title: Reconstitution of recombinant bovine A1 adenosine receptors in Sf9 cell membranes with recombinant G proteins of defined composition.
abstract: We investigated the coupling of A1 adenosine receptors to recombinant G proteins. Recombinant baculoviruses were used to express bovine A1 adenosine receptors in Sf9 insect cells that lack endogenous adenosine receptors. Binding parameters for recombinant receptors expressed in Sf9 cell membranes using the antagonist radioligand [125I]BW-A844U ([125I]8-cyclopentyl-3-iodoaminophenethyl-1-propylxanthine) are Bmax = 2-5 pmol/mg of protein and K(D) = 0.53 +/- 0.12 nM. In competition assays, the potency order of agonists is (R)-phenylisopropyladenosine > (S)-phenylisopropyladenosine > 5'-N-ethylcarboxamidoadenosine, properties characteristic of native bovine A1 adenosine receptors. The agonist radioligand 125I-N6-4-aminobenzyladenosine binds to two affinity states of the recombinant A1 adenosine receptors with K(D) values of 0.09 and 10.4 nM. The high affinity binding site represents <10% of total sites and is increased 7-fold on reconstitution with both alpha and betagamma G protein subunits but not with either subunit alone; thus, exogenous alpha and betagamma subunits do not functionally interact with endogenous Sf9 betagamma and alpha subunits, respectively. Four different alpha subunits (alpha i1, alpha i2, alpha i3, and alpha o) and six different beta gamma subunits (beta1gamma1, beta1gamma2, beta1gamma3, beta2gamma2, beta2gamma3, and bovine brain betagamma)) increased GTP-sensitive, high affinity agonist binding. The results indicate that bovine A1 adenosine receptors couple equally well to G protein alpha i and alpha o subunits in combination with betagamma subunits containing the beta1 or beta2 subunits and gamma2 or gamma3 subunits. G protein heterotrimers that contain the beta1gamma1 dimer couple with similar potency but reduced efficacy to A1 adenosine receptors.
authors.affiliation: Department of Molecular Physiology, University of Virginia Health Sciences Center, Charlottesville 22908, USA.
authors.forename: R A
authors.initials: RA
authors.lastname: Figler
authors.forename: S G
authors.initials: SG
authors.lastname: Graber
authors.forename: M A
authors.initials: MA
authors.lastname: Lindorfer
authors.forename: H
authors.initials: H
authors.lastname: Yasuda
authors.forename: J
authors.initials: J
authors.lastname: Linden
authors.forename: J C
authors.initials: JC
authors.lastname: Garrison
mesh_terms: D000818:Animals; D002417:Cattle; D002460:Cell Line; D003001:Cloning, Molecular; D019204:GTP-Binding Proteins / Q000378:metabolism*; D011869:Radioligand Assay; D018047:Receptors, Purinergic P1 / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D018411:Spodoptera
chemical_list: D018047:Receptors, Purinergic P1; D011994:Recombinant Proteins; D019204:GTP-Binding Proteins