FIGURE 10.

RET Thr⁶⁷⁵ residue regulates ligand-induced RET signaling and cell differentiation. A, analysis of the signal transduction mediated by RET^T675A receptor in response to GDNF stimulation. PC12 cells were transfected with GFP fused RET or RET^T675A and stimulated with 50 ng/ml GDNF for 5, 10 or 60 min. Immunoprecipitates or cell lysates were analyzed with the indicated antibodies. The levels of phospho-RET, phospho-Akt, and phospho-Erk1/2 were represented as the ratios of phosphoprotein over total protein. The histogram shows the phosphorylation levels of RET, Akt or Erk1/2 from three independent experiments normalized to their respective 5 min RET group. The results are represented as mean ± S.E. from three independent experiments (*, p < 0.05 versus their respective RET group; Student's t test). B, analysis of RET and RET^T675A activation in response to different GDNF concentrations. PC12 cells were transfected with GFP-fused RET or RET^T675A and stimulated by different GDNF concentrations. The levels of phospho-RET were represented as the ratios of phospho-RET versus total RET. At each GDNF concentration, phosphorylation levels of RET^T675A were normalized to that of RET. The results are represented as mean ± S.E. from three independent experiments (*, p < 0.05, **, p < 0.01 versus their respective RET group; Student's t test). C, GDNF induced differentiation of PC12 cells expressing GFP fused RET or RET^T675A. PC12 cells transfected with the indicated constructs were stimulated with 50 ng/ml GDNF for 3 days. Cells displaying neurite extension longer than twice the cell diameter were counted as differentiated cells. The results were normalized to the total number of transfected (GFP-positive) PC12 cells in each field. All of the GDNF stimulations were supplemented with 300 ng/ml soluble GFRα1. The results are represented as mean ± S.E. from three independent experiments (**, p < 0.01 versus their respective control group; #, p < 0.05 versus GDNF-treated RET group; one-way ANOVA).