Stable association of G proteins with beta 2AR is independent of the state of receptor activation

Cell Signal. 1999 Jul;11(7):523-33. doi: 10.1016/s0898-6568(99)00024-8.

Abstract

beta 2-Adrenergic receptors expressed in Sf9 cells activate endogenous Gs and adenylyl cyclase [Mouillac B., Caron M., Bonin H., Dennis M. and Bouvier M. (1992) J. Biol. Chem. 267, 21733-21737]. However, high affinity agonist binding is not detectable under these conditions suggesting an improper stoichiometry between the receptor and the G protein and possibly the effector molecule as well. In this study we demonstrate that when beta 2-adrenergic receptors were co-expressed with various mammalian G protein subunits in Sf9 cells using recombinant baculoviruses signalling properties found in native receptor systems were reconstituted. For example, when beta 2AR was co-expressed with the Gs alpha subunit, maximal receptor-mediated adenylyl cyclase stimulation was greatly enhanced (60 +/- 9.0 versus 150 +/- 52 pmol cAMP/min/mg protein) and high affinity, GppNHp-sensitive, agonist binding was detected. When G beta gamma subunits were co-expressed with Gs alpha and the beta 2AR, receptor-stimulated GTPase activity was also demonstrated, in contrast to when the receptor was expressed alone, and this activity was higher than when beta 2AR was co-expressed with Gs alpha alone. Other properties of the receptor, including receptor desensitization and response to inverse agonists were unaltered. Using antisera against an epitope-tagged beta 2AR, both Gs alpha and beta gamma subunits could be co-immunoprecipitated with the beta 2AR under conditions where subunit dissociation would be expected given current models of G protein function. A desensitization-defective beta 2AR (S261, 262, 345, 346A) and a mutant which is constitutively desensitized (C341G) could also co-immunoprecipitate G protein subunits. These results will be discussed in terms of a revised view of G protein-mediated signalling which may help address issues of specificity in receptor/G protein coupling.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenylyl Cyclases / metabolism
  • Adrenergic beta-Agonists / metabolism
  • Animals
  • Cell Line
  • GTP Phosphohydrolases / metabolism
  • GTP-Binding Protein alpha Subunits, Gi-Go / genetics
  • GTP-Binding Protein alpha Subunits, Gi-Go / metabolism
  • GTP-Binding Protein alpha Subunits, Gs / genetics
  • GTP-Binding Protein alpha Subunits, Gs / metabolism
  • GTP-Binding Protein beta Subunits*
  • GTP-Binding Protein gamma Subunits*
  • GTP-Binding Proteins / genetics
  • GTP-Binding Proteins / metabolism*
  • Heterotrimeric GTP-Binding Proteins*
  • Humans
  • Isoproterenol / metabolism
  • Receptors, Adrenergic, beta-2 / genetics
  • Receptors, Adrenergic, beta-2 / metabolism*
  • Signal Transduction
  • Spodoptera / cytology

Substances

  • Adrenergic beta-Agonists
  • G-protein Beta gamma
  • GTP-Binding Protein beta Subunits
  • GTP-Binding Protein gamma Subunits
  • Receptors, Adrenergic, beta-2
  • GTP Phosphohydrolases
  • GTP-Binding Proteins
  • GTP-Binding Protein alpha Subunits, Gi-Go
  • GTP-Binding Protein alpha Subunits, Gs
  • Heterotrimeric GTP-Binding Proteins
  • Adenylyl Cyclases
  • Isoproterenol