title: Activation of the beta 2-adrenergic receptor involves disruption of an ionic lock between the cytoplasmic ends of transmembrane segments 3 and 6.
abstract: The movements of transmembrane segments (TMs) 3 and 6 at the cytoplasmic side of the membrane play an important role in the activation of G-protein-coupled receptors. Here we provide evidence for the existence of an ionic lock that constrains the relative mobility of the cytoplasmic ends of TM3 and TM6 in the inactive state of the beta(2)-adrenergic receptor. We propose that the highly conserved Arg-131(3.50) at the cytoplasmic end of TM3 interacts both with the adjacent Asp-130(3.49) and with Glu-268(6.30) at the cytoplasmic end of TM6. Such a network of ionic interactions has now been directly supported by the high-resolution structure of the inactive state of rhodopsin. We hypothesized that the network of interactions would serve to constrain the receptor in the inactive state, and the release of this ionic lock could be a key step in receptor activation. To test this hypothesis, we made charge-neutralizing mutations of Glu-268(6.30) and of Asp-130(3.49) in the beta(2)-adrenergic receptor. Alone and in combination, we observed a significant increase in basal and pindolol-stimulated cAMP accumulation in COS-7 cells transiently transfected with the mutant receptors. Moreover, based on the increased accessibility of Cys-285(6.47) in TM6, we provide evidence for a conformational rearrangement of TM6 that is highly correlated with the extent of constitutive activity of the different mutants. The present experimental data together with the recent high-resolution structure of rhodopsin suggest that ionic interactions between Asp/Glu(3.49), Arg(3.50), and Glu(6.30) may constitute a common switch governing the activation of many rhodopsin-like G-protein-coupled receptors.
authors.affiliation: Novasite Pharmaceuticals, Inc., San Diego, California 92121, USA.
authors.forename: J A
authors.initials: JA
authors.lastname: Ballesteros
authors.forename: A D
authors.initials: AD
authors.lastname: Jensen
authors.forename: G
authors.initials: G
authors.lastname: Liapakis
authors.forename: S G
authors.initials: SG
authors.lastname: Rasmussen
authors.forename: L
authors.initials: L
authors.lastname: Shi
authors.forename: U
authors.initials: U
authors.lastname: Gether
authors.forename: J A
authors.initials: JA
authors.lastname: Javitch
mesh_terms: D000318:Adrenergic beta-Agonists / Q000493:pharmacokinetics; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001120:Arginine; D001224:Aspartic Acid; D019556:COS Cells; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D002522:Chlorocebus aethiops; D017124:Conserved Sequence; D000242:Cyclic AMP / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D018698:Glutamic Acid; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011412:Propanolamines / Q000493:pharmacokinetics; D017433:Protein Structure, Secondary; D018343:Receptors, Adrenergic, beta-2 / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014162:Transfection
chemical_list: D000318:Adrenergic beta-Agonists; D011412:Propanolamines; D018343:Receptors, Adrenergic, beta-2; D011994:Recombinant Proteins; D001224:Aspartic Acid; D018698:Glutamic Acid; D001120:Arginine; D000242:Cyclic AMP; C033394:CGP 12177