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

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 Arg1563.50 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.