ability to bind CalDAG-GEFI as well as, for some, C3G. In cellular models where Rap1 is activated via endogenous GEFs, the Rap1[S17A] mutant inhibits both the cAMP-Epac and EGF-C3G pathways, whereas Rap1[G15D] selectively interferes with the latter. Finally, Rap1[S17A] is able to act as a bona fide dominant-negative mutant in vivo since it phenocopies the eye-reducing and lethal effects of D-Rap1 deficiency in Drosophila, effects that are overcome by the overexpression of D-Epac or D-Rap1.
Dupuy
Aurélien G
AG
Inserm U528, Institut Curie, 26 rue d'Ulm, 75005 Paris, France.
L'Hoste
Sébastien
S
Cherfils
Jacqueline
J
Camonis
Jacques
J
Gaudriault
Georges
G
de Gunzburg
Jean
J
D000595:Amino Acid Sequence; D000818:Animals; D030801:Animals, Genetically Modified; D001483:Base Sequence; D001665:Binding Sites; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D003176:Complement C3 / Q000235:genetics / Q000378:metabolism*; D003179:Complement C3b; D000242:Cyclic AMP / Q000378:metabolism; D004331:Drosophila melanogaster / Q000235:genetics / Q000254:growth & development; D005124:Eye Abnormalities / Q000235:genetics; D005799:Genes, Dominant; D005804:Genes, Lethal; D020662:Guanine Nucleotide Exchange Factors / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D015398:Signal Transduction; D020827:rap1 GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*
D002352:Carrier Proteins; D003176:Complement C3; D020662:Guanine Nucleotide Exchange Factors; C116122:RAPGEF3 protein, human; C415483:RASGRP2 protein, human; C486707:Rapgef4 protein, mouse; C034772:complement C3g; D003179:Complement C3b; D000242:Cyclic AMP; D020827:rap1 GTP-Binding Proteins