title: The N-terminal coiled-coil domain of the cytohesin/ARNO family of guanine nucleotide exchange factors interacts with Galphaq.
abstract: Cytohesins are guanine-nucleotide exchange factors (GEF) for the Arf family of GTPases. One member of the Arf family, ARF6, plays an active role in the intracellular trafficking of G protein-coupled receptors. We have previously reported that Galphaq signaling leads to the activation of ARF6, possibly through a direct interaction with cytohesin-2/ARNO. Here, we report that Galphaq can directly interact with cytohesin-1, another Arf-GEF of the ARNO/cytohesin family. Cytohesin-1 preferentially associated with a constitutively active mutant of Galphaq (Galphaq-Q209L) compared to wild-type Galphaq in HEK293 cells. Stimulation of TPbeta, a Galphaq-coupled receptor, to activate Galphaq resulted in the promotion of a protein complex between Galphaq and cytohesin-1. Confocal immunofluorescence microscopy revealed that wild-type Galphaq and cytohesin-1 co-localized in intracellular compartments and at or near the plasma membrane. In contrast, expression of Galphaq-Q209L induced a drastic increase in the localization of cytohesin-1 at the plasma membrane. Expression of a dominant-negative mutant of cytohesin-1 reduced by 40% the agonist-induced internalization of TPbeta, a process that we previously demonstrated to be dependent on Galphaq-mediated signaling and Arf6 activation. Using deletion mutants, we show that cytohesin-1 interacts with Galphaq through its N-terminal coiled-coil domain. Cytohesin-1 and cytohesin-2/ARNO mutants lacking the coiled-coil domain were unable to relay Galphaq-mediated activation of Arf6. This is the first report of an interaction between the coiled-coil domain of the cytohesin/ARNO family of Arf-GEFs and a member of the heterotrimeric G proteins.
authors.affiliation: Division of Rheumatology, Department of Medicine, Faculty of Medicine, Centre de Recherche Clinique Etienne-Lebel, University of Sherbrooke, 3001, 12th Avenue North, J1H 5N4 Fleurimont, Sherbrooke, QC, Canada.
authors.forename: Geneviève
authors.initials: G
authors.lastname: Laroche
authors.forename: Patrick M
authors.initials: PM
authors.lastname: Giguère
authors.forename: Emilie
authors.initials: E
authors.lastname: Dupré
authors.forename: Gilles
authors.initials: G
authors.lastname: Dupuis
authors.forename: Jean-Luc
authors.initials: JL
authors.lastname: Parent
mesh_terms: D000089982:ADP-Ribosylation Factor 6; D020727:ADP-Ribosylation Factors / Q000235:genetics / Q000378:metabolism*; D019390:Arrestins / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D005455:Fluorescent Antibody Technique; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000235:genetics / Q000378:metabolism*; D020690:GTPase-Activating Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020662:Guanine Nucleotide Exchange Factors / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D047468:Immunoprecipitation; D007668:Kidney / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D014162:Transfection
chemical_list: D000089982:ADP-Ribosylation Factor 6; D019390:Arrestins; D020690:GTPase-Activating Proteins; D020662:Guanine Nucleotide Exchange Factors; D011994:Recombinant Proteins; C082333:arrestin3; C100723:cytohesin-1; C102474:cytohesin-2; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D020727:ADP-Ribosylation Factors; C000715348:ARF6 protein, human