Fig. 4. Rapgef2 protein abundance is enriched in neuroendocrine cells, and its exogenous expression in HEK 293T cells confers cAMP-ERK signaling.

(A) Profile of Rapgef2 protein abundance in human and rodent cell lines. Lane 1: PC12-G; lane 2: NS-1; lane 3: BCC; lane 4: 293T rPAC₁hop; lane 5: HEK 293FT; lane 6: HeLa; lane 7: 3T3 Swiss; lane 8: SH-SY5Y; lane 9: NG108-15; lane 10: NBFL; lane 11: FRTL-5; lane 12: B16-F10; lane 13: AtT20; lane 14: Chinese hamster ovary (CHO) K1. Western blotting data are representative of three independent experiments. (B) Western blotting analysis of Rapgef2 abundance in HEK 293T rPAC₁hop cells (lane 1), HEK 293T rPAC₁hop cells stably expressing hRapgef2 (lane 2), and nontransduced SH-SY5Y cells (lane 3). Western blotting data are representative of three independent experiments. (C and D) Measurement of ERK phosphorylation in (C) 293T rPAC₁hop cells and (D) 293T rPAC₁hop cells expressing hRapgef2 after treatment for 10 min with 100 nM PACAP-38, 30 μM isoproterenol, 25 μM forskolin, cholera toxin (CTX; 50 μg/ml), 0.5 mM 8-Br-cAMP, or 100 nM PMA. Bar graphs show the ratio of the abundance of pERK to that of total ERK protein for each sample relative to untreated control cells (set at 100%). *P < 0.05, **P < 0.01, ***P < 0.001; n = 4 experiments. (E) Analysis of Rap1 activation in 293T rPAC₁hop cells expressing or not expressing the retroviral vector encoding hRapgef2. Cells were treated with vehicle, 100 μM 8-CPT-cAMP, or 100 μM 8-CPT-2′-O-Me-cAMP. *P < 0.05, **P < 0.01, compared to vehicle-treated cells. In cells expressing hRapgef2, 8-CPT-cAMP conferred a significantly greater activation of Rap1 (**P < 0.01) than was observed in nontransduced cells (^#P < 0.05, Bonferroni). Data are from four to six independent experiments.