Corticotropin-releasing factor type 1 and type 2 alpha receptors regulate phosphorylation of calcium/cyclic adenosine 3 ',5 '-monophosphate response element-binding protein and activation of p42/p44 mitogen-activated proteinkinase

CRF exerts a key neuroregulatory control on the function of the hypothalami c-pituitary-adrenal axis. These effects are thought to be mediated primaril y through activation of G(s)-coupled plasma membrane receptors. In the pres ent study, we investigated the effects of activation of CRF receptors by sa uvagine on signaling pathways that converge on phosphorylation of the trans cription factor calcium/cAMP response element-binding protein (CREB). Studi es were undertaken using CHO cell lines transfected with either rat CRF-1 o r CRF-2 alpha receptors. Signaling pathways were investigated using immunoc ytochemical, Western blot, and imaging techniques. Treatment with sauvagine increased phosphorylation of p42/p44, but not of p38 or stress-activated p rotein kinase (SAPK)/JUN N-terminal kinase (JNK) mitogen-activated protein (MAP) kinases correlating with increased p42/p44 MAP kinase activity. Mobil ization of intracellular Ca2+ stores was observed in cells treated with hig h concentrations (100 nM, 1 mu M) of sauvagine. A time- and dose-dependent increase in phosphorylation of the transcription factor CREB was observed i n cultures treated with sauvagine. Phosphorylation of CREB occurred at lowe r concentrations of sauvagine than those required to mobilize intracellular calcium stores, and phosphorylation was not blocked by the mitogen-activat ed protein kinase kinase inhibitor PD98059 at a concentration (1 mu M that fully inhibited phosphorylation of MAP kinase. Cotreatment of cultures with the protein kinase A inhibitor H89 (10 mu M) blocked fully the stimulatory actions of sauvagine (0.1 nM, 1 nM) on phosphorylation of CREB, but not th ose on phosphorylation of MAP kinase. Phosphorylation of MAP kinase was par tially blocked by the phosphoinositide 3-kinase inhibitor LY294002 (5 mu M and by the phosphoinositide-phospholipase C inhibitor U73122 (10 mu M). The se data demonstrate that cAMP-, Ca2+, and MAP kinase-ependent signaling pat hways are activated by stimulation of CRF-1 and CRF-Bcr receptors. However, in these cells, only protein kinase A-dependent pathways contribute signif icantly to enhanced phosphorylation of CREB. These represent the first repo rted observations of CRF receptor-mediated phosphorylation of the transcrip tion factor CREB and activation of MAP kinase signal transduction pathways.