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The molecular mechanisms governing calcium signal transduction of corticotropin-releasing factor (CRF) receptors CRF 1 and CRF 2(a) stably expressed in human embryonic kidney (HEK) 293 cells were investigated. Calcium signaling strictly depended on intracellular calcium sources, and this is the first study to establish a prominent contribution of the three major G-protein families to CRF receptor-mediated calcium signaling. Overexpression of G␣ q/11 and G␣ 16 led to leftward shifts of the agonist concentration-response curves. Blockade of G␣ q/11 proteins by the small interfering RNA (siRNA) technology partially reduced agonist-mediated calcium responses in CRF 1 -and CRF 2(a) -expressing HEK293 cells, thereby proving a contribution of the G q protein family. A small but significant inhibition of calcium signaling was recorded by pharmacological inhibition of G i/o proteins with pertussis toxin treatment. This effect was mediated by direct binding of G␤␥ subunits to phospholipase C. G i/o inhibition also elevated cAMP responses in CRF receptor-overexpressing HEK293 cells and in Y79 retinoblastoma cells endogenously expressing human CRF 1 and CRF 2(a)

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Endogenous expression of the corticotropin‐releasing factor type 2a receptor [CRF2(a)] but not CRF2(b) and CRF2(c) was observed in higher passage cultures of human Y79 retinoblastoma cells. Functional studies further demonstrated an increase in CRF2(a) mRNA and protein levels with higher passage numbers (> 20 passages). Although the CRF1 receptor was expressed at higher levels than the CRF2(a) receptor, both receptors were easily distinguishable from one another by selective receptor ligands. CRF1‐preferring or non‐selective agonists such as CRF, urocortin 1 (UCN1), and sauvagine stimulated cAMP production in Y79 to maximal responses of ∼100 pmoles/105 cells, whereas the exclusive CRF2 receptor‐selective agonists UCN2 and 3 stimulated cAMP production to maximal responses of ∼25–30 pmoles/105 cells. UCN2 and 3‐mediated cAMP stimulation was potently blocked by the ∼300‐fold selective CRF2 antagonist antisauvagine (IC50 = 6.5 ± 1.6 nmol/L), whereas the CRF1‐selective antagonist NBI27914 only blocked cAMP responses at concentrations > 10 μmol/L. When the CRF1‐preferring agonist ovine CRF was used to activate cAMP signaling, NBI27914 (IC50 = 38.4 ± 3.6 nmol/L) was a more potent inhibitor than antisauvagine (IC50 = 2.04 ± 0.2 μmol/L). Finally, UCN2 and 3 treatment potently and rapidly desensitized the CRF2 receptor responses in Y79 cells. These data demonstrate that Y79 cells express functional CRF1 and CRF2(a) receptors and that the CRF2(a) receptor protein is up‐regulated during prolonged culture.