Alpha 2-C10 adrenergic receptors expressed in rat 1 fibroblasts can regulate both adenylylcyclase and phospholipase D-mediated hydrolysis of phosphatidylcholine by interacting with pertussis toxin-sensitive guanine nucleotide-binding proteins.
The alpha 2-C10 adrenergic receptor from human platelets was expressed permanently in Rat-1 fibroblasts. A series of clones that varied in expression of the receptor from 0 to 3.5 pmol/mg of membrane protein were isolated. We have demonstrated recently in cells of one of these clones (1C) that the alpha 2-C10 receptor interacts directly with two distinct pertussis toxin-sensitive G-proteins, Gi2 and Gi3 (Milligan, G., Carr, C., Gould, G. W., Mullaney, I., and Lavan, B.E. (1991) J. Biol. Chem. 266, 6447-6455). High affinity GTPase activity in membranes of cells from the various clones was stimulated by the addition of the alpha 2-adrenergic agonist UK14304, defining that the receptor coupled productively to the G-protein signaling system. Maximal stimulation of high affinity GTPase activity correlated with the levels of receptor expressed. Clones expressing the receptor also demonstrated agonist-mediated inhibition of adenylylcyclase. Futhermore, the alpha 2-C10 receptor in one clone (1C), but not other clones, promoted a marked stimulation in the generation of water-soluble products derived from phosphatidylcholine. The concentration of UK14304 required to produce half-maximal regulation of GTPase activity (20-30 nM), of forskolin-amplified adenylylcyclase activity (30-40 nM), and of choline generation (30-40 nM) were similar. Transphosphatidylation experiments with cells of clone 1C indicated that the receptor-mediated hydrolysis of phosphatidylcholine was via the action of a phospholipase D. All of these effects were attenuated by pretreatment of the cells with pertussis toxin. Dose-effect curves of pertussis toxin-treatment demonstrated similar effective concentrations of the toxin in causing endogenous ADP-ribosylation of both Gi2 and Gi3, inhibition of receptor-stimulated GTPase activity, and phospholipase D activity. Receptor activation of phospholipase D activity was not dependent upon prior phospholipase C-dependent activation of protein kinase C, as alpha 2-adrenergic stimulation of inositol phosphate production was negligible and the presence of the selective protein kinase C inhibitor RO-31-8220, at concentrations up to 10 microM, had no effect on UK14304-mediated production of phosphatidylbutanol. These results demonstrate that expression of the alpha 2-C10 receptor in a heterologous system can result in receptor regulation of signaling elements that appear not to be primary targets for the receptor in vivo. Such results are important in respect to recent observations that transfection of a single defined receptor into separate cell lines can lead to the regulation of distinct effector systems (Vallar, L., Muca, C., Magni, M., Albert, P., Bunzow, J., Meldolesi, J. and Civelli, O. (1990) J. Biol. Chem. 265, 10320-10326).(ABSTRACT TRUNCATED AT 400 WORDS)
MacNulty
E E
EE
Department of Biochemistry, University of Glasgow, Scotland, United Kingdom.
McClue
S J
SJ
Carr
I C
IC
Jess
T
T
Wakelam
M J
MJ
Milligan
G
G
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000262:Adenylyl Cyclases / Q000378:metabolism*; D000316:Adrenergic alpha-Agonists / Q000494:pharmacology; D000818:Animals; D000068438:Brimonidine Tartrate; D002772:Cholera Toxin / Q000494:pharmacology; D004274:DNA, Recombinant / Q000235:genetics; D016232:Endothelins / Q000494:pharmacology; D005347:Fibroblasts / Q000378:metabolism; D020558:GTP Phosphohydrolases / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D007295:Inositol Phosphates / Q000096:biosynthesis; D037342:Pertussis Toxin; D010713:Phosphatidylcholines / Q000378:metabolism*; D010739:Phospholipase D / Q000378:metabolism*; D011810:Quinoxalines / Q000494:pharmacology; D051381:Rats; D011942:Receptors, Adrenergic, alpha / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D014162:Transfection; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D000316:Adrenergic alpha-Agonists; D004274:DNA, Recombinant; D016232:Endothelins; D007295:Inositol Phosphates; D010713:Phosphatidylcholines; D011810:Quinoxalines; D011942:Receptors, Adrenergic, alpha; D010566:Virulence Factors, Bordetella; D000246:Adenosine Diphosphate Ribose; D000068438:Brimonidine Tartrate; D002772:Cholera Toxin; D037342:Pertussis Toxin; D010739:Phospholipase D; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases