Anchoring TRP to the INAD macromolecular complex requires the last 14 residues in its carboxyl terminus.

Drosophila transient-receptor-potential (TRP) is a Ca2+ channel responsible for the light-dependent depolarization of photoreceptors. TRP is anchored to a macromolecular complex by tethering to inactivation-no-afterpotential D (INAD). We previously reported that INAD associated with the carboxyl tail of TRP via its third post-synaptic density protein 95, discs-large, zonular occludens-1 domain. In this paper, we further explored the molecular basis of the INAD interaction and demonstrated the requirement of the last 14 residues of TRP, with the critical contribution of Gly1262, Val1266, Trp1274, and Leu1275. We also revealed by pull-down assays that the last 14 residues of TRP comprised the minimal sequence that competes with the endogenous TRP from fly extracts, leading to the co-purification of a partial INAD complex containing INAD, no-receptor-potential A, and eye-protein kinase C is critical for the negative regulation of the visual signaling and was shown to phosphorylate TRP in vivo. To uncover the substrates of in the INAD complex, we designed a complex-dependent eye-duanyu1531 assay, which utilized endogenous INAD complexes isolated from flies. We demonstrate that activated eye-duanyu1531 phosphorylates INAD, TRP but not no-receptor-potential A. Moreover, phosphorylation of TRP is dependent on the presence of both eye-duanyu1531 and INAD. Together, these findings indicate that stable kinase-containing protein complexes may be isolated by pull-down assays, and used in this modified kinase assay to investigate phosphorylation of the proteins in the complex. We conclude that TRP associates with INAD via its last 14 residues to facilitate its regulation by eye-duanyu1531 that fine-tunes the visual signaling.

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