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. 2010 May 1;21(9):1597–1608. doi: 10.1091/mbc.E09-12-1033

Figure 4.

Figure 4.

PACAP38-mediated Rit activation involves PACR1-dependent TrkA transactivation. (A) PC6 cells were transfected with either shCTR or shPACR1-384 and then subjected to G418 (400 μg/ml) selection for 60 h. Total RNA was isolated and RT-PCR used to monitor PACR1 silencing. (B) PC6 cells cotransfected with 3xFLAG-Rit-WT and either shCTR or shPACR1-384 were starved in serum-free DMEM (5 h) and stimulated with PACAP38 (20 nM). Cell lysates were prepared and subjected to GST-RGL3-RBD pull-down. (C) PC6 cells transfected with either shCTR or shPACR1-384 were enriched with G418 (400 μg/ml; 60 h) and serum starved (5 h) before stimulation with either PACAP38 (20 nM) or NGF (100 ng/ml). Total cell lysates (1 mg) were prepared and subjected to anti-TrkA immunoprecipitation. Anti-phosphotyrosine specific immunoblotting was used to detect activate TrkA, and the stripped membrane was reprobed with anti-TrkA antibody to demonstrate equal loading. (D) PC6 cells were pretreated with PP2 (10 μM), PP3 (10 μM), PTX (100 ng/ml), ddA (50 μM), or dimethyl sulfoxide (DMSO); stimulated with PACAP38 (20 nM); and total cell lysates were prepared. NGF stimulation (100 ng/ml) served as positive control. Endogenous TrkA was immunoprecipitated (1 mg of total cell lysate) and activated receptor identified by anti-phosphotyrosine immunoblotting. (E) PC6 cells were transfected with c-Src-WT in the presence or absence of CA-Epac2, serum starved, and then stimulated with PACAP38 (10 nM), EGF (100 ng/ml), or not treated (phosphate-buffered saline [PBS]). Total lysates (500 μg) were subjected to anti-c-Src immunoprecipitation, and active Src levels were examined by phosphotyrosine-specific immunoblotting. Note that both PACAP38 and CA-Epac2 result in c-Src activation. (F) PC6 cells expressing c-Src-WT were starved for 5 h before stimulation with 8-CPT-2-Me-cAMP (25 μM) for 5, 10, or 30 min, and the phosphotyrosine levels of Src determined as described above. (G) PC6 cells expressing c-Src-WT were starved (5 h) and pretreated with or without PTX (100 ng/ml; 30 min) before stimulation with either PACAP38 (20 nM; 10, 30, or 60 min) or CTX (2.5 μg/ml; 10, 30, or 60 min). Total cell lysates were prepared and analyzed for Src tyrosine phosphorylation analysis as described above. The data in each panel are representative of the results from a minimum of three independent experiments.