Abstract
Drosophila eye-specific protein kinase C (eye-PKC) is involved in light adaptation and deactivation. eye-PKC, NORPA (phospholipase Cbeta), and transient-receptor-potential (TRP) (calcium channel) are integral components of a signal transduction complex organized by INAD, a protein containing five PDZ domains. We previously demonstrated the direct association between the third PDZ domain of INAD with TRP in addition to the carboxyl-terminal half of INAD with the last three residues of NORPA. In this work, the molecular interaction between eye-PKC and INAD is defined via the yeast two-hybrid and ligand overlay assays. We show that the second PDZ domain of INAD interacts with the last three residues in the carboxyl-terminal tail of eye-PKC, Thr-Ile-Ile. The association between eye-PKC and INAD is disrupted by an amino acid substitution (Ile-700 to Asp) at the final residue of eye-PKC. In flies lacking endogenous eye-PKC (inaCp215), normal visual physiology is restored upon expression of wild-type eye-PKC, whereas the eye-PKCI700D mutant is completely inactive. Flies homozygous for inaCp209 and InaDp215, a mutation that causes a loss of the INAD-TRP association, were generated. These double mutants display a more severe response inactivation than either of the single mutants. Based on these findings, we conclude that the in vivo activity of eye-PKC depends on its association with INAD and that the sensitivity of photoreceptors is cooperatively regulated by the presence of both eye-PKC and TRP in the signaling complex.
Highlights
Drosophila eye-specific protein kinase C is involved in light adaptation and deactivation. eye-PKC, NORPA, and transient-receptor-potential (TRP) are integral components of a signal transduction complex organized by INAD, a protein containing five PDZ domains
Despite the uncertain role of inositol 1,4,5-trisphosphate in photoreceptors, diacylglycerol generated by NORPA is a potent activator of protein kinase C (PKC). eye-PKC encoded by the inaC locus is known to be involved in the negative feedback regulation of visual transduction in Drosophila photoreceptors [11, 12]
We initially found that INAD interacts with both full-length eye-PKC and PKC-18 as shown by LacZ reporter activity (Fig. 1B)
Summary
LOCALIZATION OF BINDING DOMAINS AND ELECTROPHYSIOLOGICAL CHARACTERIZATION OF A LOSS OF ASSOCIATION IN TRANSGENIC FLIES*. Eye-PKC has been shown to be a component of a signal transduction complex by immunoprecipitation and affinity chromatography assays [13,14,15,16] This complex is organized by an adaptor protein, INAD, that interacts with two key components of the visual cascade, TRP and NORPA [13,14,15,16,17,18]. The norpA transgenic flies display a unique electroretinogram recordings (ERG) characterized by delayed activation and slow deactivation This phenotype of slow kinetics is observed in InaD2 that contains a missense mutation in the fifth PDZ domain leading to a loss of the NORPA association [16]. Potential substrates for eye-PKC that are involved in deactivation and desensitization of the visual response are discussed
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