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.
Department of Pharmacology, The University of Tennessee Health Sciences Center, Memphis, TN 38163, USA.
Hassan
H
Hajjhussein
Lidia A
LA
Gardner
Naoaki
N
Fujii
Nancy M
NM
Anderson
Suleiman W
SW
Bahouth
D000596:Amino Acids / Q000737:chemistry* / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D019204:GTP-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions / Q000187:drug effects; D007545:Isoproterenol / Q000494:pharmacology; D020929:Mitogen-Activated Protein Kinase Kinases / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation; D010766:Phosphorylation / Q000187:drug effects; D011487:Protein Conformation; D018342:Receptors, Adrenergic, beta-1 / Q000737:chemistry* / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D013329:Structure-Activity Relationship
D000596:Amino Acids; D018342:Receptors, Adrenergic, beta-1; D043562:Receptors, G-Protein-Coupled; D020929:Mitogen-Activated Protein Kinase Kinases; D019204:GTP-Binding Proteins; D007545:Isoproterenol