Measurement of agonist efficacy using an alpha2A-adrenoceptor-Gi1alpha fusion protein

FEBS Lett. 1997 Dec 8;419(1):141-6. doi: 10.1016/s0014-5793(97)01431-2.


A fusion protein was constructed between the porcine alpha2A-adrenoceptor and a pertussis toxin-insensitive (Cys351Gly) form of the alpha subunit of the G protein Gi1. Addition of agonist ligands to membranes of COS-7 cells transiently transfected to express this construct, and treated with pertussis toxin prior to cell harvest, resulted in stimulation of both high affinity GTPase activity and enhanced binding of [35S]GTPgammaS. By considering the fusion protein as an agonist-activated enzyme and measuring Vmax of GTP hydrolysis a range of agonist ligands displayed varying efficacy in their capacity to activate the receptor-associated G protein with adrenaline = noradrenaline = alpha-methylnoradrenaline > UK14304 > BHT933 > or = xylazine = clonidine. A similar rank order was observed following independent co-expression of the alpha2A-adrenoceptor and Cys351Gly-Gi1alpha. These data demonstrate the utility and applicability of using a receptor-G protein fusion protein approach, in which the stoichiometry of receptor and G protein is fixed at 1:1, to measure and further understand the nature of agonist efficacy.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adrenergic alpha-Agonists / metabolism
  • Adrenergic alpha-Agonists / pharmacology*
  • Animals
  • COS Cells
  • GTP Phosphohydrolases / metabolism
  • GTP-Binding Protein alpha Subunits, Gi-Go / genetics
  • GTP-Binding Protein alpha Subunits, Gi-Go / metabolism*
  • Guanosine 5'-O-(3-Thiotriphosphate) / metabolism
  • Ligands
  • Pertussis Toxin
  • Rats
  • Receptors, Adrenergic, alpha-2 / genetics
  • Receptors, Adrenergic, alpha-2 / metabolism*
  • Recombinant Fusion Proteins / metabolism*
  • Swine
  • Virulence Factors, Bordetella / pharmacology


  • Adrenergic alpha-Agonists
  • Ligands
  • Receptors, Adrenergic, alpha-2
  • Recombinant Fusion Proteins
  • Virulence Factors, Bordetella
  • Guanosine 5'-O-(3-Thiotriphosphate)
  • Pertussis Toxin
  • GTP Phosphohydrolases
  • GTP-Binding Protein alpha Subunits, Gi-Go