Comparative analysis of the efficacy of A1 adenosine receptor activation of Gi/o alpha G proteins following coexpression of receptor and G protein and expression of A1 adenosine receptor-Gi/o alpha fusion proteins

Biochemistry. 1999 Feb 23;38(8):2272-8. doi: 10.1021/bi982054f.


HEK293T cells were transiently transfected to express either the human A1 adenosine receptor together with pertussis toxin-resistant cysteine-to-glycine forms of the alpha subunits of Gi1 (C351G), Gi2 (C352G), and Gi3 (C351G) and wild-type Go1alpha or fusion proteins comprising the A1 adenosine receptor and these Gi/o G proteins to compare A1 adenosine receptor agonist-mediated activation of these Gi family G proteins upon coexpression of individual Gi/o G proteins and receptor versus expression as receptor-G protein fusion proteins. Addition of the adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA) to membranes of pertussis toxin-treated cells resulted in a concentration-dependent stimulation of [35S]GTPgammaS binding with comparable amounts of NECA required to produce half-maximal stimulation following transfection of A1 adenosine receptor and Gi/o G proteins either as fusion proteins or as separate polypeptides. However, the magnitude of agonist-mediated activation of GTPgammaS binding was greatly enhanced by expressing the A1 adenosine receptor and Gi family G proteins from chimaeric open reading frames. This observation was consistent following the study of more than 40 agonists. No preferential activation of any G protein was observed with more than 40 A1 receptor agonists following cotransfection of receptor with G protein or transfection of receptor-G protein fusion proteins. These studies demonstrate the utility of using fusion proteins to study receptor-G protein interaction, show that the A1 adenosine receptor couples equally well to the Gi/o G proteins Gi1alpha, G i2alpha, Gi3alpha, and Go1alpha, and demonstrate that for a range of agonists there is no selectivity for activation of any particular A1 adenosine receptor-Gi/o G protein combination.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine-5'-(N-ethylcarboxamide) / pharmacology
  • Amino Acid Sequence
  • Animals
  • Binding Sites / drug effects
  • Cell Line
  • Embryo, Mammalian
  • GTP-Binding Protein alpha Subunits, Gi-Go / biosynthesis
  • GTP-Binding Protein alpha Subunits, Gi-Go / genetics
  • GTP-Binding Protein alpha Subunits, Gi-Go / metabolism*
  • Guanosine 5'-O-(3-Thiotriphosphate) / metabolism
  • Humans
  • Kidney
  • Molecular Sequence Data
  • Pertussis Toxin
  • Purinergic P1 Receptor Agonists
  • Rats
  • Receptors, Purinergic P1 / biosynthesis
  • Receptors, Purinergic P1 / genetics
  • Receptors, Purinergic P1 / physiology*
  • Recombinant Fusion Proteins / biosynthesis*
  • Recombinant Fusion Proteins / chemical synthesis
  • Transfection
  • Virulence Factors, Bordetella / pharmacology


  • Purinergic P1 Receptor Agonists
  • Receptors, Purinergic P1
  • Recombinant Fusion Proteins
  • Virulence Factors, Bordetella
  • Adenosine-5'-(N-ethylcarboxamide)
  • Guanosine 5'-O-(3-Thiotriphosphate)
  • Pertussis Toxin
  • GTP-Binding Protein alpha Subunits, Gi-Go