INAD Organizes Signaling Complexes

Phototransduction in Drosophila is one of the fastest G-protein-coupled signaling pathways known, taking less than 20 ms from light activation to maximum response. This high speed of signaling is primarily due to the incorporation of signaling components into multi-protein complexes. Signaling complexes bring components into close proximity, promoting rapid interaction as well as ensuring the proper sub-cellular localization of components. The central organizer of these signaling complexes is the inactivation/no-afterpotential D protein (INAD), which functions as a scaffold for complexes. The original inaD mutant, inaD215, was first isolated in a large genetic screen for mutants with a defect in light responsiveness [16]. The inaD gene was later identified as the affected gene [17]. The predicted protein sequence of INAD was found to contain five protein-protein interaction domains called postsynaptic-density-95/Discs-large/ZO1 (PDZ) domains [18]. In general, PDZ domains, like other protein-protein interaction domains, such as Src homology 2 (SH2), SH3, and phosphotyrosine binding (PTB) domains, constitute the glue that holds such macromolecular complexes together [2,5,3,19].

PDZ domains are conserved sequences of 80 to 100 amino acids that have been shown to most commonly bind C-terminal protein motifs (-S/T-X-V/I-COOH, or COOH, where $ represents a hydrophobic residue, or -X-X-C-COOH) of proteins [5,20]. Some PDZ domains, however, have been shown to bind internal sites of the protein [21,22] as well as other PDZ domains [23,24]. About 200 PDZ-domain-containing proteins have been identified in yeast, C. elegans, Drosophila, and humans [25]. Many scaffolding proteins have been reported to contain multiple PDZ domains [5,26-28]. PDZ domains are found more frequently in multiples within a single protein than any other protein-protein binding domain [5]. These findings suggest that multiple PDZ domains are a common feature of scaffold proteins.

As its sequence would suggest, the eye-specific INAD protein, which consists almost entirely of PDZ domains (PDZ1 to PDZ5 from N to C terminus), has been found to interact with multiple members of the phototransduction cascade. Studies in both Drosophila and Calliphora ery-throcephala, using co-immunoprecipitation experiments, GST-fusion "pull-down" assays, ligand overlay assays, and the yeast-two-hybrid technique, have revealed three major proteins that interact with INAD: the effector PLCP, the light-activated ion channel TRP, and the eye-PKC required for normal deactivation [1,2]. Each PDZ domain appears to be specific for a particular protein partner. PDZ3 binds to the TRP channel [18,21,29-32]. PDZ1 and PDZ5 bind to the extreme C terminus (-F-C-A-COOH) and an internal sequence, respectively, of PLCP [22,33]. PDZ2 and PDZ4 have been reported to bind eye-PKC [18,34]. The interaction of PDZ4 with eye-PKC depends on the C-terminal-T-I-I-COOH of eye-PKC [34].

To understand the functional significance of INAD in photoreceptors, a null mutant of inaD (inaD1) was isolated and examined [18]. In inaD1 null mutants, three key observations were made: (1) PLCP, TRP, and eye-PKC are all mislocalized in young flies; (2) light-responsiveness in these young flies is severely impaired; and (3) with age, flies display a further degradation of PLCP, TRP, and eye-PKC. In wild-type flies, phototransduction components, including INAD-signaling complexes, are exclusively localized to the rhabdomere (see Fig. 1); the rhabdomere is a specialized subcellular compartment of photoreceptors that contains « 60,000 tightly packed microvilli, a structure analogous to the membranous discs of the vertebrate rod photoreceptor. In the inaD1 null mutant, PLCP, TRP, and eye-PKC are all mislocalized from the time of eclosion (adult fly emergence from its pupal stage): PLCP and eye-PKC are found in the cytoplasm of the cell body, and TRP localizes to the plasma membrane of the cell body outside of the rhabdomere [18]. While these core components of the INAD-signaling complex are mislocalized, other phototransduction components,

LIGHT rhabdomere

Figure 1 Illustration shows a Drosophila photoreceptor with the rhabdomere of the cell indicated. An expanded view of a microvillus membrane from the rhabdomere is shown. INAD-signaling complexes are clustered in the membrane, while rhodopsin molecules (Rh1, gray balls) are distributed randomly at a high density throughout the membrane. (From Tsunoda, S. and Zuker, C. S., Cell Calcium, 26(5), 166, 1999. With permission.)

Figure 1 Illustration shows a Drosophila photoreceptor with the rhabdomere of the cell indicated. An expanded view of a microvillus membrane from the rhabdomere is shown. INAD-signaling complexes are clustered in the membrane, while rhodopsin molecules (Rh1, gray balls) are distributed randomly at a high density throughout the membrane. (From Tsunoda, S. and Zuker, C. S., Cell Calcium, 26(5), 166, 1999. With permission.)

such as rhodopsin, Gaq, and TRPL, remain normally localized in the rhabdomeres of inaD1 null photoreceptors. These results show that INAD is essential for localizing its target proteins to the rhabdomere.

Light responsiveness of photoreceptors was examined in newly eclosed flies by electroretinogram (ERG) recordings and whole-cell voltage-clamp recordings; inaD1 null flies were found to be nearly blind, displaying very small responses with slowed activation and deactivation kinetics [18,35]. Although newly eclosed inaD1 null flies have somewhat reduced levels of PLCp, TRP, and eye-PKC, their mislocalization most likely produces the defective signaling observed. The placement of signaling complexes at the appropriate subcellular location has proven to be critical for signaling in other systems as well. The role INAD plays in assembling signaling complexes, targeting and/or anchoring components to the rhabdomere is just beginning to be explored (see below).

As inaD1 null flies age, levels of PLCp, TRP, and eye-PKC all become progressively reduced, while rhodopsin, Gaq, and TRPL protein levels are unaffected [18,36]. In addition, point mutations in single PDZ domains of INAD affect steady-state protein levels as well as the localization of the corresponding target protein, but not those of any other target proteins [18,30,36]. Thus, each target protein depends on its interaction with the scaffold protein INAD for both its subcellular localization as well as its stability.

In addition to PLCp, TRP, and eye-PKC, there may be other protein partners that transiently or constitutively interact with INAD. INAD has been reported to bind to rhodopsin, TRPL, calmodulin, and the eye-specific unconventional myosin III, neither-inactivation-nor-afterpotential C (NINA C) [30,37,38]. These interactions are not observed under all conditions and are present in co-immunoprecipitates in much lower levels than INAD, TRP, PLCp, and eye-PKC [39]. In addition, the localization and stability of rhodopsin, TRPL, calmodulin, and NINA C are unaffected in inaD1 null mutants [30,38]. Thus, it may be that these interactions are transient in nature. Future studies may reveal their significance.

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