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

Systematic analysis of structural changes induced by activating mutations h as been frequently utilized to study activation mechanisms of G-protein-cou pled receptors (GPCRs). In the thyrotropin receptor and the lutropin recept or (LHR), a large number of naturally occurring mutations leading to consti tutive receptor activation were identified. Saturating mutagenesis studies of a highly conserved Asp in the junction of the third intracellular loop a nd 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 mot if at the N-terminal ends of alpha -helices terminating and stabilizing the helical structure (helix capping). Since Asp/Glu residues in the third int racellular loop/transmembrane domain 6 junction are not only preserved in g lycoprotein hormone receptors but also in other GPCRs we speculated that th is 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 po ly-Ala substitutions in the LHR and thyrotropin receptor. CD and NMR studie s of peptides derived from the juxtamembrane portion of the LHR confirmed t he helix extension by the poly-Ala substitution and provided further eviden ce for an involvement of Asp in a helix-capping structure. Our data implica te that in addition to well established interhelical interactions the inact ive conformation of GPCRs is also stabilized by specific intrahelical struc tures.