B-Raf and CRHR1 Internalization Mediate Biphasic ERK1/2 Activation by CRH in Hippocampal HT22 Cells

Abstract

CRH is a key regulator of neuroendocrine, autonomic, and behavioral response to stress. CRH-stimulated CRH receptor 1 (CRHR1) activates ERK1/2 depending on intracellular context. In a previous work, we demonstrated that CRH activates ERK1/2 in limbic areas of the mouse brain (hippocampus and basolateral amygdala). ERK1/2 is an essential mediator of hippocampal physiological processes including emotional behavior, synaptic plasticity, learning, and memory. To elucidate the molecular mechanisms by which CRH activates ERK1/2 in hippocampal neurons, we used the mouse hippocampal cell line HT22. We document for the first time that ERK1/2 activation in response to CRH is biphasic, involving a first cAMP- and B-Raf-dependent early phase and a second phase that critically depends on CRHR1 internalization and β-arrestin2. By means of mass-spectrometry-based screening, we identified B-Raf-associated proteins that coimmunoprecipitate with endogenous B-Raf after CRHR1 activation. Using molecular and pharmacological tools, the functional impact of selected B-Raf partners in CRH-dependent ERK1/2 activation was dissected. These results indicate that 14-3-3 proteins, protein kinase A, and Rap1, are essential for early CRH-induced ERK1/2 activation, whereas dynamin and vimentin are required for the CRHR1 internalization-dependent phase. Both phases of ERK1/2 activation depend on calcium influx and are affected by calcium/calmodulin-dependent protein kinase II inactivation. Thus, this report describes the dynamics and biphasic nature of ERK1/2 activation downstream neuronal CRHR1 and identifies several new critical components of the CRHR1 signaling machinery that selectively controls the early and late phases of ERK1/2 activation, thus providing new potential therapeutic targets for stress-related disorders.

Don’t stress about CRF: assessing the translational failures of CRF1antagonists

“…To the degree that the biology of human vs rodent CRF 1 receptors differ (or that of several, molecular partners that influence CRF 1 signaling; Dunn et al 2016; Bonfiglio et al 2013; Bangasser et al 2010 Walther et al 2015), cryptic species differences also may be impeding therapeutic silencing of CRF 1 receptors in humans. Furthermore, selective CRF 1 antagonists that have been tested to date have no activity at CRF 2 receptors or the CRE-binding protein (CRF-BP).…”

The activity of the glucocorticoid receptor is regulated by SUMO conjugation to FKBP51

“…20 Therefore, we used HT22 cells, a murine hippocampal neuronal cell line, 21 to study SUMOylation of FKBP51 in this context. As shown in Figure 1h, FKBP51 is SUMOylated in HT22 cells; and the expression of SENP1 decreases its conjugation to SUMO.…”

CRF1 Receptor Signaling via the ERK1/2-MAP and Akt Kinase Cascades: Roles of Src, EGF Receptor, and PI3-Kinase Mechanisms

“…Regarding β-arrestin regulation of CRF/CRF 1 R-mediated ERK1/2 activation, β-arrestin-2-mediation of CRF 1 R internalization participates in the late phase of sustained ERK1/2 activation after G protein activation and B-Raf mediate the early phase of ERK1/2 activation (88). However, overexpression of PDS-95 in HEK293 cells, a CRF 1 R-interacting protein, inhibited CRF-induced-CRF 1 R internalization in a PDZ-binding motifdependent manner by suppressing β-arrestin-2 recruitment.…”