From Molecular Details of the Interplay between Transmembrane Helices of the Thyrotropin Receptor to General Aspects of Signal Transduction in Family A G-protein-coupled Receptors (GPCRs)

Gerd Krause,Inna Hoyer,Catherine L. Worth,Ann-Karin Haas,Ralf Schülein,Jens Furkert,Claudia Rutz,Gunnar Kleinau,Annika Kreuchwig

doi:10.1074/jbc.m110.196980

Abstract

Transmembrane helices (TMHs) 5 and 6 are known to be important for signal transduction by G-protein-coupled receptors (GPCRs). Our aim was to characterize the interface between TMH5 and TMH6 of the thyrotropin receptor (TSHR) to gain molecular insights into aspects of signal transduction and regulation. A proline at TMH5 position 5.50 is highly conserved in family A GPCRs and causes a twist in the helix structure. Mutation of the TSHR-specific alanine (Ala-593⁵·⁵⁰) at this position to proline resulted in a 20-fold reduction of cell surface expression. This indicates that TMH5 in the TSHR might have a conformation different from most other family A GPCRs by forming a regular α-helix. Furthermore, linking our own and previous data from directed mutagenesis with structural information led to suggestions of distinct pairs of interacting residues between TMH5 and TMH6 that are responsible for stabilizing either the basal or the active state. Our insights suggest that the inactive state conformation is constrained by a core set of polar interactions among TMHs 2, 3, 6, and 7 and in contrast that the active state conformation is stabilized mainly by non-polar interactions between TMHs 5 and 6. Our findings might be relevant for all family A GPCRs as supported by a statistical analysis of residue properties between the TMHs of a vast number of GPCR sequences.

Highlights

G-protein-coupled receptors (GPCRs)3 are mediators between extracellular stimuli and intracellular signaling cascades
Side Chain Variations of Alanine 5935.50 to Proline, Valine, and Glycine Cause Fold Defects or Constitutive Activation of Signaling—In most of the family A GPCRs, a proline is found at position 5.50 in TMH5
One of the important questions of signal transduction by GPCRs relates to the details of the structural-functional interplay between TMH5 and TMH6

Summary

Introduction

G-protein-coupled receptors (GPCRs) are mediators between extracellular stimuli and intracellular signaling cascades. Our aim was to characterize the interface of amino acids between TMH5 and TMH6 of the thyrotropin receptor (TSHR) in detail to gain deeper insight into properties that are responsible for the inactive (or basal) and the active conformations This we studied by site-directed mutations, structural homology modeling, and by statistical analysis of a vast number of GPCR sequences. In combination with previous findings, our data provide (i) hints for details of interacting partners at transmembrane helices 5 and 6, (ii) insight into general aspects and specific details of signaling mechanisms during TSHR activation, and (iii) structural and functional consequences of variations at conserved positions in family A GPCRs. this study focused on the causal relationship among amino acid sequence, the three-dimensional structural properties, and the particular functions proceeding at the interfaces along the transmembrane helices of both the TSHR and other family A GPCRs

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