BIOLOGICAL OVERVIEW

Drosophila visual signal transduction, the process by which incoming light is converted to neural signals that can be passed to the brain, provides an ideal system for the molecular dissection of the process by which extracellular signals are transduced across the plasma membrane leading to neuron activation. Inactivation no afterpotential D (InaD) holds together a protein complex involved in visual signal transduction. Before more detail is provided about InaD, a brief word on the visual transduction process is in order.

In the visual signal transduction pathway, light stimulates rhodopsin, which activates an eye-specific G protein (Galphaq). Activated Galphaq triggers NORPA (No receptor potential A), a phospholipase C-beta that catalyzes the breakdown of phospholipids and generates inositol trisphosphate (IP₃) and diacylglycerol. Diacylglycerol is a potential precursor for several polyunsaturated fatty acids, such as arachidonic acid and linolenic acid. Both TRP (transient receptor potential) and TRPL (TRP-like) are cation channels that are activated in the visual transduction process. These two proteins share homology with alpha-subunits of voltage-gated calcium and sodium channels in vertebrates. The rise in IP₃ has been thought to result in the release of Ca²⁺ from the internal Ca²⁺ stores. However, the release of Ca²⁺ has been shown not to involved the Inositol 1,4,5,-tris-phosphate receptor, leaving unanswered questions as to the source and regulation of the initial Ca²⁺ current (Acharya, 1997). It has now been shown, however, that polyunsaturated fatty acids activate the Drosophila light-sensitive channels TRP and TRPL. As arachidonic acid may not be found in Drosophila, it is suggested that another polyunsaturated fatty acid, such as linolenic acid, may be a messenger of excitation in Drosophila photoreceptors (Chyb, 1999).

Inactivation no afterpotential D (InaD) was identified on the basis of an abnormal electroretinogram (Pak, 1979 ) and was later shown to display a slow deactivation of the light-induced current (Shieh, 1995). Unlike the response in wild-type flies, inaD mutants lack the prolonged depolarizing afterpotential, following stimulation by a pulse of intense blue light (Pak, 1979 and Shieh, 1995). By whole-cell patch-clamp recordings, inaD photoreceptors show a slow deactivation of light-induced currents. This abnormal visual response depends on Ca²⁺ influx; removal of extracellular Ca²⁺ masks the defective phenotype. Furthermore, inaD cells exhibit increased sensitivity toward dim light stimulation. Most of the InaD is associated with rhabdomeres, the organelles in which components of visual signaling are localized (Shieh, 1995).

Inactivation-no-afterpotential D is a photoreceptor-specific protein containing five repeated protein interaction motifs known as PDZ repeats. PDZ domains are composed of ~90 amino acid modules, identified initially in PSD-95, Discs large and ZO-1 (Drosophila homolog: Polychaetoid), which mediate protein-protein interactions by binding to the C-terminal ends of their targets. InaD lacks an SH2 domain and a guanylate kinase domain found in the MAGUK family of PDZ-containing proteins such as Discs large. InaD is an adaptor protein that homomultimerizes and has the ability to interact with multiple components of the signal transduction pathway, including Rhodopsin, NORPA, PKC, Calmodulin, TRP and TRPL (Shieh, 1997 and references and Xu, 1998a and references). The view has been widely held for many years that signaling through G protein-coupled cascades occurs via random stochastic collisions between membrane receptors and effector molecules. However, alternative proposals suggesting that signaling cascades are comprised of components that are physically coupled have been presented but have received less attention. The major conclusion from the work showing the multiple interactions of InaD is that Drosophila vision is mediated by a massive supramolecular complex and that assembly of such a complex is facilitated by homomultimerization of the scaffold protein InaD. Thus, most of the proteins critical in phototransduction appear to couple directly to InaD. The InaD supramolecular complex may not be a particle, consisting of a single InaD monomer to which a maximum of five target proteins bind. Instead, the visual cascade appears to be mediated through a more complicated higher order signaling web or complex (signalplex) consisting of an extended network of InaD homomultimers to which more than five targets bind. Most of these targets appear to bind to more than one PDZ module and several targets appear to associate with InaD via the same PDZ domains. Thus, the nature of the InaD signalplex appears to be more complicated than a single particle held together by a scaffolding protein (Xu, 1998a and references).

An intriguing question concerns the function of the complex between TRP, NORPA, and rhodopsin RH1. The release of Ca²⁺ from Ca²⁺ stores is proposed to activate TRP through a conformational coupling mechanism or via a diffusible messenger. However, this model for Drosophila phototransduction does not require a single complex containing NORPA, RH1, and TRP. An attractive possibility, which is favored, is that TRP is complexed with NORPA, RH1, and possibly INAC (eye protein kinase C) to facilitate negative feedback regulation, such as occurs in adaptation or inactivation. InaD may serve as an adapter to link TRP with NORPA and RH1 to facilitate modulation of NORPA and/or RH1 activity by TRP activity. Consistent with this proposal, disruption of the TRP-InaD interaction and mislocalization of TRP in inaD^P215 flies results in a slow deactivation of the light-induced current. Furthermore, whole cell recording studies have demonstrated that the inaD^P215 phenotype requires Ca²⁺ entry (Shieh, 1995). Under conditions in which there is no Ca²⁺ entry, the light response of inaD^P215 photoreceptor cells is indistinguishable from wild type. Further support for the proposal that TRP is associated with the signaling complex for feedback regulation of phototransduction is lent by the observation that the other proteins in the TRP signaling complex are affected by Ca²⁺. NORPA has been shown to be activated maximally at low concentrations of Ca²⁺, suggesting that the TRP-mediated Ca²⁺ flux serves a negative regulatory function. In addition, Ca²⁺ has been shown to enhance dephosphorylation of Drosophila rhodopsin and phosphorylation of Drosophila arrestin, a protein that binds to rhodopsin and plays a role in the termination mechanism of rhodopsin. Since InaD associates with calmodulin, in a Ca2+-dependent manner, InaD may be modulated by Ca2+ fluxes as well. A calcium channel, such as TRP, may be complexed with other signaling proteins, rather than at a distance in the membrane, to facilitate rapid responses to local changes in Ca2+ concentration, which might not be possible otherwise. It will be interesting to learn whether other ion channels linked to proteins containing PDZ domains are also associated with, and modulate the activities of, proteins that act upstream in signaling cascades (Chevesich, 1997).

PROTEIN STRUCTURE

Amino Acids - 674

Structural Domains

InaD is extremely rich in both basic (15.4%) and acidic (14.1%) amino acid residues, with a predicted pH of 8.86. Most of these charged amino acids are distributed throughout the entire molecule, with the exception of a stretch of 14 amino acids (amino acids 460-473), which is made exclusively of lysine and glutamic acid residues. Repeats consisting of GQ(M) are present between amino acids 142 and 158. Sequence analysis also reveals potential phosphorylation sites of cAMP- and cGMP-dependent protein kinases (one site), tyrosine kinases (one site), and protein kinases C (eight sites) suggesting that InaD may be potentially regulated by these modifications. Also of considerable interest is the finding that two repeats (as reported by Scott, 1998; others report five) of 40 amino acids of InaD show sequence similarity to repeats in a family of proteins that includes Drosophila Discs-large; the rat post-synaptic density protein and the vertebrate tight junction protein ZO-1 The identities between the first repeat of InaD and the first repeat of these other proteins ranges from 32% to 51% (Shieh, 1995).

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