Fig. 3. Re-expression of wild-type ß-arrestin 2 GFP in ß-arrestin knockout MEFs restores LPA₁ signal attenuation and receptor endocytosis. (A) ß-arrestin 1/2 double knockout MEFs were transiently transfected with plasmids encoding LPA₁ and wild-type ß-arrestin-2-GFP. Cells were then incubated with 10 µM LPA for 0, 2, or 30 minutes prior to fixation and indirect immunofluorescence. The inset shows a magnified image of the boxed region and the arrows indicate punctate structures that co-label for both LPA₁ and ß-arrestin-2-GFP. Note that the recruitment of ß-arrestin2-GFP to these punctate structures is transient, observable after 2 minutes of LPA treatment but not after 30 minutes of LPA treatment. (B) MEFs derived from wild type (WT) or ß-arrestin 1/2 null (ßArr 1/2KO) mice were transfected with plasmid encoding wild-type LPA₁ receptors; ß-arrestin 1/2 null MEFs were also co-transfected with plasmids encoding LPA₁ and wild-type ß-arrestin 2 (ßArr 1/2KO + LPA₁ + WT ßArr2). Cells were then labeled with [³H]myo-inositol overnight in serum-free medium and incubated for 1 hour in the absence (Untreated) or presence of 10 µM LPA prior to analysis of phosphoinositide hydrolysis, as described in Materials and Methods. The radioactivity recovered in the different samples was normalized to total cellular protein and the data are presented as the mean±s.e.m. of triplicate measurements from a representative experiment that was repeated three times. ^**P<0.01, comparison of LPA-stimulated phosphoinositide hydrolysis in ß-arrestin 1/2 KO MEFs to that observed in WT MEFs. Bar, 10 µm.