LOCALIZATION/SIGNAL TRANSDUCTION

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DESCRIPTION (Taken from application) This proposal is directed at defining the molecular mechanisms involved in the regulation of signal transduction and cellular growth by the heptahelical, G-protein-coupled, PACAP receptor (PACAP-R). PACAP-Rs are a useful model to study receptor coupling to intracellular effectors because following agonist stimulation these receptors are capable of activating both adenylate cyclase (AC) and phospholipase C (PLC). Some tumor cells expressing PACAP-Rs also secrete PACAP suggesting a potential autocrine effect of the hormone on tumor growth. The gene for the human PACAP-R was recently cloned and shown to contain two exons encoding the 3rd intracellular loop that can be alternatively spliced into four distinct cDNA splice variants. Although each splice variants is capable of activating both (AC) and (PLC), two (SV2, and SV-3) show greater efficacy for PLC activation and the induction of immediate early genes. Therefore, the first specific aim of this proposal is to characterize the tissue-distribution of PACAPR splice variants and the signaling pathways to which they are coupled. Tissue distribution will be determined using RT-PCR and by using recently developed PACAP-R antibodies for immunohistochemistry. Preliminary studies suggest that PACAP receptor splice variants are coupled to specific G-proteins such as Gas and Gaq. The regions of the PACAP-R that are involved in G-protein coupling will be determined by receptor mutagenesis. The G-proteins involved in coupling to PACAP-R splice variants will be studied by examining the ligand-induced PLC response in NIH/3T3 cells cotransfected with the cDNA of hPACAP-R splice variants and either Gas or Gaq. By a second approach the effects of recombinant antisense gene constructs of Gas and Gaq on receptor-mediated stimulation of PLC and AC will be studied. The second specific aim is to characterize PACAP-mediated expression of the immediate early genes, c-fos, c-myc and c-jun in NIH/3T3 cells stably expressing hPACAP-R splice variants. Agonist induced effects on immediate early gene expression for each hPACAP-R splice variant will be performed using RT/PCR and northern blot analysis. These changes will be correlated with observed differences in biological response by using growth assays. These studies presented here will advance our understanding of the localization of PACAP receptors, lead to a greater understanding of their unique signal transduction properties and their role on cellular growth and de-differentiation.