R. Jockers et al., SPECIES-DIFFERENCE IN THE G-PROTEIN SELECTIVITY OF THE HUMAN AND BOVINE A(1)-ADENOSINE RECEPTOR, The Journal of biological chemistry, 269(51), 1994, pp. 32077-32084
The purified bovine brain A(1)-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 codin
g for the human A(1)-adenosine receptor was inserted into a plasmid pl
acing the synthesis of the receptor protein under the control of the M
alE promoter, Following induction by maltose, active receptor accumula
ted in Escherichia coli membranes, Binding of the antagonist 8-[H-3]cy
clopentyl-1,3-dipropylxanthine to E. coli membranes (K-D similar to 2
nM, B-max similar to 0.2-0.4 pmol/mg) showed the appropriate pharmacol
ogical profile, Incubation of E. coli membranes with purified G(o,i)-r
econstituted guanine nucleotide-sensitive high affinity binding of the
agonist (-)[I-125] N-6-3-(iodo-4-hydroxyphenylisopropyl) adenosine to
the receptor (K-D similar to 1 nM), In the presence of purified beta
gamma-subunit, the recombinant receptor interacted equally well with t
he recombinant G protein alpha-subunits G(i alpha-1), G(i alpha-2), G(
i alpha-3), G(o alpha) displayed a lower affinity for the receptor whi
le G(5 alpha) was inactive, Parallel experiments were carried out in b
ovine and human brain membranes pretreated with N-ethylmaleimide to in
activate the endogenous G(o)/G(i) proteins; G(i alpha-3) was most pote
nt in reconstituting I-125-HPIA binding to bovine membranes, while G(i
alpha-1), G(i alpha-2), and G(o alpha) displayed similar affinities,
However, in human membranes, G(i alpha-1), G(i alpha-2), and G(i alpha
-3), were equipotent and high concentrations of G(o alpha) were requir
ed to promote I-125-HPIA binding. These observations show (i) that fun
ctional human A(1)-adenosine receptors were synthesized in E. coli; (i
i) that the pattern of G protein coupling is identical, for the recomb
inant human A(1)-receptor and its counterpart in the native membrane;
(iii) and that species differences between bovine and human receptor e
xist not only in their pharmacological profile but also in their G pro
tein specificity suggesting that species homologues of receptors may u
se different signaling mechanisms.