Activation of phospholipase D by the LHRH receptor and associated mechanisms
The αT3 -1 gonadotroph- derived cell line was used as a model system to study intracellular signalling systems associated with the LHRH receptor, with particular interest in phospholipase D (PLD) and mechanisms involved in its regulation, such as protein kinase C (PKC) and non -receptor tyrosine kinases.Phospholipase D activation in aT3 -1 cells proceeded after a delay when stimulated by LHRH, or by the phorbol ester, phorbol 12,13 -dibutyrate (PDBu). LHRH -stimulated PLD activity was fully resistant to desensitisation over a 40 min time course. The Cat+ ionophore, ionomycin was unable to stimulate PLD activity. LHRH -stimulated PLD activity was inhibited by PKC downregulation or by bisindolylmaleimide PKC inhibitors at an approximately 8 -fold higher concentration than that seen for the PDBu- stimulated PLD activity. Another PKC inhibitor, H7, inhibited PDBu- stimulated PLD activity in a manner that indicated multiple components to the inhibition and inhibited LHRH -stimulated PLD activity with a low potency. This is consistent with the possibility that one mediator of the LHRH response is a form of PKC which is relatively resistant to certain PKC inhibitors and insensitive to phorbol esters, or that some other unknown kinase is involved. The tyrosine kinase inhibitors, lavendustin A and genistein were able to selectively inhibit the LHRH -stimulated PLD activity. Phospholipase D activation stimulated by LHRH was insensitive to pertussis toxin pretreatment, indicating that the heterotrimeric G- proteins, Gi and Go are not involved.LHRH induced tyrosine phosphorylation of a number of proteins in aT3 -1 cells; a similar phosphorylation profile was also produced by stimulation with PDBu. Tyrosine phosphorylation of proteins in aT3 -1 cells 4 was reduced by ionomycin. Two members of the src- family of non -receptor tyrosine kinases, src and fyn, were detected at significant levels in these cells by immunoblot. Using immunoprecipitation techniques we have shown that the enzymic activity of fyn is increased within 10 min of LHRH stimulation.The LHRH -stimulated PLD activity could not be detectably enhanced by a conditioning preincubation with LHRH, such as brings about the priming effect of LHRH ( a phenomenon of cellular sensitisation elicited by LHRH receptor activation). Removal of steroids from the growth medium severely impaired the PLD activation due to LHRH; this response was largely restored by addition of oestradiol -1713 but not progesterone. This may have implications for the LHRH priming effect in vivo, which is dependent upon the level of circulating steroid hormones.The murine LHRH receptor was transiently expressed in COS 7 cells from the cDNA in a pcDNA vector using a DEAE dextran transfection procedure. It was demonstrated that LHRH -stimulated PLD activation displayed similar sensitivity to the bisindolylmaleimide PKC inhibitor, Ro 31 -8220, to that seen in aT3 -1 cells. It was not however sensitive to tyrosine kinase inhibitors or pertussis toxin. Using site -directed mutagenesis of residues 87 and 318 in the transmembrane 2 and 7 domains of the LHRH receptor (which represents an inversion of the consensus motif in the G- protein coupled receptor family), it was found that mutation of Asp 318 to Asn was alone sufficient to produce a rapidly desensitising PLD response to LHRH, in contrast to the non -desensitising wild type.