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

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 159^3.53 to glutamic acid and isoleucine160^3.54 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 Tyr166^3.60 in intracellular loop 2. The A159E/I160R ß₁-AR was uncoupled from G_s as determined by cyclic AMP/adenylyl cyclase assays and by FRET-based proximity measurements between the ß₁-AR and G_sα. 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 ERK_1/2 in cells expressing the WT ß₁-AR and this effect was dependent on the activation of the G_s-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 ERK_1/2. These results indicate that mutations in the G_sα-binding pocket of the GPCR interfered with receptor coupling to G_s and with its downstream signaling cascades.

• Adrenergic receptor
• FRET
• GPCR
• MAP kinases
• signal transduction
• 3-D modeling

Acknowledgements

We thank the following investigators for kindly providing the following reagents: Zhijun Luo, at Boston University Medical Center for WT B-Raf and B-Raf K482M, Catherine Berlot, Weis Center for research, Geisinger Clinic, Danville, PA, for G_sα-CFP, Robert Lefkowitz, HHMI, Duke University for the human ß₂-AR and ß-Arrestin₂-GFP and Stanley Mcknight, University of Washington, Seattle, for MT-REV_AB.