Abstract
The molecular mechanisms governing calcium signal transduction of corticotropin-releasing factor (CRF) receptors CRF1 and CRF2(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 Gαq/11 and Gα16 led to leftward shifts of the agonist concentration-response curves. Blockade of Gαq/11 proteins by the small interfering RNA (siRNA) technology partially reduced agonist-mediated calcium responses in CRF1- and CRF2(a)-expressing HEK293 cells, thereby proving a contribution of the Gq protein family. A small but significant inhibition of calcium signaling was recorded by pharmacological inhibition of Gi/o proteins with pertussis toxin treatment. This effect was mediated by direct binding of Gβγ subunits to phospholipase C. Gi/o inhibition also elevated cAMP responses in CRF receptor-overexpressing HEK293 cells and in Y79 retinoblastoma cells endogenously expressing human CRF1 and CRF2(a) receptors, thereby demonstrating natural coupling of Gi proteins to both CRF receptors. The strongest reduction of CRF receptor-mediated calcium mobilization was noted when blocking the Gs 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 Gs and Gq on the calcium signaling and, hence, that they act in parallel. On the other hand, Gi coupling required prior Gs activation.
Footnotes
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ABBREVIATIONS: CRF, corticotropin-releasing factor; 2-APB, 2-aminoethoxyphenyl borate; CTX, cholera toxin; GRK2ct, carboxyl terminus of the G-protein kinase 2; IP3R, inositol 1,4,5 triphosphate receptor; PKA, protein kinase A; PLC, phospholipase C; PTX, pertussis toxin; UCN, urocortin; ANOVA, analysis of variance; HEK, human embryonic kidney; siRNA, small interfering RNA; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; bp, base pair; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; PBS, phosphate-buffered saline; BSA, bovine serum albumin; QT-PCR, quantitative reverse transcriptase-polymerase chain reaction; PBS-T, phosphate-buffered saline/Tween 20.
- Received July 22, 2008.
- Accepted December 19, 2008.
- The American Society for Pharmacology and Experimental Therapeutics
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