The role of specific receptor domains in signal transduction by the VIP2 receptor
MacKenzie, Christopher John
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Receptors for the neuropeptides VIP and P ACAP belong to a novel sub-family of G protein-coupled receptors, the secretin/ calcitonin/ parathyroid hormone receptor family. The rat VIP2 receptor was recently cloned in this laboratory and the present project was carried out to characterise the signalling mechanisms used by this receptor and define the role of crucial receptor domains. These studies have primarily involved the transient expression of receptors in host cells. The results demonstrated for the first time that both the VIP1 and VIP2 receptors can stimulate phospholipase C (PLC) in addition to adenylate cyclase (AC) and that this stimulation occurs by a pertussis toxin(PTx)-sensitive mechanism. Correspondingly, GTP,.S modulation of ligand-binding to the VIP2 receptor in COS 7 cell membranes was shown to be partially PTx-sensitive, suggesting an interaction of the receptor with a PTx-sensitive G protein. An epitope-tagged human VIP2 receptor was expressed in COS 7 cells and imrnunoprecipitated with its associated G proteins for Western-blotting studies. Immunoreactivity for Gaq, Ga5 and a member of the G<Xif o/ t/ z family, other than Gan or G<Xi2, appeared to be associated with the receptor. The use of specific calcium channel blockers identified a role for receptor-mediated calcium influx in VIP2 receptormediated PLC stimulation. The P ACAP and VIP2 receptors contain many common structural features but have their own distinct pharmacological profiles. The function of specific domains of the VIP2 receptor was investigated by creating P ACAP /VIP2 receptor chimaerics and C-terminal truncations of the Vll'2 receptor. These constructs allowed identification of the primary region of the receptor responsible for ligand-binding and the probable site of interaction with a PTx-sensitive G protein and also assessment of any contribution of the C-terminus to AC and PLC stimulation. A number of further studies were carried out on native receptors. The signalling pathways activated by the endogenous Vll'2 receptor in the Gfl3 rat pituitary tumour cell line were investigated. Consistent with previous evidence that a Vll' receptor in other cells can stimulate nitric oxide production, VIP2 receptor-mediated stimulation of nitric oxide synthase, PLC and AC was demonstrated in GHJ cells. As a result of reports describing the effects of VIP and P ACAP on cerebral blood flow the Reverse Transcriptase-Polymerase Chain Reaction technique was used to determine the type of VIP receptor present in rat cerebral microvessels.