Figure 1. GIPC directly interacts with the C-terminal PDZ binding motif of LPA₁ but not with other LPA receptors.

A, Endogenous GIPC and GIPC-GFP co-immunoprecipitate with FLAG- LPA₁ from HEK cells expressing FLAG- LPA₁ (arrowhead, lane 4) but not control HEK cells (lane 3). C-terminally tagged GIPC-GFP and N-terminally tagged FLAG-LPA₁ were transiently coexpressed in HEK293 cells, and immunoprecipitation was carried out on cell lysates with mouse anti-FLAG IgG followed by immunoblotting with mouse anti-FLAG (LPA₁) and rabbit anti-GIPC IgG. Lanes were cropped from a single exposure of a continuous membrane. The lower panel shows the amount of IgG light-chain (IgG-LC) in each IP. Lanes 1–2: Input showing the amounts of LPA₁ and GIPC present in the lysates used for the IP. B, Upper panel: GIPC binds GST-LPA₁ (GST fused to the cytoplasmic tail of mouse LPA₁ (aa 311–364), lane 3) but not to GST alone (lane 2) or GST-LPA₁ΔC (lacking the last three C-terminal amino acids, lane 4). Immobilized recombinant GST, GST-LPA₁ and GST- LPA₁ΔC were incubated 4–15 h with lysates from HEK293 cells transiently transfected with FLAG-GIPC. Proteins bound to immobilized fusion proteins were eluted with 2X sample buffer for SDS-PAGE and immunoblotted with anti-GIPC IgG. Lane 1: input, showing the amount of GIPC in 1% of the lysate used for the assay. Lower panel : Ponceau staining demonstrating the amount of GST proteins used in each assay. C, Upper panel: Autoradiography showing that in vitro translated, [³⁵S]GIPC PDZ domain binds to GST-LPA₁ (lane 3) but not to GST alone (lane 2), GST- LPA₁AAA (last three amino acids mutated to alanine, lane 4), or GST-LPA₂ (lane 5). GST fusion proteins were immobilized on glutathione-agarose beads as in “B” and incubated with in vitro translated [³⁵S]Met-labeled, GIPC PDZ domain (aa 125–225). Bound proteins were separated by SDS-PAGE and detected by autoradiography. Lane 1: 5% of the in vitro translated protein. Lower panel : Coomassie Blue staining showing the GST proteins used for the assay. D, Upper panel: Autoradiography showing that in vitro translated, [³⁵S] GIPC-PDZ interacts with the C-terminal PDZ binding motif of LPA₁ (SVV, lane 3) and with GST-GAIP (lane 8, used as a positive control [7] but shows little or no interaction with GST alone (lane 2) or GST-LPA₁ mutants in which the three C-terminal amino acids were modified to those of LPA₂ (STL, lane 4), LPA₃ (NGS, lane 5), LPA₄ (STF, lane 6) or LPA₅ (SAL, lane 7). Immobilized GST fusion proteins were incubated with in vitro translated [³⁵S]Met-labeled GIPC PDZ and analyzed as in C. Lower panel: Coomassie Blue staining showing the amounts of GST proteins used.