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Intracellular Mechanisms Regulating Corticotropin-Releasing Hormone Receptor-2β Endocytosis and Interaction with Extracellularly Regulated Kinase 1/2 and p38 Mitogen-Activated Protein Kinase Signaling Cascades

Endocrinology and Metabolism, Warwick Medical School, University of Warwick, Coventry CV4 7AL, United Kingdom

Address all correspondence and requests for reprints to: Prof. D. K Grammatopoulos, Sir Quinton Hazell Molecular Medicine Research Centre, Department of Biological Sciences, The University of Warwick, Gibbet Hill Road, Coventry CV4 7AL, United Kingdom. E-mail: d.grammatopoulos{at}warwick.ac.uk.

Many important physiological roles of the urocortin (UCN) family of peptides as well as CRH involve the type 2 CRH receptor (CRH-R2) and downstream activation of multiple pathways. To characterize molecular determinants of CRH-R2 functional activity, we used HEK293 cells overexpressing recombinant CRH-R2β and investigated mechanisms involved in attenuation of CRH-R2 signaling activity and uncoupling from intracellular effectors. CRH-R2β-mediated adenylyl cyclase activation was sensitive to homologous desensitization induced by pretreatment with either UCN-II or the weaker agonist CRH. CRH-R2β activation induced transient β-arrestin1 and β-arrestin2, as well as clathrin, recruitment to the plasma membrane. β-Arrestin2 appeared to be the main β-arrestin subtype associated with the receptor. This was followed by CRH-R2β endocytosis in a mechanism that exhibited distinct agonist-dependent temporal characteristics. CRH-R2β also induced transient activation of the ERK1/2 and p38MAPK signaling cascades that peaked at 5 min and returned to basal within 20–30 min. Unlike p38MAPK, activated ERK1/2 was localized both in the cytoplasm and nucleus. Experiments employing inhibitors of receptor endocytosis showed that CRH-R2β-MAPK interaction does not require β-arrestin, clathrin, or receptor endocytosis. Site-directed mutagenesis studies on CRH-R2β C terminus showed that the amino acid cassette TAAV at the end of the C terminus is important for CRH-R2β signaling because loss of a potential phospho-acceptor site in mutant receptors containing deletion or Ala substitution of the cassette TAAV resulted in reduced ERK1/2 activation and accelerated receptor internalization. These findings provide new insights about the signaling mechanisms regulating CRH-R2β functional activity and determining its biological responses.

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