Interaction of eye protein kinase C and INAD in Drosophila. Localization of binding domains and electrophysiological characterization of a loss of association in transgenic flies.
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.
Adamski
F M
FM
Department of Pharmacology and Center for Molecular Neuroscience, Vanderbilt University, Nashville, Tennessee 37232-6600, USA.
Zhu
M Y
MY
Bahiraei
F
F
Shieh
B H
BH
D000595:Amino Acid Sequence; D000818:Animals; D030801:Animals, Genetically Modified; D004330:Drosophila / Q000235:genetics*; D029721:Drosophila Proteins; D005074:Evoked Potentials, Visual; D005123:Eye / Q000201:enzymology / Q000378:metabolism*; D005136:Eye Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D017956:Photoreceptor Cells, Invertebrate / Q000378:metabolism*; D011485:Protein Binding; D011493:Protein Kinase C / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics; D017386:Sequence Homology, Amino Acid
D029721:Drosophila Proteins; D005136:Eye Proteins; C091568:inaD protein, Drosophila; D011493:Protein Kinase C