The Calliphora rpa mutant lacks the PDZ domain‐assembled INAD signalling complex

Abstract

The visual transduction cascade of fly photoreceptors is a G protein-coupled phospholipase C-signalling pathway which is assembled into a supramolecular signalling complex by the PDZ (postsynaptic density protein-95, discs large, Z0-1) domain protein INAD (inactivation no afterpotential D). The norpA-encoded phospholipase Cbeta, the light-activated transient receptor potential (TRP) Ca2+ channel and an eye-specific protein kinase C are bound to INAD and together form the core of the signalling complex. In the present study we show that the Calliphora rpa mutant, which has previously been hypothesized to represent an equivalent of Drosophila norpA mutants, has normal amounts of norpA mRNA but fails to express inaD mRNA. Electrophysiological recordings from the eyes of the rpa mutant reveal that the electroretinogram is reduced (about 12% of wild type) but not completely absent, and that it exhibits markedly prolonged deactivation kinetics. Furthermore, rpa mutants display a slow, light-dependent degeneration of the photoreceptor cells. With respect to the INAD signalling complex, the rpa mutant is similar to the Drosophila inaD null mutant: not only INAD itself, but also the other core components of the INAD signalling complex, are reduced or absent in photoreceptor membranes of rpa flies. Residual TRP is localized throughout the plasma membrane of the photoreceptor cell, rather than being restricted to the microvillar photoreceptor membrane. [35S]methionine-labelling of newly synthesized retinal proteins reveals that TRP is synthesized in the rpa mutant at wild-type level, but is transported to or incorporated into the microvillar photoreceptor membrane at a much lower rate. We thus suggest, that the formation of the INAD signalling complex is required for specifically targeting its components to the photoreceptor membrane.

Scaffolding proteins organize multimolecular protein complexes for sensory signal transduction

“…Similar results were reported for Drosophila inaD P215 (Chevesich et al. , 1997) and for a mutant of the larger fly species Calliphora which also lacks the INAD protein (Huber et al. , 2000).…”

“…The presence of a multiprotein-signalling complex in the photoreceptive membrane raises the question of how such a complex is assembled and maintained. It was shown previously that the core components, TRP, PLCb and ePKC require INAD for correct localization in the rhabdomeral photoreceptor membrane (Tsunoda et al, 1997;Xu et al, 1997;Huber et al, 2000). In INAD null mutants these ligands are no longer con®ned to the photoreceptor membrane and are degraded.…”

“…, 1997; Xu et al. , 1997; Huber et al ., 2000). In INAD null mutants these ligands are no longer confined to the photoreceptor membrane and are degraded.…”

“…These findings indicate that TRP is initially targeted to the photoreceptor membrane independently of INAD but is not retained in this membrane when the INAD signalling complex cannot be formed. In a Calliphora mutant which lacks INAD, newly synthesized TRP, which had been labelled by injection of radioactive methionine into the eye, could not be detected in the photoreceptive membrane 2.5 h after injection of the tracer, but as in Drosophila inaD null mutants, residual amounts of TRP were found to be present in the photoreceptive membrane of this mutant, suggesting that at least some TRP molecules are targeted correctly (Huber et al ., 2000). Indeed, when longer incubation times for labelling newly synthesized TRP molecules were employed, newly synthesized TRP could be detected in the photoreceptor membrane of this Calliphora mutant, suggesting INAD independent targeting of TRP to the photoreceptor membrane (Huber & Bähner, unpublished).…”

The latency of the light response is modulated by the phosphorylation state of Drosophila TRP at a specific site

“…The effect of light intensity on the latency was statistically significant (P < 0.0001) and no statistically significant interaction was found (P D 0.9121). Tukey's multiple comparison test revealed statistical significance between the trp S936A and trp S936D transgenic flies at light intensities of 3 £ 10 3 and between the trp S936A and trp WT Antibodies -For Western blot analyses, the following antibodies were used: polyclonal a-RH1 antibody, 16 monoclonal a-TRP antibody (MAb83F6, Developmental Studies Hybridoma Bank), polyclonal a-TRP antibody, 17 monoclonal a-b-tubulin antibody (E7, Developmental Studies Hybridoma Bank), polyclonal a-PLC antibody, 18 polyclonal a-TRPL antibody, 19 polyclonal a-Gqa antibody 20 and polyclonal a-Moesin antibody. To generate polyclonal a-INAD and a-PKC antibodies, fragments spanning the last A two-way ANOVA was conducted that examined the effect of fly strain and light intensity on response latency.…”