Anti-INAD (aa 659-674) polyclonal antibody

Calcium signaling is an important mediator of neuropeptide-stimulated fluid transport by Drosophila Malpighian (renal) tubules. We demonstrate the first epithelial role, in vivo, for members of the TRP family of calcium channels. RT-PCR revealed expression of trp, trpl, and trp gamma in tubules. Use of antipeptide polyclonal antibodies for TRP, TRPL, and TRP gamma showed expression of all three channels in type I (principal) cells in the tubule main segment. Neuropeptide (CAP(2b))-stimulated fluid transport rates were significantly reduced in tubules from the trpl(302) mutant and the double mutant, trpl(302); trp(343). However, a trp null, trp(343), had no impact on stimulated fluid transport. Measurement of cytosolic calcium concentrations ([Ca2+](i)) in tubule principal cells using an aequorin transgene in trp and trpl mutants showed a reduction in calcium responses in trpl(302). Western blotting of tubule preparations from trp and trpl mutants revealed a correlation between TRPL levels and CAP(2b)-stimulated fluid transport and calcium signaling. Rescue of trpl(302) with a trpl transgene under heat-shock control resulted in a stimulated fluid transport phenotype that was indistinguishable from wild-type tubules. Furthermore, restoration of normal stimulated rates of fluid transport by rescue of trpl(302) was not compromised by introduction of the trp null, trp(343). Thus, in an epithelial context, TRPL is sufficient for wild-type responses. Finally, a scaffolding component of the TRPL/TRP-signaling complex, INAD, is not expressed in tubules, suggesting that inaD is not essential for TRPL/TRP function in Drosophila tubules.

In Drosophila photoreceptors the multivalent PDZ protein INAD organizes the phototransduction cascade into a macromolecular signaling complex containing the effector PLC, the light-activated TRP channels, and a regulatory PKC. Previously, we showed that the subcellular localization of INAD signaling complexes is critical for signaling. Now we have examined how INAD complexes are anchored and assembled in photoreceptor cells. We find that trp mutants, or transgenic flies expressing inaD alleles that disrupt the interaction between INAD and TRP, cause the mislocalization of the entire transduction complex. The INAD-TRP interaction is not required for targeting but rather for anchoring of complexes, because INAD and TRP can be targeted independently of each other. We also show that, in addition to its scaffold role, INAD functions to preassemble transduction complexes. Preassembly of signaling complexes helps to ensure that transduction complexes with the appropriate composition end up in the proper location. This may be a general mechanism used by cells to target different signaling machinery to the pertinent subcellular location.