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The N-terminal coiled-coil domain of the cytohesin/ARNO family of guanine nucleotide exchange factors interacts with Gαq

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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 Gαq signaling leads to the activation of ARF6, possibly through a direct interaction with cytohesin-2/ARNO. Here, we report that Gαq can directly interact with cytohesin-1, another Arf-GEF of the ARNO/cytohesin family. Cytohesin-1 preferentially associated with a constitutively active mutant of Gαq (Gαq-Q209L) compared to wild-type Gαq in HEK293 cells. Stimulation of TPβ, a Gαq-coupled receptor, to activate Gαq resulted in the promotion of a protein complex between Gαq and cytohesin-1. Confocal immunofluorescence microscopy revealed that wild-type Gαq and cytohesin-1 co-localized in intracellular compartments and at or near the plasma membrane. In contrast, expression of Gαq-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 TPβ, a process that we previously demonstrated to be dependent on Gαq-mediated signaling and Arf6 activation. Using deletion mutants, we show that cytohesin-1 interacts with Gαq through its N-terminal coiled-coil domain. Cytohesin-1 and cytohesin-2/ARNO mutants lacking the coiled-coil domain were unable to relay Gαq-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.

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Abbreviations

GPCR:

G protein-coupled receptor

TP:

Thromboxane A2 receptor

Arf:

ADP-ribosylation factor

ARNO:

Arf nucleotide-binding-site opener

GEF:

Guanine nucleotide-exchange factor

GGA3:

Golgi-localized γ-ear homology domain Arf-binding protein-3

ELISA:

Enzyme-linked immunosorbent assay

PBS:

Phosphate-buffered saline

TBS:

Tris-buffered saline

BSA:

Bovine serum albumin

HA:

Hemagglutinin

GST:

Glutathione S-transferase

IP:

Immunoprecipitation

IB:

Immunoblotting

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Acknowledgments

This work was supported by a grant to J.L.P. from the Canadian Institutes of Health Research (CIHR). G.L received a studentship from the Heart & Stroke Foundation of Canada. During this work, J.L.P. received salary support from a New Investigator Award from CIHR and from a “Chercheur-boursier Junior 2” scholarship from the Fonds de la Recherche en Santé du Québec.

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Authors and Affiliations

  1. 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

    Geneviève Laroche, Patrick M. Giguère, Émilie Dupré, Gilles Dupuis & Jean-Luc Parent

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  1. Geneviève Laroche
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  2. Patrick M. Giguère
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  3. Émilie Dupré
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  4. Gilles Dupuis
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  5. Jean-Luc Parent
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Correspondence to Jean-Luc Parent.

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Laroche, G., Giguère, P.M., Dupré, É. et al. The N-terminal coiled-coil domain of the cytohesin/ARNO family of guanine nucleotide exchange factors interacts with Gαq. Mol Cell Biochem 306, 141–152 (2007). https://doi.org/10.1007/s11010-007-9564-9

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