Mutagenesis within helix 6 of the human β1-adrenergic receptor identifies lysine324 as a residue involved in imparting the high-affinity binding state of agonists

Omeima Zeitoun, Noel M. Delos Santos, Lidia A. Gardner, Stephen W. White, Suleiman Bahouth

Research output: Contribution to journalArticle

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Abstract

Competition binding isotherms for agonists to G protein-coupled receptors (GPCR) display high and low binding affinities. Mutagenesis of lysine at position 324 in helix 6 of the wild-type (WT) human β1- adrenergic receptor (β1-AR) generated mutant receptors that had GTP-insensitive single low-affinity binding sites for agonists and reduced potencies of full or partial agonists in stimulating adenylyl cyclase. Unlike the WT β1-AR, intrinsic activities of full and partial agonists in activating the Lys324→Ala β1-AR (K324A) mutant were correlated with their binding affinities to the K324A mutant. In assays, such as agonist-mediated phosphorylation and recycling, the K324A mutant and the WT β1-AR behaved similarly. However, in fluorescence resonance energy transfer assays that determined the proximity between the WT β1-AR or the K324A mutant to Gsα, there were significant differences. The conceptual framework of the ternary complex model could not adequately account for the behavior of the K324A mutant except under assumptions of low receptor-G protein binding affinities. The single low-affinity binding site of the K324A mutant to isoproterenol was converted by the C-terminal 11-amino-acid peptide of Gsα, which acts a GDP-bound Gsα mimic, to high- and low-affinity sites. Based upon the three-dimensional architecture of the human β1-AR, the distance between Lys324 and the Asp/Glu-Arg-Tyr motif in helix 3 was the shortest among the various amino acids in helix 6. These findings indicate that Lys324 lies in a groove between helices 3 and 6, and its mutagenesis generates a mutant receptor with very low binding affinity for the GDP-bound isoform of Gs.

Original languageEnglish (US)
Pages (from-to)838-850
Number of pages13
JournalMolecular Pharmacology
Volume70
Issue number3
DOIs
StatePublished - Aug 28 2006

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Mutagenesis
Adrenergic Receptors
Binding Sites
Amino Acids
Fluorescence Resonance Energy Transfer
Recycling
G-Protein-Coupled Receptors
Guanosine Triphosphate
Isoproterenol
GTP-Binding Proteins
Adenylyl Cyclases
Protein Binding
Lysine
Protein Isoforms
Phosphorylation
Peptides

All Science Journal Classification (ASJC) codes

  • Pharmacology

Cite this

Mutagenesis within helix 6 of the human β1-adrenergic receptor identifies lysine324 as a residue involved in imparting the high-affinity binding state of agonists. / Zeitoun, Omeima; Delos Santos, Noel M.; Gardner, Lidia A.; White, Stephen W.; Bahouth, Suleiman.

In: Molecular Pharmacology, Vol. 70, No. 3, 28.08.2006, p. 838-850.

Research output: Contribution to journalArticle

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abstract = "Competition binding isotherms for agonists to G protein-coupled receptors (GPCR) display high and low binding affinities. Mutagenesis of lysine at position 324 in helix 6 of the wild-type (WT) human β1- adrenergic receptor (β1-AR) generated mutant receptors that had GTP-insensitive single low-affinity binding sites for agonists and reduced potencies of full or partial agonists in stimulating adenylyl cyclase. Unlike the WT β1-AR, intrinsic activities of full and partial agonists in activating the Lys324→Ala β1-AR (K324A) mutant were correlated with their binding affinities to the K324A mutant. In assays, such as agonist-mediated phosphorylation and recycling, the K324A mutant and the WT β1-AR behaved similarly. However, in fluorescence resonance energy transfer assays that determined the proximity between the WT β1-AR or the K324A mutant to Gsα, there were significant differences. The conceptual framework of the ternary complex model could not adequately account for the behavior of the K324A mutant except under assumptions of low receptor-G protein binding affinities. The single low-affinity binding site of the K324A mutant to isoproterenol was converted by the C-terminal 11-amino-acid peptide of Gsα, which acts a GDP-bound Gsα mimic, to high- and low-affinity sites. Based upon the three-dimensional architecture of the human β1-AR, the distance between Lys324 and the Asp/Glu-Arg-Tyr motif in helix 3 was the shortest among the various amino acids in helix 6. These findings indicate that Lys324 lies in a groove between helices 3 and 6, and its mutagenesis generates a mutant receptor with very low binding affinity for the GDP-bound isoform of Gs.",
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