title: Light-driven activation of beta 2-adrenergic receptor signaling by a chimeric rhodopsin containing the beta 2-adrenergic receptor cytoplasmic loops.
abstract: Structure-function studies of rhodopsin indicate that both intradiscal and transmembrane (TM) domains are required for retinal binding and subsequent light-induced structural changes in the cytoplasmic domain. Further, a hypothesis involving a common mechanism for activation of G-protein-coupled receptor (GPCR) has been proposed. To test this hypothesis, chimeric receptors were required in which the cytoplasmic domains of rhodopsin were replaced with those of the beta(2)-adrenergic receptor (beta(2)-AR). Their preparation required identification of the boundaries between the TM domain of rhodopsin and the cytoplasmic domain of the beta(2)-AR necessary for formation of the rhodopsin chromophore and its activation by light and subsequent optimal activation of beta(2)-AR signaling. Chimeric receptors were constructed in which the cytoplasmic loops of rhodopsin were replaced one at a time and in combination. In these replacements, size of the third cytoplasmic (EF) loop critically determined the extent of chromophore formation, its stability, and subsequent signal transduction specificity. All the EF loop replacements showed significant decreases in transducin activation, while only minor effects were observed by replacements of the CD and AB loops. Light-dependent activation of beta(2)-AR leading to Galphas signaling was observed only for the EF2 chimera, and its activation was further enhanced by replacements of the other loops. The results demonstrate coupling between light-induced conformational changes occurring in the transmembrane domain of rhodopsin and the cytoplasmic domain of the beta(2)-AR.
authors.affiliation: Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, USA.
authors.forename: Jong-Myoung
authors.initials: JM
authors.lastname: Kim
authors.forename: John
authors.initials: J
authors.lastname: Hwa
authors.forename: Pere
authors.initials: P
authors.lastname: Garriga
authors.forename: Philip J
authors.initials: PJ
authors.lastname: Reeves
authors.forename: Uttam L
authors.initials: UL
authors.lastname: RajBhandary
authors.forename: H Gobind
authors.initials: HG
authors.lastname: Khorana
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D019556:COS Cells; D002417:Cattle; D002460:Cell Line; D002522:Chlorocebus aethiops; D006224:Cricetinae; D003593:Cytoplasm / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008027:Light; D008969:Molecular Sequence Data; D011487:Protein Conformation; D017433:Protein Structure, Secondary / Q000235:genetics; D018343:Receptors, Adrenergic, beta-2 / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism; D012243:Rhodopsin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017299:Rod Opsins / Q000737:chemistry / Q000378:metabolism; D015398:Signal Transduction / Q000235:genetics*
chemical_list: D018343:Receptors, Adrenergic, beta-2; D011993:Recombinant Fusion Proteins; D017299:Rod Opsins; D012243:Rhodopsin