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.