title: Requirement of specific intrahelical interactions for stabilizing the inactive conformation of glycoprotein hormone receptors.
abstract: 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.
authors.affiliation: Institut für Pharmakologie, Universitätsklinikum Benjamin Franklin, Freie Universität Berlin, Thielallee 69-73, 14195 Berlin, Germany.
authors.forename: A
authors.initials: A
authors.lastname: Schulz
authors.forename: K
authors.initials: K
authors.lastname: Bruns
authors.forename: P
authors.initials: P
authors.lastname: Henklein
authors.forename: G
authors.initials: G
authors.lastname: Krause
authors.forename: M
authors.initials: M
authors.lastname: Schubert
authors.forename: T
authors.initials: T
authors.lastname: Gudermann
authors.forename: V
authors.initials: V
authors.lastname: Wray
authors.forename: G
authors.initials: G
authors.lastname: Schultz
authors.forename: T
authors.initials: T
authors.lastname: Schöneberg
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001216:Asparagine / Q000378:metabolism; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011974:Receptors, LH / Q000737:chemistry / Q000378:metabolism*; D011989:Receptors, Thyrotropin / Q000737:chemistry / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
chemical_list: D011974:Receptors, LH; D011989:Receptors, Thyrotropin; D001216:Asparagine