The Regulation of Agonist-Stimulated Phospholipase D Activity in Swiss 3T3 Fibroblasts

Abstract

The work in this thesis aimed to characterise the regulation of phospholipase D (PLD) activity by receptor occupancy in Swiss 3T3 fibroblasts. Bombesin-stimulated PLD activity had previously been found to be partially dependent on protein kinase C (PKC) suggesting that activation of PLD was to some extent down stream of the G-protein coupled receptor activated phosphatidylinositol 4,5, bisphosphate (PtdIns(4,5)P2) hydrolysis (Cook et al., 1992). Using a permeabilised cell system to introduce non-hydrolysable analogues of guanine-nucleotides, bombesin-stimulated PLD activity was found to be indirectly regulated by a G-protein. The phosphorylation of proteins on tyrosine was determined using immunoblotting with a monoclonal anti-phosphotyrosine antibody. The elevation of tyrosine phosphorylation using pervanadate, an inhibitor of tyrosine phosphatases, was found to activate PLD activity. PLD activity was found to be reduced by pretreatment with the tyrosine kinase inhibitor Genistein. Pretreatment with both Genistein and the selective PKC inhibitor Ro-31-8220 reduced bombesin-stimulated PLD activity to basal. Elevation of Ca2+ from intracellular pools using thapsigargin, an inhibitor of the Ca2+-ATPase on the intracellular stores was found to be insufficient to activate PLD alone nor did it affect bombesin-stimulated PLD activity. Elevation of cyclic AMP using forskolin and dibutyrylcyclicAMP did not affect bombesin-stimulated PLD activity nor did it alone affect the enzyme. A common event in cells exposed to agonist is a loss of responsiveness to the stimulant, referred to as desensitisation. Bombesin-stimulated PLD activity was found to desensitise rapidly with a complete loss in the response after a 40 second agonist exposure. Desensitisation was reversible and occurred within 4.5 minutes of agonist removal, however was never complete. In the continuous presence of agonist resensitisation of PLD activity occurred. The resensitised rate was reduced from that generated in response to the initial stimulus but activity continued over a period of an hour. The recovery of bombesin-stimulated total inositol phosphate generation commenced later than that of bombesin-stimulated PLD activation and reached completion within 11.5 minutes after removal of the desensitising stimulus. Radio-receptor binding studies showed that there was a transient loss of binding sites from the cell surface which was due to internalisation and recycling of the receptor. Although the rate of receptor loss and recovery paralleled that of the desensitisation and resensitisation of PLD activity, differences were observed in the extent of the two processes. Readdition of bombesin was essential for a stimulation of PLD activity to be observed on resensitisation of the enzyme. Hence degradation of the ligand was occurring before the receptor was recycled. Desensitisation and resensitisation was independent of PKC activation and a desensitising pretreatment with bombesin did not affect the PMA- stimulated PLD activity. A23187-stimulated PLD activity also underwent homologous desensitisation and could not stimulate enzyme activity after prior desensitisation by bombesin pretreatment. Thus desensitisation was not due to a decrease in intracellular Ca2+ levels. Bombesin also induced heterologous desensitisation. Pretreatment with bombesin or vasopressin reduced a subsequent stimulation with the other agonist by approximately 70%. A desensitising pretreatment with either agonist attenuated the GTP?S-stimulated PLD activity in permeabilised cells by approximately 50%. It was thus proposed that desensitisation not only affected the cell surface receptors but also occurred at or distal to the receptor-coupled G-protein. These findings support a role for PLD-derived PtdOH in mitogenic signalling, in view of the resensitisation and subsequent maintenance of PLD activity in an early mitogenic situation when the agonist is continually present. The kinetics of the agonist-stimulated PLD activity are discussed with relation to the regulatory pathways of PLD activation and mitogenic signalling.