B-Raf and CRHR1 internalization mediate biphasic ERK1/2 activation by CRH in hippocampal HT22 Cells.
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.
Instituto de Investigación en Biomedicina de Buenos Aires (IBioBA), CONICET, Partner Institute of the Max Planck Society, Godoy Cruz 2390, C1425FQA Buenos Aires, Argentina.
Juan J
JJ
Bonfiglio
Carolina
C
Inda
Sergio
S
Senin
Giuseppina
G
Maccarrone
Damián
D
Refojo
Damiana
D
Giacomini
Christoph W
CW
Turck
Florian
F
Holsboer
Eduardo
E
Arzt
Susana
S
Silberstein
D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D019390:Arrestins / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000378:metabolism; D003346:Corticotropin-Releasing Hormone / Q000494:pharmacology*; D000242:Cyclic AMP / Q000378:metabolism; D004705:Endocytosis / Q000187:drug effects*; D004789:Enzyme Activation / Q000187:drug effects; D006624:Hippocampus / Q000166:cytology / Q000201:enzymology*; D006801:Humans; D051379:Mice; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism*; D008954:Models, Biological; D048493:Proto-Oncogene Proteins B-raf / Q000378:metabolism*; D051381:Rats; D018019:Receptors, Corticotropin-Releasing Hormone / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D013347:Subcellular Fractions / Q000187:drug effects / Q000378:metabolism; D013997:Time Factors; D014746:Vimentin / Q000378:metabolism; D000071557:beta-Arrestins; D000099052:CRF Receptor, Type 1
D019390:Arrestins; D018019:Receptors, Corticotropin-Releasing Hormone; D014746:Vimentin; D000071557:beta-Arrestins; D000099052:CRF Receptor, Type 1; D003346:Corticotropin-Releasing Hormone; D000242:Cyclic AMP; D048493:Proto-Oncogene Proteins B-raf; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D000262:Adenylyl Cyclases; D002118:Calcium