Figure 5.
5-HT4R-mediated activation of p-ERK1/2 is independent of PLC, Gi/Go, and EGF receptors. (A) 5-HT4R–mediated ERK activation does not require PLC activation. Serum-starved HEK293 cells expressing 5-HT4R were pretreated with 10 μM U 73122, a PLC inhibitor, or by its inactive analogue U 73343 for 30 min before stimulation with 10 μM 5-HT for 5 min. Total lysates were analyzed by immunoblotting with antibody to p-ERK1/2. (B) In parallel, HEK293 cells coming from the same transfection as described in A for each condition were seeded into 24-well plates, pretreated by LiCl 10 min before a 30-min stimulation by 10 μM 5-HT. Quantification of IP production was performed by HTRF by using the IP-One assay as described in Materials and Methods. IP levels accumulated during the stimulation time are expressed as the percentage of maximum IP response to 10 μM 5-HT, and they are reported in the absence or presence of pretreatment with U 73122 and U 73343. Values are the mean ± SEM of four independent experiments. **p < 0.01, significantly different from the corresponding cells before U 73122-treatment. (C) 5-HT4R–mediated ERK activation does not require G protein PTX sensitive (Gi/Go). HEK293 cells expressing 5-HT4R were pretreated with 100 ng/ml PTX overnight before treatment with 10 μM 5-HT for 5 min. Total lysates were analyzed by immunoblotting with antibody to p-ERK1/2. (D) 5-HT4R–mediated ERK activation does not require transactivation of EGF-R tyrosine kinase. HEK293 cells expressing 5-HT4R were pretreated with 250 nM tyrphostin/AG1478 for 30 min before stimulation with 10 μM 5-HT. Total lysates were analyzed by immunoblotting with antibody to p-ERK 1/2. A representative blot of each experiment is shown in A, C, and D.