Abstract
Gs protein-coupled receptors regulate many vital body functions by activation of cAMP response elements (CRE) via cAMP-dependent kinase A (PKA)-mediated phosphorylation of the CRE binding protein (CREB). Melanocortin 4 receptors (MC4R) are prototypical Gs-coupled receptors that orchestrate the hypothalamic control of food-intake and metabolism. Remarkably, the significance of PKA for MC4R-induced CRE-dependent transcription in hypothalamic cells has not been rigorously interrogated yet. In two hypothalamic cell lines, we observed that blocking PKA activity had only weak or no effects on reporter gene expression. In contrast, inhibitors of exchange factors directly activated by cAMP-1/2 (EPAC-1/2) mitigated MC4R-induced CRE reporter activation and mRNA induction of the CREB-dependent genes c-fos and thyrotropin-releasing hormone. Furthermore, we provide first evidence that extracellular-regulated kinases-1/2 (ERK-1/2) activated by EPACs and not PKA are the elusive CREB kinases responsible for MC4R-induced CREB/CRE activation in hypothalamic cells. Overall, these data emphasize the pivotal role of EPACs rather than PKA in hypothalamic gene expression elicited by a prototypical Gs-coupled receptor.
Highlights
Gs protein-coupled receptors regulate many vital body functions by activation of cAMP response elements (CRE) via cAMP-dependent kinase A (PKA)-mediated phosphorylation of the CRE binding protein (CREB)
This scenario is remarkable because 1) extracellular-regulated kinases-1/2 (ERK-1/2) kinases have been implicated in Melanocortin 4 receptors (MC4R)-induced hypothalamic CREB phosphorylation in vivo and 2) exchange factors directly activated by cAMP induce CREB phosphorylation via ERK-1/2 when dopamine or cell-permeable cAMP analogues were applied to PC-12 or pituitary cells[26,27,28,29,30,31]
HEK-293-MC4R cells reacted to α-melanocyte-stimulating hormone (α-MSH) with increased cAMP accumulation (Fig. 1A) and concentration-dependent CRE activation after transfection of cells with a CRE-dependent reporter plasmid (Fig. 1B)
Summary
Gs protein-coupled receptors regulate many vital body functions by activation of cAMP response elements (CRE) via cAMP-dependent kinase A (PKA)-mediated phosphorylation of the CRE binding protein (CREB). We transfected the CRE reporter into GT1-7 cells and analysed the effects of PKA and EPAC-1/2 inhibitors on α-MSH-induced CRE activation S3), both EPAC-1/2 inhibitors blocked ~80% of α-MSH-induced CRE activation in mHypoA-2/10-CRE cells, in accord with our data obtained in HEK-293 and GT1-7 cells.
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