Involvement of G-protein betagamma subunits in coupling the adenosine A1 receptor to phospholipase C in transfected CHO cells.
In transfected Chinese hamster ovary (CHO-A1) cells the human adenosine A1 receptor directly stimulates pertussis toxin-sensitive increases in inositol phosphate production and potentiates (synergistically) the inositol phosphate responses mediated by Gq-coupled P2Y2 purinoceptor and CCK(A) receptors. In the present study we have investigated the role of Gbetagamma subunits in mediating adenosine A1 receptor effects on phospholipase C activation (both direct and synergistic) by transiently transfecting CHO-A1 cells with a scavenger of Gbetagamma subunits: the C-terminus of beta-adrenoceptor kinase 1 (beta ark1 residues 495-689). [3H]inositol phosphate responses to the selective adenosine A1 receptor agonist N6-cyclopentyladenosine (CPA; 1 microM) were inhibited (41 +/- 1%) in CHO-A1 cells transiently transfected with the Gbetagamma scavenger, beta ark1 (495-689). Expression of beta ark1 (495-689) protein was confirmed by Western blotting. In contrast, adenosine A1 receptor-mediated inhibition of forskolin stimulated [3H]cyclic AMP accumulation was unaffected by transient expression of beta ark1 (495-689). Beta ark1 (495-689) expression had no significant effect on the [3H]inositol phosphate responses produced by activation of the endogenous P2Y2 purinoceptor (100 microM UTP; 92 +/- 0.8% of control). [3H]inositol phosphate accumulation in response to adenosine A receptor activation was also attenuated in CHO-K1 cells co-transfected with the beta ark1 (495-689) minigene (59 +/- 4% inhibition of control response to 1 microM CPA). Finally, transient expression of beta ark1 (495-689) in CHO-A1 cells inhibited the augmentation of [3H]inositol phosphate responses resulting from co-activation of adenosine A1 receptors and P2Y2 purinoceptors. These experiments indicate that Gbetagamma subunits are involved in the direct coupling the adenosine A1 receptor to phospholipase C and that they also participate in the augmentation of P2Y2 purinoceptor-mediated [3H]inositol phosphate responses by the adenosine A1 receptor.
Dickenson
J M
JM
Institute of Cell Signalling, School of Biomedical Sciences, University of Nottingham, Queen's Medical Centre, UK.
Hill
S J
SJ
D000241:Adenosine / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells; D006224:Cricetinae; D017868:Cyclic AMP-Dependent Protein Kinases / Q000235:genetics / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007295:Inositol Phosphates / Q000378:metabolism*; D010446:Peptide Fragments / Q000235:genetics / Q000494:pharmacology; D058906:Purinergic P1 Receptor Agonists; D058910:Purinergic P2 Receptor Agonists; D011943:Receptors, Adrenergic, beta / Q000235:genetics / Q000378:metabolism; D018047:Receptors, Purinergic P1 / Q000187:drug effects / Q000378:metabolism*; D011994:Recombinant Proteins; D014162:Transfection; D010738:Type C Phospholipases / Q000378:metabolism*
D007295:Inositol Phosphates; D010446:Peptide Fragments; D058906:Purinergic P1 Receptor Agonists; D058910:Purinergic P2 Receptor Agonists; D011943:Receptors, Adrenergic, beta; D018047:Receptors, Purinergic P1; D011994:Recombinant Proteins; C048599:N(6)-cyclopentyladenosine; D017868:Cyclic AMP-Dependent Protein Kinases; C114043:BARKct protein, recombinant; D010738:Type C Phospholipases; D019204:GTP-Binding Proteins; D000241:Adenosine