The hydrophobic amino acid cluster at the cytoplasmic end of transmembrane helix III modulates the coupling of the ß1-adrenergic receptor to Gs

Hassan Hajjhussein, Lidia A. Gardner, Naoaki Fujii, Nancy M. Anderson, Suleiman Bahouth

Research output: Contribution to journalArticle

3 Citations (Scopus)

Abstract

A cluster of hydrophobic amino acids at the cytoplasmic end of trans-membranal helix III (TM-III) is a common feature among class-A of G protein-coupled receptors (GPCR). We mutagenized alanine 1593.53 to glutamic acid and isoleucine1603.54 to arginine (A159E/I160R) in TM-III of the human ß1-adrenergic receptor (ß1-AR) to disrupt the function of the hydrophobic cluster. Structurally, the combined mutations of A159E/I160R caused an almost 90° tilt in the rotation of Arg156 3.50 in the E/DRY motif of TM-III and displaced Tyr1663.60 in intracellular loop 2. The A159E/I160R ß1-AR was uncoupled from Gs as determined by cyclic AMP/adenylyl cyclase assays and by FRET-based proximity measurements between the ß1-AR and Gsα. Isoproterenol induced ß-arrestin trafficking in cells expressing both the wild-type ß1-AR and the A159E/I160R ß1-AR. Isoproterenol markedly increased the phosphorylation of ERK1/2 in cells expressing the WT ß1-AR and this effect was dependent on the activation of the Gs-cyclic AMP-dependent protein kinase→Rap→B-raf axis. However, in cells bearing the A159E/I160R ß1-AR, isoproterenol failed to increase the phosphorylation of ERK1/2. These results indicate that mutations in the Gsα-binding pocket of the GPCR interfered with receptor coupling to Gs and with its downstream signaling cascades.

Original languageEnglish (US)
Pages (from-to)79-88
Number of pages10
JournalJournal of Receptors and Signal Transduction
Volume33
Issue number2
DOIs
StatePublished - Apr 1 2013

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Adrenergic Receptors
Amino Acids
Isoproterenol
Phosphorylation
G-Protein-Coupled Receptors
Cyclic AMP
Bearings (structural)
Cells
Arrestin
Mutation
Distance measurement
Adenylyl Cyclases
Alanine
Adrenergic Agents
Arginine
Glutamic Acid
Assays
Chemical activation
Proteins

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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The hydrophobic amino acid cluster at the cytoplasmic end of transmembrane helix III modulates the coupling of the ß1-adrenergic receptor to Gs. / Hajjhussein, Hassan; Gardner, Lidia A.; Fujii, Naoaki; Anderson, Nancy M.; Bahouth, Suleiman.

In: Journal of Receptors and Signal Transduction, Vol. 33, No. 2, 01.04.2013, p. 79-88.

Research output: Contribution to journalArticle

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abstract = "A cluster of hydrophobic amino acids at the cytoplasmic end of trans-membranal helix III (TM-III) is a common feature among class-A of G protein-coupled receptors (GPCR). We mutagenized alanine 1593.53 to glutamic acid and isoleucine1603.54 to arginine (A159E/I160R) in TM-III of the human {\ss}1-adrenergic receptor ({\ss}1-AR) to disrupt the function of the hydrophobic cluster. Structurally, the combined mutations of A159E/I160R caused an almost 90° tilt in the rotation of Arg156 3.50 in the E/DRY motif of TM-III and displaced Tyr1663.60 in intracellular loop 2. The A159E/I160R {\ss}1-AR was uncoupled from Gs as determined by cyclic AMP/adenylyl cyclase assays and by FRET-based proximity measurements between the {\ss}1-AR and Gsα. Isoproterenol induced {\ss}-arrestin trafficking in cells expressing both the wild-type {\ss}1-AR and the A159E/I160R {\ss}1-AR. Isoproterenol markedly increased the phosphorylation of ERK1/2 in cells expressing the WT {\ss}1-AR and this effect was dependent on the activation of the Gs-cyclic AMP-dependent protein kinase→Rap→B-raf axis. However, in cells bearing the A159E/I160R {\ss}1-AR, isoproterenol failed to increase the phosphorylation of ERK1/2. These results indicate that mutations in the Gsα-binding pocket of the GPCR interfered with receptor coupling to Gs and with its downstream signaling cascades.",
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