title: Mapping structural determinants within third intracellular loop that direct signaling specificity of type 1 corticotropin-releasing hormone receptor.
abstract: The type 1 corticotropin-releasing hormone receptor (CRH-R1) influences biological responses important for adaptation to stressful stimuli, through activation of multiple downstream effectors. The structural motifs within CRH-R1 that mediate G protein activation and signaling selectivity are unknown. The aim of this study was to gain insights about important structural determinants within the third intracellular loop (IC3) of the human CRH-R1α important for cAMP and ERK1/2 pathways activation and selectivity. We investigated the role of the juxtamembrane regions of IC3 by mutating amino acid cassettes or specific residues to alanine. Although simultaneous tandem alanine mutations of both juxtamembrane regions Arg(292)-Met(295) and Lys(311)-Lys(314) reduced ligand binding and impaired signaling, all other mutant receptors retained high affinity binding, indistinguishable from wild-type receptor. Agonist-activated receptors with tandem mutations at the proximal or distal terminal segments enhanced activation of adenylyl cyclase by 50-75% and diminished activation of inositol trisphosphate and ERK1/2 by 60-80%. Single Ala mutations identified Arg(292), Lys(297), Arg(310), Lys(311), and Lys(314) as important residues for the enhanced activation of adenylyl cyclase, partly due to reduced inhibition of adenylyl cyclase activity by pertussis toxin-sensitive G proteins. In contrast, mutation of Arg(299) reduced receptor signaling activity and cAMP response. Basic as well as aliphatic amino acids within both juxtamembrane regions were identified as important for ERK1/2 phosphorylation through activation of pertussis toxin-sensitive G proteins as well as G(q) proteins. These data uncovered unexpected roles for key amino acids within the highly conserved hydrophobic N- and C-terminal microdomains of IC3 in the coordination of CRH-R1 signaling activity.
authors.affiliation: Department of Endocrinology and Metabolism, Clinical Sciences Research Institute, Warwick Medical School, University of Warwick, Coventry, United Kingdom.
authors.forename: Anu
authors.initials: A
authors.lastname: Punn
authors.forename: Jing
authors.initials: J
authors.lastname: Chen
authors.forename: Maria
authors.initials: M
authors.lastname: Delidaki
authors.forename: Jiyou
authors.initials: J
authors.lastname: Tang
authors.forename: George
authors.initials: G
authors.lastname: Liapakis
authors.forename: Hendrik
authors.initials: H
authors.lastname: Lehnert
authors.forename: Michael A
authors.initials: MA
authors.lastname: Levine
authors.forename: Dimitris K
authors.initials: DK
authors.lastname: Grammatopoulos
mesh_terms: D000595:Amino Acid Sequence; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism; D004789:Enzyme Activation; D006801:Humans; D020935:MAP Kinase Signaling System; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D008969:Molecular Sequence Data; D011485:Protein Binding; D017433:Protein Structure, Secondary; D018019:Receptors, Corticotropin-Releasing Hormone / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D000099052:CRF Receptor, Type 1
chemical_list: D018019:Receptors, Corticotropin-Releasing Hormone; D000099052:CRF Receptor, Type 1; D000242:Cyclic AMP; C535150:MAPK1 protein, human; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3