Figure 2. Trafficking of LPA₁ and GIPC to Endosomes.

A, Upper panel: In serum starved cells stably expressing LPA₁ (asterisks), GIPC is concentrated at the plasma membrane (arrowheads) where it colocalizes with LPA₁. Middle panels : 2 min following stimulation with LPA, LPA₁ colocalizes with APPL and GIPC (arrowheads) in endocytic vesicles at the cell periphery. Lower panel : 30 min following stimulation, LPA₁ colocalizes with EEA1 in early endosomes concentrated in the juxtanuclear region (arrowheads). Boxed regions are enlarged (2.2×) in the insets. Bar = 10 µm. HEK- LPA₁ cells were serum starved for 4 h, incubated on ice with mouse anti-FLAG IgG to label FLAG-LPA₁ at the cell surface, washed in PBS and shifted to fresh medium containing LPA (10 µM) 2 or 5 min before fixation. Cells were processed for immunofluorescence using affinity purified rabbit anti-GIPC, anti-APPL1 or anti-EEA1 IgG, followed by goat anti-rabbit Alexa-488 and goat-anti-mouse Alexa-594 F(ab’)₂ (the latter to detect FLAG-LPA₁). Images were taken with a PerkinElmer UltraView Vox Spinning Disk Confocal unit connected to an Olympus IX81 inverted microscope and a EMCCD camera (Hamamatsu) using a 60X oil immersion lens (1.42 NA). B, GIPC co-immunoprecipitates with FLAG-LPA₁ from serum-starved cells (lane 7) at all time points after LPA stimulation, but the interaction gradually decreases after LPA stimulation (lanes 8–10). The relative abundance of GIPC that coprecipitated with FLAG-LPA₁ is indicated beneath each band. As expected, both LPA₁ and GIPC are absent from immunoprecipitates of cells transiently transfected with empty vector instead of FLAG- LPA₁ (lane 6, vector control). HEK293 cells were transiently co-transfected with full-length GIPC and FLAG-LPA₁ (lanes 2–5 and 7–10) or GIPC alone (lanes 1 and 6). Cells were serum starved overnight (lanes 1, 2, 6 and 7) or starved and stimulated with 10 µM LPA for 5 (lanes 3 and 8), 15 (lanes 4 and 9) or 30 min (lanes 5 and 10) before lysis. IP was carried out on cell lysates using mouse anti-FLAG IgG and immunoblotted as is Fig. 1A. The abundance of GIPC and LPA₁ in each immunoprecipitation reaction was quantified using the LICOR imaging system, and the GIPC abundance relative to LPA₁ was calculated for each reaction. Similar results were obtained in 2 additional experiments. Input (lanes 1–5): Lysates (2%) are shown to verify comparable expression levels.