Intracellular mechanisms regulating corticotropin-releasing hormone receptor-2beta endocytosis and interaction with extracellularly regulated kinase 1/2 and p38 mitogen-activated protein kinase signaling cascades.
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-R2beta and investigated mechanisms involved in attenuation of CRH-R2 signaling activity and uncoupling from intracellular effectors. CRH-R2beta-mediated adenylyl cyclase activation was sensitive to homologous desensitization induced by pretreatment with either UCN-II or the weaker agonist CRH. CRH-R2beta activation induced transient beta-arrestin1 and beta-arrestin2, as well as clathrin, recruitment to the plasma membrane. Beta-arrestin2 appeared to be the main beta-arrestin subtype associated with the receptor. This was followed by CRH-R2beta endocytosis in a mechanism that exhibited distinct agonist-dependent temporal characteristics. CRH-R2beta 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-R2beta-MAPK interaction does not require beta-arrestin, clathrin, or receptor endocytosis. Site-directed mutagenesis studies on CRH-R2beta C terminus showed that the amino acid cassette TAAV at the end of the C terminus is important for CRH-R2beta 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-R2beta functional activity and determining its biological responses.
Markovic
Danijela
D
Endocrinology and Metabolism, Warwick Medical School, University of Warwick, Coventry CV4 7AL, United Kingdom.
Punn
Anu
A
Lehnert
Hendrik
H
Grammatopoulos
Dimitris K
DK
D020816:Amino Acid Motifs; D019390:Arrestins / Q000378:metabolism; D002460:Cell Line; D002966:Clathrin / Q000378:metabolism; D003346:Corticotropin-Releasing Hormone / Q000378:metabolism / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D004705:Endocytosis / Q000502:physiology; D004789:Enzyme Activation; D006801:Humans; D020935:MAP Kinase Signaling System; D008856:Microscopy, Fluorescence; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D034741:RNA, Small Interfering / Q000235:genetics; D018019:Receptors, Corticotropin-Releasing Hormone / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D054832:Urocortins / Q000378:metabolism / Q000494:pharmacology; D000071557:beta-Arrestins
D019390:Arrestins; C101531:CRF receptor type 2; D002966:Clathrin; D034741:RNA, Small Interfering; D018019:Receptors, Corticotropin-Releasing Hormone; C426705:UCN2 protein, human; D054832:Urocortins; D000071557:beta-Arrestins; D003346:Corticotropin-Releasing Hormone; D000242:Cyclic AMP; D020928:Mitogen-Activated Protein Kinases