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First published online 1 November 2005
doi: 10.1242/jcs.02634

This Article Figures Only Full Text Full Text (PDF) All Versions of this Article: jcs.02634v1 118/22/5291    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Related articles in JCS Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Urs, N. M. Articles by Radhakrishna, H. Search for Related Content PubMed PubMed Citation Articles by Urs, N. M. Articles by Radhakrishna, H.

A requirement for membrane cholesterol in the ß-arrestin- and clathrin-dependent endocytosis of LPA₁ lysophosphatidic acid receptors

¹ School of Biology, Georgia Institute of Technology, 315 Ferst Drive, Atlanta, GA 30332, USA
² School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA 30332, USA
³ Department of Pharmacology, University of North Carolina, Chapel Hill, NC 27599, USA
⁴ Petit Institute for Bioengineering and Biosciences, Georgia Institute of Technology, 315 Ferst Drive, Atlanta, GA 30332, USA

^* Author for correspondence (e-mail: harish.radhakrishna{at}biology.gatech.edu)

Accepted 12 August 2005

Lysophosphatidic acid (LPA) stimulates heterotrimeric G protein signaling by activating three closely related receptors, termed LPA₁, LPA₂ and LPA₃. Here we show that in addition to promoting LPA₁ signaling, membrane cholesterol is essential for the association of LPA₁ with ß-arrestin, which leads to signal attenuation and clathrin-dependent endocytosis of LPA₁. Reduction of clathrin heavy chain expression, using small interfering RNAs, inhibited LPA₁ endocytosis. LPA₁ endocytosis was also inhibited in ß-arrestin 1 and 2-null mouse embryo fibroblasts (ß-arrestin 1/2 KO MEFs), but was restored upon re-expression of wild-type ß-arrestin 2. ß-arrestin attenuates LPA signaling as LPA₁-dependent phosphoinositide hydrolysis was significantly elevated in ß-arrestin 1/2 KO MEFs and was reduced to wild-type levels upon re-expression of wild-type ß-arrestin. Interestingly, extraction of membrane cholesterol with methyl-ß-cyclodextrin inhibited LPA₁ signaling, ß-arrestin membrane recruitment and LPA₁ endocytosis. Cholesterol repletion restored all of these functions. However, neither the stimulation of phosphoinositide hydrolysis by the M₁ acetylcholine receptor nor its endocytosis was affected by cholesterol extraction. LPA treatment increased the detergent resistance of LPA₁ and this was inhibited by cholesterol extraction, suggesting that LPA₁ localizes to detergent-resistant membranes upon ligand stimulation. These data indicate that although LPA₁ is internalized by clathrin- and ß-arrestin dependent endocytosis, membrane cholesterol is critical for LPA₁ signaling, membrane recruitment of ß-arrestins and LPA₁ endocytosis.

Key words: LPA, ß-arrestin, Lipid raft, Endocytosis, G-protein-coupled receptor

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