Reconstitution of beta2-adrenoceptor-GTP-binding-protein interaction in Sf9 cells--high coupling efficiency in a beta2-adrenoceptor-G(s alpha) fusion protein

Eur J Biochem. 1998 Jul 15;255(2):369-82. doi: 10.1046/j.1432-1327.1998.2550369.x.


In most studies, coupling of the beta2-adrenoceptor (beta2AR) to the stimulatory, heterotrimeric GTP-binding protein of adenylyl cyclase the (Gs) is studied indirectly by measuring adenylyl cyclase activation. The aim of this study was to establish a model system in which beta2AR-Gs interactions can be studied directly at the level of the G-protein. We expressed the beta2AR alone, in combination with the alpha-subunit of Gs (G(s alpha)), and as fusion protein with G(s alpha) (beta2AR-G(s alpha)) in Sf9 insect cells. The beta2AR expressed alone couples poorly to the endogenous G(s alpha)-like G-protein of Sf9 cells since no high-affinity agonist binding could be detected, and the effects of agonist and inverse agonist on adenylyl cyclase, high-affinity GTPase and guanosine 5'-O-(3-thiotriphosphate) (GTP[S]) binding were small. Beta2AR-G(s alpha) reconstituted high-affinity agonist binding and regulated adenylyl cyclase more effectively than the beta2AR co-expressed with a large excess of G(s alpha). In membranes expressing beta2AR-G(s alpha), highly effective agonist- and inverse agonist regulation of high-affinity GTP hydrolysis and GTP[S] binding was observed. In contrast, agonist and inverse agonist regulation of GTP hydrolysis and GTP[S] binding in membranes expressing beta2AR and G(s alpha) as separate proteins was difficult to detect. Our data show that the beta2AR interacts with G(s alpha) more efficiently when expressed as a fusion protein than when expressed with an excess of non-fused G(s alpha). The beta2AR-G(s alpha) fusion protein provides a very sensitive model system to study the regulation of Gs function by beta2AR agonists and inverse agonists directly at the level of the G-protein.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Adenylyl Cyclases / metabolism
  • Adrenergic beta-Antagonists / pharmacology
  • Animals
  • Binding, Competitive
  • Cell Line
  • Cell Membrane / metabolism
  • Cyclic AMP-Dependent Protein Kinases / biosynthesis
  • Cyclic AMP-Dependent Protein Kinases / drug effects
  • Cyclic AMP-Dependent Protein Kinases / physiology*
  • Dihydroalprenolol / metabolism
  • GTP Phosphohydrolases / metabolism
  • GTP-Binding Proteins / biosynthesis
  • GTP-Binding Proteins / metabolism*
  • Guanosine 5'-O-(3-Thiotriphosphate) / metabolism
  • Guanosine Diphosphate / pharmacology
  • Guanosine Triphosphate / metabolism
  • Isoproterenol / pharmacology
  • Kinetics
  • Polymerase Chain Reaction
  • Propanolamines / pharmacology
  • Rats
  • Receptors, Adrenergic, beta / drug effects
  • Receptors, Adrenergic, beta / physiology
  • Recombinant Fusion Proteins / biosynthesis
  • Recombinant Fusion Proteins / metabolism
  • Spodoptera
  • Transfection
  • beta-Adrenergic Receptor Kinases


  • Adrenergic beta-Antagonists
  • Propanolamines
  • Receptors, Adrenergic, beta
  • Recombinant Fusion Proteins
  • Guanosine Diphosphate
  • Guanosine 5'-O-(3-Thiotriphosphate)
  • ICI 118551
  • Dihydroalprenolol
  • Guanosine Triphosphate
  • Cyclic AMP-Dependent Protein Kinases
  • beta-Adrenergic Receptor Kinases
  • GTP Phosphohydrolases
  • GTP-Binding Proteins
  • Adenylyl Cyclases
  • Isoproterenol