FIG. 2.

A Y314F mutation in the vGPCR ITIM motif does not adversely affect vGPCR expression, localization, or ligand binding. (A) HEK293 cells were transfected with pcDNA3.1-vGPCR or pcDNA3.1-ΔITIM as indicated. Immunoblotting for vGPCR (antibody was a gift of Gary Hayward) was done 24 h later. The blot was stripped and reprobed for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (top). In the bottom panel, lysates were blotted with antibody to phosphorylated tyrosines (Millipore catalogue no. 05-1050X). (B) Cells expressing hemagglutinin (HA)-tagged vGPCR or an HA-tagged Y314F ITIM mutant (ΔITIM). At 24 h, confocal images were obtained. (C) HEK293 cells were transfected with increasing amounts of plasmid encoding vGPCR or the ΔITIM mutant; after 24 h, binding assays using I¹²⁵-labeled growth-regulated oncogene alpha (Groα) were performed. Results are normalized to untransfected cells.