A hybrid system was used to further explore the relationship between Nf1 and the PKA pathway. PACAP38 is mammalian protein that belongs to the vasoactive intestinal polypeptide-secretin-glucagon peptide family. Mutations in Drosophila genes rutabaga, Ras1 and Raf1 eliminate the response of flies to PACAP38. PACAP38 functions as a ligand for G protein-coupled receptors in vertebrates and in flies is known to stimulate cAMP synthesis inducing a 100-fold enhancement in K+ currents by coactivating both Rutabaga-adenylyl cyclase-cAMP and Ras-Raf kinase pathways (Zhong, 1995a). Mutations in rutabaga, Ras1 and Raf1 eliminate the response to PACAP38. Activation of both cAMP and Ras-Raf pathways together, but not alone, mimics the PACAP38 response (Zhong, 1995a). The involvement of Ras in the PACAP38 response has led to an investigation into the effect of Nf1 mutations. The purpose of this study was to further test whether Nf1 acts in the Ras or PKA pathways (Guo, 1997).
The PACAP38 enhancement of potassium currents is eliminated in Nf1 mutants. Perfusion of PACAP38 to the neuromuscular junction induces an inward current followed by a 100-fold enhancement of K+ currents in wild-type larvae. In Nf1 mutants, the inward current remains mostly intact, but the enhancement of K+ currents is abolished. Because the inward current is not affected in Nf1 mutants, it appears that PACAP38 receptors are normally activated by the peptide in these mutants. To control for the involvement of potential developmental effects of Nf1 mutants, wild type heat shock inducable Nf1 was induced in Nf1 mutants and the response to PACAP38 was studied. The PACAP mediated enhancement requires heat shock induction of Nf1. Because PACAP38 is a vertebrate peptide, the response induced by endogenous PACAP38-like neuropeptide (Zhong, 1995b) was tested. High-frequency stimulation (40Hz) applied to motor axons through a suction pipette increases K+ currents, presumably by causing the release of PACAP38-like peptides (Zhong, 1995b). The evoked PACAP38-like response is also eliminated in NF1 mutants. Induced expression of constitutively active Ras or active Raf neither blocks nor mimicks the PACAP38 response, suggesting that failure to negatively regulate Ras-Raf signaling does not explain the defective PACAP38 response in Nf1 mutants (Guo, 1997).
What is the role of the cyclic AMP pathway in PACAP38 responses? Application of cAMP analogs to Nf1 mutants restores the normal response to PACAP38. The cAMP analogs are effective if applied any time before or within 2 min after application of PACAP38. Application of cAMP analogs also restores the response to PACAP38 in rutabaga mutants, but not in Ras mutants. Thus, Nf1 appears to regulate the rutabaga-encoded adenylyl cyclase rather than the Ras-Raf pathway. Moreover, the Nf1 defect is rescued by the exposure of cells to pharmacological treatment that increased concentrations of cAMP, such as forskolin, which stimulates G-protein coupled adenylyl cyclase activity. Exploration of the mechanism by which NF1 influences G protein-mediated activation of adenylyl cyclase may lead to new insights into the mechanisms of G protein-mediated signal transduction and the pathogenesis, and possibly the treatment, of human type 1 neurofibromatosis (Guo, 1997).
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