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. 2017 Jun 9;292(29):12178–12191. doi: 10.1074/jbc.M117.780304

Figure 4.

Figure 4.

GPR124 interacts with intersectins via its C-terminal tail, which exhibits affinity for ITSN SH3 modules. A, schematic representation GPR124 in complex with ITSN. B, GPR124 localizes with ITSN at cell projections of adherent cells. Endogenous GPR124 and ITSN were visualized by immunostaining in HUVECs left to adhere for 30 min. Analysis by confocal microscopy showed that GPR124 localized with ITSN at the cell membrane during cell attachment (white arrowheads). Co-localization was measured, selecting three different areas at cell protrusions from three independent experiments. Similar results were observed in 65 of 124 cells. Scale bar, 20 μm. C, GPR124 interacts with ITSN1 and -2. HEK293T cells were co-transfected with GST–GPR124 C terminus (GPR124C-ter) and full-length HA–intersectin 1 (HA–ITSN1L) or 2 (HA–ITSN2L). GST pulldown assays were performed, and ITSN1 and ITSN2 associated with GPR124 were revealed by Western blotting. GST was used as a control (Ctrl). D, the GPR124 C terminus interacts with SH3 domains of ITSNs. HEK293T cells were co-transfected with SH3A–E domains of ITSN1 or ITSN2 and GST–GPR124 C terminus. GST pulldown assays were performed, and SH3A–E domains of both ITSNs were able to specifically associate with the GPR124 C terminus. GST–vector was used as a control (GST +). E, full-length GPR124 interacts with ITSN1/2-SH3A–E domains in HEK293T cells. Cells were transfected with FLAG-tagged full-length GPR124 and GST–ITSN1–SH3A–E or GST–ITSN2–SH3A–E. GST pulldown experiments were performed, and full-length GPR124 was detected associated with SH3A–E domains of both ITSNs. GST was used as a control. Expression of transfected proteins was confirmed in total cell lysates (TCL). F, the GPR124 C terminus directly interacts with all five SH3 domains of ITSN1. HEK293T cells were transfected with FLAG–GPR124 C terminus. GST pull-down assays were performed using individual GST–recombinant SH3 domains (A–E; shown at the bottom, stained with Ponceau) of ITSN1 or GST–vector, used as control. All ITSN1-SH3 domains interacted with the GPR124 C terminus, the ITSN1-SH3D domain being a more effective interactor. G, full-length GPR124 preferentially interacts with ITSN1-SH3D domain. Cells were transfected with full-length FLAG–GPR124. GST pull down assays using the individual recombinant GST–ITSN1-SH3 domains (A–E) or GST–vector, used as control, showed that GPR124 preferentially interacts with ITSN1-SH3D. Error bars, S.E.