EGFR mainly phosphorylates the tyrosine residue Y317/Y320 of GPRC5A. A, Sequence alignment of the C-terminal tails of GPRC5A from different species. B, The constructs of GPRC5A mutants used in this study. C,D , E,F, HEK293T cells were co-transfected with plasmids encoding EGFR or vector plus either GPRC5A-wild type (WT) or Y317/320 F or Y347/350 F or 4 F mutants as indicated. Cells were treated with or without EGF as indicated. Cell lysates were harvested for Western blots using the antibodies as indicated. G, HEK293T cells were co-transfected with the plasmids containing EGFR plus GPRC5A-WT or a series of GPRC5A mutants as indicated. Cellular proteins were analyzed by Western blot using antibodies as indicated.