Reconstitution of alpha2D-adrenergic receptor coupling to phospholipase D in a PC12 cell lysate.
We have previously shown that alpha2-adrenergic receptor-mediated coupling to phospholipase D (PLD) in vascular tissues requires a tyrosine kinase activity (Jinsi, A., Paradise, J., and Deth, R. C. (1996) Eur. J. Pharmacol. 302, 183-190). To further clarify this mode of regulation we reconstituted alpha2A/D-adrenergic receptor-stimulated PLD activity in PC12 cells expressing the cloned receptor. [3H]Myristic acid-labeled cells were lysed by nitrogen cavitation, and aliquots of subnuclear fraction were utilized in the PLD assay. Agonist-stimulated PLD activity was measured in the presence of 0.4% butanol as [3H]phosphatidylbutanol formation. Both GTP and its non-hydrolyzable analog guanosine 5'-O-(thiotriphosphate) stimulated PLD activity in a concentration- and time-dependent manner that required co-activation of protein kinase C by phorbol dibutyrate. Addition of epinephrine produced a 3-fold stimulation of PLD activity in the presence of GTP and GDP. This agonist-stimulated PLD activity was completely blocked by the alpha2-adrenergic receptor antagonist rauwolscine and by Clostridium botulinum toxin as well as by antibodies directed against either pp60(src), RhoA, or Ras GTPase-activating protein. These results indicate that coupling of the alpha2A/D-adrenergic receptor to PLD is complexly regulated by both the tyrosine kinase pp60(src) and the low molecular weight G protein RhoA.
Jinsi-Parimoo
A
A
Department of Pharmaceutical Sciences, Northeastern University, Boston, Massachusetts 02115, USA.
Deth
R C
RC
D000818:Animals; D002474:Cell-Free System; D003001:Cloning, Molecular; D004789:Enzyme Activation; D004837:Epinephrine / Q000494:pharmacology*; D019204:GTP-Binding Proteins / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006153:Guanosine Diphosphate / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000494:pharmacology; D008954:Models, Biological; D019814:Myristic Acid; D009227:Myristic Acids / Q000378:metabolism; D016716:PC12 Cells; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D010739:Phospholipase D / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism; D051381:Rats; D018341:Receptors, Adrenergic, alpha-2 / Q000096:biosynthesis / Q000502:physiology*; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection
D009227:Myristic Acids; D018341:Receptors, Adrenergic, alpha-2; D011994:Recombinant Proteins; D019814:Myristic Acid; D006153:Guanosine Diphosphate; D015240:Phorbol 12,13-Dibutyrate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D011493:Protein Kinase C; D010739:Phospholipase D; D019204:GTP-Binding Proteins; D004837:Epinephrine