Mapping Structural Determinants within Third Intracellular Loop That Direct Signaling Specificity of Type 1 Corticotropin-releasing Hormone Receptor

Anu Punn,Jing Chen,Maria Delidaki,Jiyou Tang,George Liapakis,Hendrik Lehnert,Michael A Levine,Dimitris K Grammatopoulos

doi:10.1074/jbc.m111.272161

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.

Highlights

The CRH-R1 is key for mammalian adaptation to stress; the structural determinants of signal transduction are unknown
Binding Properties of Mutant CRH-R1␣ Receptors—To identify residue(s) in the CRH-R1␣ N- and C-terminal regions of IC3 involved in receptor signaling, we used a tandem alanine scanning mutagenesis strategy, in which four consecutive amino acid residues were replaced by alanines (Fig. 1)
Because our previous observations suggested that specific amino acids within Arg292-Lys297 and Arg310-Lys314 terminal regions as well as Arg299, were important for CRH-R1␣ coupling to pertussis toxin (PTX)-sensitive G proteins, we investigated the role of Gi/o proteins in wild-typeand mutant receptor-mediated ERK1/2 activation

Summary

Introduction

The CRH-R1 is key for mammalian adaptation to stress; the structural determinants of signal transduction are unknown. The type 1 corticotropin-releasing hormone receptor (CRHR1) influences biological responses important for adaptation to stressful stimuli, through activation of multiple downstream effectors. 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. 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

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