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

Abstract The human corticotropin-releasing factor (hCRF) receptors CRF 1 and CRF 2(a) couple to the G s 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 G s 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 hCRF 1 and hCRF 2(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 hCRF 1 and hCRF 2(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 (IP 3 ) accumulation in hCRF 1 -and hCRF 2(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.