title: Structural requirements for the activation of the human growth hormone secretagogue receptor by peptide and nonpeptide secretagogues.
abstract: Antibodies raised against an intracellular and extracellular domain of the GH secretagogue receptor (GHS-R) confirmed that its topological orientation in the lipid bilayer is as predicted for G protein-coupled receptors with seven transmembrane domains. A strategy for mapping the agonist-binding site of the human GHS-R was conceived based on our understanding of ligand binding in biogenic amine and peptide hormone G protein-coupled receptors. Using site-directed mutagenesis and molecular modeling, we classified GHS peptide and nonpeptide agonist binding in the context of its receptor environment. All peptide and nonpeptide ligand classes shared a common binding domain in transmembrane (TM) region 3 of the GHS-R. This finding was based on TM-3 mutation E124Q, which eliminated the counter-ion to the shared basic N+ group of all GHSs and resulted in a nonfunctional receptor. Restoration of function for the E124Q mutant was achieved by a complementary change in the MK-0677 ligand through modification of its amine side-chain to the corresponding alcohol. Contacts in other TM domains [TM-2 (D99N), TM-5 (M213K, S117A), TM-6 (H280F), and extracellular loop 1 (C116A)] of the receptor revealed specificity for the different peptide, benzolactam, and spiroindolane GHSs. GHS-R agonism, therefore, does not require identical disposition of all agonist classes at the ligand-binding site. Our results support the hypothesis that the ligand-binding pocket in the GHS-R is spatially disposed similarly to the well characterized catechol-binding site in the beta2-adrenergic receptor.
authors.affiliation: Department of Biochemistry and Physiology, Merck Research Laboratories, Rahway, New Jersey 07065, USA.
authors.forename: S D
authors.initials: SD
authors.lastname: Feighner
authors.forename: A D
authors.initials: AD
authors.lastname: Howard
authors.forename: K
authors.initials: K
authors.lastname: Prendergast
authors.forename: O C
authors.initials: OC
authors.lastname: Palyha
authors.forename: D L
authors.initials: DL
authors.lastname: Hreniuk
authors.forename: R
authors.initials: R
authors.lastname: Nargund
authors.forename: D
authors.initials: D
authors.lastname: Underwood
authors.forename: J R
authors.initials: JR
authors.lastname: Tata
authors.forename: D C
authors.initials: DC
authors.lastname: Dean
authors.forename: C P
authors.initials: CP
authors.lastname: Tan
authors.forename: K K
authors.initials: KK
authors.lastname: McKee
authors.forename: J W
authors.initials: JW
authors.lastname: Woods
authors.forename: A A
authors.initials: AA
authors.lastname: Patchett
authors.forename: R G
authors.initials: RG
authors.lastname: Smith
authors.forename: L H
authors.initials: LH
authors.lastname: Van der Ploeg
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D019204:GTP-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D019382:Human Growth Hormone / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000502:physiology*; D051381:Rats; D011956:Receptors, Cell Surface / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled; D054440:Receptors, Ghrelin; D013552:Swine
chemical_list: D010455:Peptides; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; D054440:Receptors, Ghrelin; D019382:Human Growth Hormone; D019204:GTP-Binding Proteins