Abstract

The molecular mechanisms governing calcium signal transduction of corticotropin-releasing factor (CRF) receptors CRF₁ 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 Gα_q/11 and Gα₁₆ led to leftward shifts of the agonist concentration-response curves. Blockade of Gα_q/11 proteins by the small interfering RNA (siRNA) technology partially reduced agonist-mediated calcium responses in CRF₁- 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 Gβγ 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 CRF₁ and CRF_2(a) receptors, thereby demonstrating natural coupling of G_i proteins to both CRF receptors. The strongest reduction of CRF receptor-mediated calcium mobilization was noted when blocking the G_s signaling protein either by cholera toxin or by siRNA. It is noteworthy that simultaneous inhibition of two G-proteins shed light on the additive effects of G_s and G_q on the calcium signaling and, hence, that they act in parallel. On the other hand, G_i coupling required prior G_s activation.