Figure 3.

MET^PRC mutants are constitutively coupled to signal transducers via two tyrosines located in the tail of the receptor. (A) NIH 3T3 fibroblasts were transfected with either wild-type or PRC-mutated MET receptor mutants to generate stable cell lines. FF indicates MET-receptor variants in which tyrosines Y¹³⁴⁹ and Y¹³⁵⁶ were converted into phenylalanines. The amount of Met proteins and the level of phosphorylation was evaluated by immunoprecipitation with anti-Met antibodies followed by Western blotting with anti-Met or anti-phosphotyrosine (pTyr) antibodies, respectively. (B) Lysates of the same cells were immunoprecipitated with anti-Gab1 antibodies and analyzed by Western immunoblotting with anti-pTyr antibodies. The identity of the phosphorylated protein coimmunoprecipitated with Gab1 was confirmed by reprobing the same blot with an anti-Met antibody (data not shown). (C) Lysates of COS cells expressing either the wild-type (wt), the D1228H mutant, or its double phenylalanine counterpart D1228H (FF) were immunoprecipitated and analyzed by Western blotting with anti-Met antibodies. The gap between the arrows indicates the electrophoretic shift caused by differences in phosphorylation. (D) Lysates of NIH 3T3 cells expressing the D1228H mutant were incubated with the Y^pVNV phosphopeptide corresponding to the receptor’s docking site and immunoprecipitated with anti-Gab1 antibodies. The amount of associated receptor was determined by Western blot with anti-Met antibodies. The nonphosphorylated phenylalanine peptide analogue (FVNV) was used as a negative control. The arrows indicate the position of the 145-kDa MET receptor β-chain (p145^Met).