A requirement for membrane cholesterol in the -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 LPA₁, LPA₂ and LPA₃. Here we show that in addition to promoting LPA₁ signaling, membrane cholesterol is essential for the association of LPA₁ with -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 -arrestin 1 and 2-null mouse embryo fibroblasts (-arrestin 1/2 KO MEFs), but was restored upon re-expression of wild-type -arrestin 2. -arrestin attenuates LPA signaling as LPA₁-dependent phosphoinositide hydrolysis was significantly elevated in -arrestin 1/2 KO MEFs and was reduced to wild-type levels upon re-expression of wild-type -arrestin. Interestingly, extraction of membrane cholesterol with methyl--cyclodextrin inhibited LPA₁ signaling, -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 -arrestin dependent endocytosis, membrane cholesterol is critical for LPA₁ signaling, membrane recruitment of -arrestins and LPA₁ endocytosis.