SPECIES-DIFFERENCE IN THE G-PROTEIN SELECTIVITY OF THE HUMAN AND BOVINE A(1)-ADENOSINE RECEPTOR

Citation
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
Citations number
53
Categorie Soggetti
Biology
ISSN journal
00219258
Volume
269
Issue
51
Year of publication
1994
Pages
32077 - 32084
Database
ISI
SICI code
0021-9258(1994)269:51<32077:SITGSO>2.0.ZU;2-D
Abstract
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.