Cell-type specific calcium signaling by corticotropin-releasing factor type 1 (CRF1) and 2a (CRF2(a)) receptors: phospholipase C-mediated responses in human embryonic kidney 293 but not SK-N-MC neuroblastoma cells.

The human corticotropin-releasing factor (hCRF) receptors CRF1 and CRF2(a) couple to the Gs protein. It has been postulated that CRF receptors may also signal through phospholipase C (PLC). To test this hypothesis, binding and signaling properties were determined for both receptor subtypes stably expressed in human embryonic kidney 293 (HEK293) and human SK-N-MC neuroblastoma cells. CRF receptors were highly expressed and strongly coupled to Gs in HEK293 and SK-N-MC cells. However, when the calcium mobilization pathway was investigated, marked differences were observed. In SK-N-MC cells, neither CRF receptor stimulated calcium mobilization in the fluorometric imaging plate reader (FLIPR) assay, whereas activation of orexin type 1 and 2 receptors stably expressed in SK-N-MC cells revealed robust calcium responses. In contrast, intracellular calcium was strongly mobilized by agonist stimulation of hCRF1 and hCRF2(a) receptors in HEK293 cells. In HEK293 cells, potency rank orders for calcium and cAMP responses were identical for both receptors, despite a rightward shift of the dose-response curves. Complete inhibition of calcium signaling of both hCRF1 and hCRF2(a) receptors was observed in the presence of the PLC inhibitor U-73,122 whereas ryanodine, an inhibitor of calcium release channels and the protein kinase A inhibitor Rp-cAMPS were ineffective. Finally, CRF agonists produced a small but significant stimulation of inositol 1,4,5-triphosphate (IP3) accumulation in hCRF1-and hCRF2(a)-transfected HEK293 cells. These data clearly show that phospholipase C-mediated signaling of CRF receptors is dependent upon the cellular background and that in HEK293 cells human CRF receptors robustly respond in the FLIPR format.