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

• Published in: Biochemical pharmacology 2004-11, Vol.68 (9), p.1833-1844
• Main Authors: Dautzenberg, Frank M., Gutknecht, Eric, Linden, Ilse Van der, Olivares-Reyes, J. Alberto, Dürrenberger, Franz, Hauger, Richard L.
• Format: Article
• Language: English
• Subjects: Biological and medical sciences; Ca 2+ mobilization; Calcium - metabolism; Calcium Signaling - physiology; Cell Line; Cell receptors; Cell structures and functions; Corticotropin-Releasing Hormone - metabolism; CRF receptor signaling; Cyclic AMP; Fundamental and applied biological sciences. Psychology; Hormone receptors. Growth factor receptors. Cytokine receptors. Prostaglandin receptors; Humans; Inositol 1,4,5-triphosphate; Kidney; Ligand binding; Molecular and cellular biology; Neuroblastoma - pathology; Phospholipase C; Receptors, Corticotropin-Releasing Hormone - metabolism; Receptors, Corticotropin-Releasing Hormone - physiology; Tumor Cells, Cultured; Type C Phospholipases - physiology
• ISSN: 0006-2952; 1873-2968
• DOI: 10.1016/j.bcp.2004.07.013

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.