Localization of RhoA GTPase to Endothelial Caveolae-Enriched Membrane Domains

doi:10.1006/bbrc.1998.8885

Biochemical and Biophysical Research Communications

Volume 247, Issue 3, 29 June 1998, Pages 888-893

https://doi.org/10.1006/bbrc.1998.8885 Get rights and content

Abstract

Caveolae are small microdomains of the plasma membrane that are thought to play important roles in signal transduction processes. In this work, we have investigated the association of Rho proteins with caveolae-enriched membrane domains isolated from cultured endothelial cells. Fractionation of ECV304 cells by sucrose gradient density centrifugation in the absence of detergent resulted in the co-sedimentation of a significant proportion of RhoA and Cdc42 with known caveolae marker proteins, including caveolin, but not with other non-caveolae membrane proteins such as the angiotensin-converting enzyme. Immunoprecipitation experiments carried on crude endothelial cell lysates as well as with solubilized caveolae-enriched membrane domains showed the coimmunoprecipitation of caveolin with RhoA but not with Cdc42. Incubation of endothelial cell lysates with a glutathione-S-transferase (GST)-RhoA fusion protein resulted in the specific precipitation of caveolin, while addition of GST-caveolin-1 to the lysates promoted the precipitation of RhoA. Moreover, incubation of bacterially expressed RhoA with GST-caveolin-1 resulted in the precipitation of RhoA, indicating that RhoA directly interacts with caveolin-1. This interaction was found to be nucleotide-independent and was not affected by prior modification of RhoA with the C3 exoenzyme fromC. botuliniumor with the cytotoxic necrotinizing factor fromE. coli.Taken together, these results suggest the association of RhoA with endothelial caveolae-enriched membrane domains, likely through physical interaction with caveolin-1. These findings may provide new insights into the functions played by Rho proteins and caveolae in signal transduction events.

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^☆: Abbreviations used in this paper: CNF1, cytotoxic necrotinizing factor from E. coli; FAK, focal adhesion kinase; GST, glutathione-S-transferase; GTPase, guanosine triphosphatase; GTPγS, guanosine-5′-O-thio-triphosphate; LPA, lysophosphatidic acid; rhoGDI, rho GDP-dissociation inhibitor

¹: To whom correspondence should be addressed at Département de chimie-biochimie, Université du Québec à Montrél, C.P. 8888, Succ. Centre-ville, Montrél, Québec H3C 3P8, Canada. Fax: (514) 987-0246; E-mail:[email protected].

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Regular ArticleLocalization of RhoA GTPase to Endothelial Caveolae-Enriched Membrane Domains☆

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Regular Article
Localization of RhoA GTPase to Endothelial Caveolae-Enriched Membrane Domains☆