The hydrophobic amino acid cluster at the cytoplasmic end of transmembrane helix III modulates the coupling of the β(1)-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 β(1)-adrenergic receptor (β(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 Tyr166(3.60) in intracellular loop 2. The A159E/I160R β(1)-AR was uncoupled from G(s) as determined by cyclic AMP/adenylyl cyclase assays and by FRET-based proximity measurements between the β(1)-AR and G(s)α. Isoproterenol induced β-arrestin trafficking in cells expressing both the wild-type β(1)-AR and the A159E/I160R β(1)-AR. Isoproterenol markedly increased the phosphorylation of ERK1/2 in cells expressing the WT β(1)-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 β(1)-AR, isoproterenol failed to increase the phosphorylation of ERK(1/2). These results indicate that mutations in the G(s)α-binding pocket of the GPCR interfered with receptor coupling to G(s) and with its downstream signaling cascades.

Citation

Hassan Hajjhussein, Lidia A Gardner, Naoaki Fujii, Nancy M Anderson, Suleiman W Bahouth. The hydrophobic amino acid cluster at the cytoplasmic end of transmembrane helix III modulates the coupling of the β(1)-adrenergic receptor to G(s). Journal of receptor and signal transduction research. 2013 Apr;33(2):79-88

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PMID: 23351074

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