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Mechanisms of Phospholipase C Activation by the Vasoactive Intestinal Polypeptide/Pituitary Adenylate Cyclase-Activating Polypeptide Type 2 Receptor

Medical Research Council Membrane and Adapter Proteins Co-operative Group, Membrane Biology Group, Department of Biomedical Sciences, University of Edinburgh, Edinburgh, United Kingdom EH8 9XD; and Medical Research Council Brain Metabolism Unit, Edinburgh, United Kingdom EH8 9JZ

Address all correspondence and requests for reprints to: Dr. R. Mitchell, Medical Research Council Membrane and Adapter Proteins Co-operative Group, Membrane Biology Group, Department of Biomedical Sciences, University of Edinburgh, Hugh Robson Building, George Square, Edinburgh, United Kingdom EH8 9XD. E-mail: rory.mitchell{at}ed.ac.uk

The vasoactive intestinal polypeptide/pituitary adenylate cyclase-activating polypeptide type 2 (VPAC₂) receptor was shown to induce both [³H]inositol phosphate ([³H]InsP)and cAMP production in transfected COS7 cells and in GH₃ cells where it is natively expressed. Neither cholera toxin nor forskolin could elicit an equivalent [³H]InsP response, suggesting independent coupling of the two pathways. The VPAC₂ receptor-mediated [³H]InsP response was partially inhibited by pertussis toxin (Ptx) and by the G-sequestering C-terminal fragment of GRK2 (GRK2-ct) in COS7 and GH₃ cells, whereas responses of control receptors were unaffected. Blockers of receptor-activated Ca²⁺ influx pathways (Co²⁺ and SKF 96365) also partially inhibited VPAC₂ receptor-mediated [³H]InsP responses. This inhibition was not present in the component of the response remaining after Ptx treatment. A range of blockers of voltage-sensitive Ca²⁺ channels were ineffective, consistent with the reported lack of these channels in COS7 cells. The data suggest that the VPAC₂ receptor may couple to phospholipase C through both Ptx-insensitive and Ptx-sensitive G proteins (G_q/11 and G_i/o, respectively) to generate [³H]InsP. In addition to G, G_i/o activation appears to require receptor-activated Ca²⁺ entry. This is consistent with the possibility that not only G_q/11-responsive and G-responsive isoforms of phospholipase C but also Ca²⁺-responsive forms may contribute to the overall [³H]InsP response.