Association of INAD with NORPA is essential for controlled activation and deactivation of Drosophila phototransduction in vivo

Abstract

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.

Figure 1

INAD interacts with NORPA and TRP by ligand overlay assay. Shown on the left is an autoradiogram of [³⁵S]INAD overlay assay using wt, trp^p301, and norpA^p16 retinal extracts. Shown on the right is a Western blot of wt and norpA^p16 retinal extracts probed with anti-NORPA antibodies.

Figure 2

Stable association between INAD and NORPA in wt photoreceptors. (A) Anti-INAD antibodies immunoprecipitate both INAD and NORPA. Shown are Western blots of anti-INAD immunoprecipitation assays in wt and norpA^p16 extracts. (B) Anti-NORPA antisera immunoprecipitate both NORPA and INAD. Shown are Western blots of anti-NORPA immunoprecipitation assays. Preimmune antisera were used as a negative control (lanes 1 and 3). M, protein size standards.

Figure 3

Localization of the NORPA-interacting domain in INAD. (A) The C-terminal half of INAD interacts with NORPA. Shown is a summary of various INAD sequences tested for the interaction with NORPA by overlay assay. (B) The full-length INAD binds to retinal NORPA with high affinity and INAD(399–674) associates with a reduced affinity. The five PDZ repeats of INAD are depicted as arrows and the TRP-interacting region spanning residues 347–450 is indicated as a box.

Figure 4

INAD interacts with the C-terminal region of NORPA. (A) Shown on the left is Coomassie staining of a protein gel containing bacterial extracts expressing several overlapping NORPA sequences as fusion proteins of T7 gene 10 or alone. Each fusion protein is indicated with a star on the left. The corresponding autoradiogram of [³⁵S]INAD overlay assay is shown on the right. (B) Shown is a summary of [³⁵S]INAD overlay results.

Figure 5

Transgenic flies expressing norpA^C1094S display abnormal ERG with slower kinetics. (A) Shown are ERG recordings of norpA^p24, transgenic flies expressing wt norpA, and norpA^C1094S in norpA^p24 or wt genetic background in response to a 2-sec pulse of the orange light. Three independent lines were analyzed, and consistent results were obtained. The kinetics of ERG were examined using the following parameters: latency, defined as time elapsed between stimulation and initiation of ERG (B); initial slope of the response, defined as the rate to reach 50% of the maximal response following the initiation of the response (C); and time to repolarization, time to reach resting potential following the termination of stimulation (D) in wt norpA and norpA^C1094S;norpA^p24. Both latency (B) and time to repolarization (D) are lengthened, and initial slope (C) is reduced in norpA^C1094S;norpA^p24 lines.

Figure 7

Expression and subcellular distribution of NORPA^C1094S. (A) Expression of NORPA^C1094S in transgenic flies. Shown are Western blots of retinal extracts from norpA^C1094S transgenic lines in norpA^p24 (lanes 1 and 2) or wt background (lane 3), and extracts from wt (wt) and various mutants probed with antibodies as indicated. Each lane represents extracts from one fly head. M, prestained protein molecular weight standards, 150, 112, 84 kDa. (B) Random distribution of NORPA^C1094S in the compound eye. Shown is immunolocalization of NORPA and INAD in wt, norpA^p24 and norpA^C1094S;norpA^p24 eyes. In wt retinas, both NORPA and INAD are co-localized in the rhabdomere. However, NORPA^C1094S appears to be distributed in cytoplasm as well as in rhabdomeres of norpA^C1094S following heat shock treatment.

Figure 6

Double mutants analysis of norpA^C1094S by ERG. norpA^C1094S was crossed into inaC^p209, InaD^p215, and trp^p301 mutant backgrounds and analyzed. (A) inaC^p209 (Upper), norpA^C1094S;inaC^p209 (Lower) (B) trp^p301 (Upper), norpA^C1094S; trp^p301 (Lower). (C) InaD^p215 (Upper), norpA^C1094S;InaD^p215 (Lower) (D) Superimposition of ERG from norpA^C1094S, norpA^C1094S;InaD^p215, and norpA^C1094S;trp^p301. Flies of 1-day posteclosion were subject to heat shock and analyzed. Flies were dark adapted for 3 min prior to the recording using a 2-sec orange light. Multiple animals were recorded and consistent results obtained. Note the difference in the time course and amplitude of the response in trp^p301 and norpA^C1094S;trp^p301.

Figure 8

A model for visual transduction in Drosophila. Shown is a simplified visual cascade highlighting the protein–protein interactions in the INAD complex. In this model, G protein activates NORPA which leads to generation of diacylglycerol and opening of TRP. An increase in cytoplasmic calcium may activate calmodulin and Ca-dependent processes to modulate the visual response. Eye-PKC, activated by Ca²⁺ and diacylglycerol, may phosphorylate NORPA and TRP to terminate the activity. How TRPL is gated is not known.