Requirement of specific intrahelical interactions for stabilizing the inactive conformation of glycoprotein hormone receptors.

Systematic analysis of structural changes induced by activating mutations has been frequently utilized to study activation mechanisms of G-protein-coupled receptors (GPCRs). In the thyrotropin receptor and the lutropin receptor (LHR), a large number of naturally occurring mutations leading to constitutive receptor activation were identified. Saturating mutagenesis studies of a highly conserved Asp in the junction of the third intracellular loop and transmembrane domain 6 suggested a participation of this anionic residue in a salt bridge stabilizing the inactive receptor conformation. However, substitution of all conserved cationic residues at the cytoplasmic receptor surface did not support this hypothesis. Asp/Glu residues are a common motif at the N-terminal ends of alpha-helices terminating and stabilizing the helical structure (helix capping). Since Asp/Glu residues in the third intracellular loop/transmembrane domain 6 junction are not only preserved in glycoprotein hormone receptors but also in other GPCRs we speculated that this residue probably participates in an N-terminal helix-capping structure. Poly-Ala stretches are known to form and stabilize alpha-helices. Herein, we show that the function of the highly conserved Asp can be mimicked by poly-Ala substitutions in the LHR and thyrotropin receptor. CD and NMR studies of peptides derived from the juxtamembrane portion of the LHR confirmed the helix extension by the poly-Ala substitution and provided further evidence for an involvement of Asp in a helix-capping structure. Our data implicate that in addition to well established interhelical interactions the inactive conformation of GPCRs is also stabilized by specific intrahelical structures.