FIGURE 3:

Full-length and ΔSP versions of CRF₂βR exhibit similar subcellular localization and downstream cAMP and Ca²⁺ responses. HEK293 cells stably expressing HA-CRF₂βR or Flag-CRF₂βRΔSP were seeded on coverslips and immunostained using anti CRFR_1/2 antibody. Stimulation with 100 nM of agonists Ucn1 or Ucn2 resulted in internalization (arrowheads) of both full-length (A) and ΔSP (B) versions of CRF₂βRs from the cell surface (arrows). Representative images are shown (n = 2 coverslips per condition, and each experiment was performed three times). Scale bar: 10 μm. (C) Quantification of images in row 1 of A and B. The percentage of total fluorescence at the cell surface for both versions of CRF₂βRs was quantified. Cell surface expression was found to be similar between CRF₂βR and CRF₂βRΔSP (n = 24 cells for each condition; two-tailed unpaired Student’s t test: n.s.). (D) Western blot analysis of HEK293 cells expressing HA-CRF₂βR or Flag-CRF₂βRΔSP showed similar receptor expression levels with both constructs. Untransfected cells were used as negative control. (E) cAMP levels increased significantly from baseline values upon stimulation with either Ucn1 or Ucn2 in a dose-dependent manner (p < 0.05 vs. baseline values with ligand concentrations >1.6 nM; two-tailed unpaired Student’s t test, mean ± SEM of three experiments in triplicate) in both full-length and ΔSP versions of CRF₂βRs. Importantly, both versions of CRF₂βRs showed similar increases in cAMP levels at all doses tested (two-tailed unpaired Student’s t test: n.s.). (F) Peak [Ca²⁺]_i increased in response to 100 nM Ucn1 or Ucn2 stimulations in both full-length and ΔSP versions of CRF₂βRs to a similar degree (normalized to peak ionomycin responses). Data are mean ± SEM. No significant differences were found between CRF₂βR and CRF₂βRΔSP (two-tailed unpaired Student’s t test: n.s.). Representative traces of [Ca²⁺]_i are shown (n = 3 wells per condition, and each experiment was performed three times).