title: Molecular determinants and feedback circuits regulating type 2 CRH receptor signal integration.
abstract: In most target tissues, the adenylyl cyclase/cAMP/PKA, the extracellular signal regulated kinase and the protein kinase B/Akt are the main pathways employed by the type 2 corticotropin-releasing hormone receptor to mediate the biological actions of urocortins (Ucns) and CRH. To decipher the molecular determinants of CRH-R2 signaling, we studied the signaling pathways in HEK293 cells overexpressing recombinant human CRH-R2β receptors. Use of specific kinase inhibitors showed that the CRH-R2β cognate agonist, Ucn 2, activated extracellular signal regulated kinase in a phosphoinositide 3-kinase and cyclic adenosine monophosphate/PKA-dependent manner with contribution from Epac activation. Ucn 2 also induced PKA-dependent association between AKAP250 and CRH-R2β that appeared to be necessary for extracellular signal regulated kinase activation. PKB/Akt activation was also mediated via pertussis toxin-sensitive G-proteins and PI3-K activation but did not require cAMP/PKA, Epac or protein kinase C for optimal activation. Potential feedback mechanisms that target the CRH-R2β itself and modulate receptor trafficking and endocytosis were also investigated. Indeed, our results suggested that inhibition of either PKA or extracellular signal regulated kinase pathway accelerates CRH-R2β endocytosis. Furthermore, Ucn 2-activated extracellular signal regulated kinase appeared to target β-arrestin1 and modulate, through phosphorylation at Ser412, β-arrestin1 translocation to the plasma membrane and CRH-R2β internalization kinetics. Loss of this "negative feedback" mechanism through inhibition of the extracellular signal regulated kinase activity resulted in significant attenuation of Ucn 2-induced cAMP response, whereas Akt phosphorylation was not affected by altered receptor endocytosis. These findings reveal a complex interplay between the signaling molecules that allow "fine-tuning" of CRH-R2β functional responses and regulate signal integration. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.
authors.affiliation: Division of Endocrinology and Metabolism, Clinical Sciences Research Institute, Warwick Medical School, University of Warwick Gibbet Hill Road, Coventry, CV4 7AL, UK.
authors.forename: Danijela
authors.initials: D
authors.lastname: Markovic
authors.forename: Anu
authors.initials: A
authors.lastname: Punn
authors.forename: Hendrik
authors.initials: H
authors.lastname: Lehnert
authors.forename: Dimitris K
authors.initials: DK
authors.lastname: Grammatopoulos
mesh_terms: D054758:A Kinase Anchor Proteins / Q000378:metabolism; D019390:Arrestins / Q000378:metabolism; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002867:Chromones / Q000494:pharmacology; D000242:Cyclic AMP / Q000096:biosynthesis; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D004705:Endocytosis / Q000187:drug effects; D004789:Enzyme Activation / Q000187:drug effects; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D025461:Feedback, Physiological / Q000187:drug effects; D057809:HEK293 Cells; D006801:Humans; D008954:Models, Biological; D009025:Morpholines / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D021381:Protein Transport / Q000187:drug effects; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D018019:Receptors, Corticotropin-Releasing Hormone / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D054832:Urocortins / Q000494:pharmacology; D000071557:beta-Arrestins
chemical_list: D054758:A Kinase Anchor Proteins; C105137:AKAP1 protein, human; D019390:Arrestins; C101531:CRF receptor type 2; D002867:Chromones; D009025:Morpholines; D047428:Protein Kinase Inhibitors; D018019:Receptors, Corticotropin-Releasing Hormone; D054832:Urocortins; D000071557:beta-Arrestins; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D000242:Cyclic AMP; D019869:Phosphatidylinositol 3-Kinases; D051057:Proto-Oncogene Proteins c-akt; D017868:Cyclic AMP-Dependent Protein Kinases; D048049:Extracellular Signal-Regulated MAP Kinases