Association of INAD with NORPA is essential for controlled activation and deactivation of Drosophila phototransduction in vivo.
Visual transduction in Drosophila is a G protein-coupled phospholipase C-mediated process that leads to depolarization via activation of the transient receptor potential (TRP) calcium channel. Inactivation-no-afterpotential D (INAD) is an adaptor protein containing PDZ domains known to interact with TRP. Immunoprecipitation studies indicate that INAD also binds to eye-specific protein kinase C and the phospholipase C, no-receptor-potential A (NORPA). By overlay assay and site-directed mutagenesis we have defined the essential elements of the NORPA-INAD association and identified three critical residues in the C-terminal tail of NORPA that are required for the interaction. These residues, Phe-Cys-Ala, constitute a novel binding motif distinct from the sequences recognized by the PDZ domain in INAD. To evaluate the functional significance of the INAD-NORPA association in vivo, we generated transgenic flies expressing a modified NORPA, NORPAC1094S, that lacks the INAD interaction. The transgenic animals display a unique electroretinogram phenotype characterized by slow activation and prolonged deactivation. Double mutant analysis suggests a possible inaccessibility of eye-specific protein kinase C to NORPAC1094S, undermining the observed defective deactivation, and that delayed activation may similarly result from NORPAC1094S being unable to localize in close proximity to the TRP channel. We conclude that INAD acts as a scaffold protein that facilitates NORPA-TRP interactions required for gating of the TRP channel in photoreceptor cells.
Shieh
B H
BH
Department of Pharmacology, Vanderbilt University, Nashville, TN 37232-6600, USA. shiehb@ctrvax.vanderbilt.edu
Zhu
M Y
MY
Lee
J K
JK
Kelly
I M
IM
Bahiraei
F
F
D000818:Animals; D030801:Animals, Genetically Modified; D004330:Drosophila / Q000502:physiology*; D029721:Drosophila Proteins; D005136:Eye Proteins / Q000502:physiology*; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D054799:Phospholipase C beta; D015398:Signal Transduction; D010738:Type C Phospholipases / Q000502:physiology*; D014785:Vision, Ocular / Q000502:physiology*
D029721:Drosophila Proteins; D005136:Eye Proteins; C091568:inaD protein, Drosophila; D010738:Type C Phospholipases; C468371:NorpA protein, Drosophila; D054799:Phospholipase C beta; D043265:Phosphatidylinositol Diacylglycerol-Lyase