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

Lysophosphatidic acid (LPA) stimulates heterotrimeric G protein signaling by activating three closely related receptors, termed LPA(1), LPA(2) and LPA(3). Here we show that in addition to promoting LPA(1) signaling, membrane cholesterol is essential for the association of LPA(1) with beta-arrestin, which leads to signal attenuation and clathrin-dependent endocytosis of LPA(1). Reduction of clathrin heavy chain expression, using small interfering RNAs, inhibited LPA(1) endocytosis. LPA(1) endocytosis was also inhibited in beta-arrestin 1 and 2-null mouse embryo fibroblasts (beta-arrestin 1/2 KO MEFs), but was restored upon re-expression of wild-type beta-arrestin 2. beta-arrestin attenuates LPA signaling as LPA(1)-dependent phosphoinositide hydrolysis was significantly elevated in beta-arrestin 1/2 KO MEFs and was reduced to wild-type levels upon re-expression of wild-type beta-arrestin. Interestingly, extraction of membrane cholesterol with methyl-beta-cyclodextrin inhibited LPA(1) signaling, beta-arrestin membrane recruitment and LPA(1) endocytosis. Cholesterol repletion restored all of these functions. However, neither the stimulation of phosphoinositide hydrolysis by the M(1) acetylcholine receptor nor its endocytosis was affected by cholesterol extraction. LPA treatment increased the detergent resistance of LPA(1) and this was inhibited by cholesterol extraction, suggesting that LPA(1) localizes to detergent-resistant membranes upon ligand stimulation. These data indicate that although LPA(1) is internalized by clathrin- and beta-arrestin dependent endocytosis, membrane cholesterol is critical for LPA(1) signaling, membrane recruitment of beta-arrestins and LPA(1) endocytosis.