title: Species difference in the G protein selectivity of the human and bovine A1-adenosine receptor.
abstract: The purified bovine brain A1-adenosine receptor has previously been shown to discriminate among closely related G protein alpha-subunits. To obtain analogous information for the human receptor, the cDNA coding for the human A1-adenosine receptor was inserted into a plasmid placing the synthesis of the receptor protein under the control of the MalE promoter. Following induction by maltose, active receptor accumulated in Escherichia coli membranes. Binding of the antagonist 8-[3H]cyclopentyl-1,3-dipropylxanthine to E. coli membranes (KD approximately 2 nM, Bmax approximately 0.2-0.4 pmol/mg) showed the appropriate pharmacological profile. Incubation of E. coli membranes with purified Go,i-reconstituted guanine nucleotide-sensitive high affinity binding of the agonist (-)[125I] N6-3-(iodo-4-hydroxyphenylisopropyl)adenosine to the receptor (KD approximately 1 nM). In the presence of purified beta gamma-subunit, the recombinant receptor interacted equally well with the recombinant G protein alpha-subunits Gi alpha-1, Gi alpha-2, Gi alpha-3; G(o) alpha displayed a lower affinity for the receptor while Gs alpha was inactive. Parallel experiments were carried out in bovine and human brain membranes pretreated with N-ethylmaleimide to inactivate the endogenous G(o)/Gi proteins; Gi alpha-3 was most potent in reconstituting 125I-HPIA binding to bovine membranes, while Gi alpha-1, Gi alpha-2, and G(o) alpha displayed similar affinities. However, in human membranes, Gi alpha-1, Gi alpha-2, and Gi alpha-3, were equipotent and high concentrations of G(o) alpha were required to promote 125I-HPIA binding. These observations show (i) that functional human A1-adenosine receptors were synthesized in E. coli; (ii) that the pattern of G protein coupling is identical for the recombinant human A1-receptor and its counterpart in the native membrane; (iii) and that species differences between bovine and human receptor exist not only in their pharmacological profile but also in their G protein specificity suggesting that species homologues of receptors may use different signaling mechanisms.
authors.affiliation: Institut Cochin de Génétique Moléculaire, Paris, France.
authors.forename: R
authors.initials: R
authors.lastname: Jockers
authors.forename: M E
authors.initials: ME
authors.lastname: Linder
authors.forename: M
authors.initials: M
authors.lastname: Hohenegger
authors.forename: C
authors.initials: C
authors.lastname: Nanoff
authors.forename: B
authors.initials: B
authors.lastname: Bertin
authors.forename: A D
authors.initials: AD
authors.lastname: Strosberg
authors.forename: S
authors.initials: S
authors.lastname: Marullo
authors.forename: M
authors.initials: M
authors.lastname: Freissmuth
mesh_terms: D000818:Animals; D001483:Base Sequence; D001921:Brain / Q000378:metabolism; D002417:Cattle; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D011485:Protein Binding; D018047:Receptors, Purinergic P1 / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013045:Species Specificity
chemical_list: D017931:DNA Primers; D018047:Receptors, Purinergic P1; D011994:Recombinant Proteins; D019204:GTP-Binding Proteins