Punn, Anu, Chen, Jing, Delidaki, Maria, Tang, Jiyou, Liapakis, George, Lehnert, Hendrik, Levine, Michael A. and Grammatopoulos, Dimitris K.. (2012) Mapping structural determinants within third intracellular loop that direct signaling specificity of type 1 corticotropin-releasing hormone receptor. Journal of Biological Chemistry, Volume 287 (Number 12). pp. 8974-8985. ISSN 0021-9258

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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 Arg292-Met295 and Lys311-Lys314 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 Arg292, Lys297, Arg310, Lys311, and Lys314 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 Arg299 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 Gq 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.

Item Type:	Journal Article
Divisions:	Faculty of Medicine > Warwick Medical School > Translational & Systems Medicine > Metabolic and Vascular Health Faculty of Medicine > Warwick Medical School
Journal or Publication Title:	Journal of Biological Chemistry
Publisher:	American Society for Biochemistry and Molecular Biology
ISSN:	0021-9258
Official Date:	16 March 2012
Volume:	Volume 287
Number:	Number 12
Page Range:	pp. 8974-8985
Identification Number:	10.1074/jbc.M111.272161
Status:	Peer Reviewed
Publication Status:	Published
Access rights to Published version:	Restricted or Subscription Access
URI:	http://wrap.warwick.ac.uk/id/eprint/47833

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