Multiple interactions of the Asp(2.61(98)) side chain of the gonadotropin-releasing hormone receptor contribute differentially to ligand interaction

Colleen A. Flanagan, Vladimir Rodic, Karel Konvicka, Tony Yuen, Ling Chi, Jean E. Rivier, Robert P. Millar, Harel Weinstein, Stuart C. Sealfon

Research output: Contribution to journalArticlepeer-review

64 Scopus citations

Abstract

Mutation of Asp(2.61(98)) at the extracellular boundary of transmembrane helix 2 of the gonadotropin-releasing hormone (GnRH) receptor decreased the affinity for GnRH. Using site-directed mutagenesis, ligand modification, and computational modeling, different side chain interactions of Asp(2.61(98)) that contribute to high-affinity binding were investigated. The conservative Asp(2.61(98)) Glu mutation markedly decreased the affinity for a series of GnRH analogues containing the native His2 residue. This mutant showed smaller decreases in affinity for His2-substituted ligands. The loss of preference for His2-containing ligands in the mutant receptor shows that Asp(2.61(98)) determines the specificity for His2. Analysis of the affinities of a series of position 2-substituted ligands suggests that a hydrogen bond forms between Asp(2.61(98)) and the δ NH group of His2 and that Asp(2.61(98)) forms a second hydrogen bond with the ligand. Substitution of Asp(2.61(98)) with an uncharged residue further decreased the affinity for all ligands and also decreased receptor expression. Computational modeling indicates an intramolecular ionic interaction of Asp(2.61(98)) with Lys(3.32(121)) in transmembrane helix 3. The uncharged, Lys(3.32(121))Gln mutation also markedly decreased agonist affinity. The modeling and the similar phenotypes of mutants with uncharged substitutions for Asp(2.61(98)) or Lys(3.32(121)) are consistent with the presence of this helix 2-helix 3 interaction. These studies support a dual role for Asp(2.61(98)): formation of an interhelical interaction with Lys(3.32(121)) that contributes to the structure of the agonist binding pocket and an interaction with His2 of GnRH that helps stabilize agonist complexing.

Original languageEnglish
Pages (from-to)8133-8141
Number of pages9
JournalBiochemistry
Volume39
Issue number28
DOIs
StatePublished - 18 Jul 2000

Fingerprint

Dive into the research topics of 'Multiple interactions of the Asp(2.61(98)) side chain of the gonadotropin-releasing hormone receptor contribute differentially to ligand interaction'. Together they form a unique fingerprint.

Cite this