Functional coupling of adenosine A2a receptor to inhibition of the mitogen-activated protein kinase cascade in Chinese hamster ovary cells.
Activation of Gs-coupled receptors enhances the increase in cyclic AMP mediated by adenylate cyclases. As it has been shown that cyclic AMP inhibits the epidermal growth factor-activated mitogen-activated protein kinase (MAPK) signalling pathway, stimulation of Gs-coupled receptors may lead to the inhibition of MAPK activation. To investigate the effect of a Gs-coupled receptor on the MAPK cascade, we cloned the adenosine (Ado) A2a receptor from a guinea-pig leucocyte cDNA library, and established Chinese hamster ovary (CHO) cells stably expressing the receptor (CHOAdoA2R). The [3H]5'-N-ethylcarbamoyladenosine (NECA) binding characteristics (Kd = 91.0 +/- 5.4 nM, Bmax = 707 +/- 11 fmol/mg of protein, n = 3) and NECA-induced cyclic AMP production indicate that the cloned Ado A2a receptor was functionally expressed in the cells. In CHO cells, thrombin induced intracellular Ca2+ increase and MAPK activation through the intrinsic G-coupled receptor. In CHOAdoA2R cells, NECA partially inhibited thrombin-elicited MAPK activation. When combining NECA-treatment with 1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid acetoxymethyl ester (BAPTA-AM) loading, a nearly complete inhibition of the MAPK activation occurred. Forskolin also partially inhibited the MAPK activation and synergized with BAPTA-AM, suggesting that partial inhibition of MAPK activation by NECA results from cyclic AMP production via Ado A2a receptor activation. The same synergism of MAPK inhibition between wortmannin and BAPTA-AM was observed, but not between wortmannin and NECA. These results suggest that cyclic AMP production through Ado A2a receptor inhibits thrombin-elicited MAPK activation by a Ca(2+)-independent/wortmannin-sensitive pathway in CHO cells.
Hirano
D
D
Life Science Research Laboratory, Japan Tobacco Inc., Kanagawa, Japan.
Aoki
Y
Y
Ogasawara
H
H
Kodama
H
H
Waga
I
I
Sakanaka
C
C
Shimizu
T
T
Nakamura
M
M
D000241:Adenosine / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D019830:Adenosine-5'-(N-ethylcarboxamide); D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism; D001483:Base Sequence; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D003001:Cloning, Molecular; D005576:Colforsin / Q000494:pharmacology; D006224:Cricetinae; D000242:Cyclic AMP / Q000378:metabolism; D017931:DNA Primers; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006168:Guinea Pigs; D007700:Kinetics; D007962:Leukocytes / Q000378:metabolism; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D058906:Purinergic P1 Receptor Agonists; D018047:Receptors, Purinergic P1 / Q000096:biosynthesis / Q000502:physiology*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000187:drug effects / Q000378:metabolism; D015398:Signal Transduction; D013917:Thrombin / Q000494:pharmacology; D014162:Transfection; D000077191:Wortmannin
D000730:Androstadienes; D017931:DNA Primers; D004791:Enzyme Inhibitors; D058906:Purinergic P1 Receptor Agonists; D018047:Receptors, Purinergic P1; D011994:Recombinant Proteins; D005576:Colforsin; D016718:Arachidonic Acid; D019830:Adenosine-5'-(N-ethylcarboxamide); D004533:Egtazic Acid; D000242:Cyclic AMP; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D013917:Thrombin; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D000241:Adenosine; D002118:Calcium; D000077191:Wortmannin