Constitutive Activation of Angiotensin II Type 1 Receptor Alters the Orientation of Transmembrane Helix-2

Shin-Ichiro Miura,Sadashiva S Karnik

doi:10.1074/jbc.m202743200

Abstract

A key step in transmembrane (TM) signal transduction by G-protein-coupled receptors (GPCRs) is the ligand-induced conformational change of the receptor, which triggers the activation of a guanine nucleotide-binding protein. GPCRs contain a seven-TM helical structure essential for signal transduction in response to a large variety of sensory and hormonal signals. Primary structure comparison of GPCRs has shown that the second TM helix contains a highly conserved Asp residue, which is critical for agonist activation in these receptors. How conformational changes in TM2 relate to signal transduction by a GPCR is not known, because activation-induced conformational changes in TM2 helix have not been measured. Here we use modification of reporter cysteines to measure water accessibility at specific residues in TM2 of the type 1 receptor for the octapeptide hormone angiotensin II. Activation-dependent changes in the accessibility of Cys76 on TM2 were measured in constitutively activated mutants. These changes were directly correlated with measurement of function, establishing the link between physical changes in TM2 and function. Accessibility changes were measured at several consecutive residues on TM2, which suggest that TM2 undergoes a transmembrane movement in response to activation. This is the first report of in situ measurement of TM2 movement in a GPCR.

Highlights

G-protein-coupled receptors (GPCRs)1 comprise a large family of cell surface receptors that mediate a diverse response to a large variety of sensory and hormonal signals [1]
Reporter Cys residues introduced at certain TM2 positions (74, 75, and 76 but not 73 and 77) reacted with positively charged MTS reagents and inhibited 125I[Sar1,Ile8]angiotensin II (Ang II) binding to the AT1 receptor
Comparative analysis of these Cys reporters in the genetic background of WT and of the gain-of-function mutant, N111G, further established alteration of the TM2 orientation. It is evident from the data obtained that the TM2 region examined here is ␣-helical in both genetic backgrounds. These findings are consistent with earlier studies, which indicate that reporter cysteine accessibility mapping (RCAM) is extremely sensitive to conformational changes and is an effective approach to measuring relative changes in a residue’s position

Summary

Introduction

G-protein-coupled receptors (GPCRs) comprise a large family of cell surface receptors that mediate a diverse response to a large variety of sensory and hormonal signals [1]. They contain a common structural feature characterized by seven transmembrane (TM) helices essential for signal transduction. The molecular mechanism of activation of AT1 receptor has been proposed to involve conformational changes in the TM domain similar to those in other GPCRs [13] This was based on the finding that Ang II-binding involves interactions with TM3, TM5, TM6, and TM7 (Fig. 1). We observed a decrease in the accessibility of TM2 to water in the constitutively activated mutants of the AT1 receptor, suggesting that activation of the receptor leads to changes in water accessibility of TM2

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Conclusion