In the Drosophila visual cascade, the transient receptor potential (TRP) calcium channel, phospholipase C (no-receptor-potential A), and an eye-specific isoform of protein kinase C (eye-PKC) comprise a multimolecular signaling complex via their interaction with the scaffold protein INAD. Previously, we showed that the interaction between INAD and eye-PKC is a prerequisite for deactivation of a light response, suggesting eye-PKC phosphorylates proteins in the complex. To identify substrates of eye-PKC, we immunoprecipitated the complex from head lysates using anti-INAD antibodies and performed in vitro kinase assays. Wild-type immunocomplexes incubated with [ 32 P]ATP revealed phosphorylation of TRP and INAD. In contrast, immunocomplexes from inaC mutants missing eye-PKC, displayed no phosphorylation of TRP or INAD. We also investigated protein phosphatases that may be involved in the dephosphorylation of proteins in the complex. Dephosphorylation of TRP and INAD was partially suppressed by the protein phosphatase inhibitors okadaic acid, microcystin, and protein phosphatase inhibitor-2. These phosphatase activities were enriched in the cytosol of wild-type heads, but drastically reduced in extracts prepared from glass mutants, which lack photoreceptors. Our findings indicate that INAD functions as RACK (receptor for activated PKC), allowing eye-PKC to phosphorylate INAD and TRP. Furthermore, dephosphorylation of INAD and TRP is catalyzed by PP1/PP2A-like enzymes preferentially expressed in photoreceptor cells. Protein kinase C (PKC)1 modulates various biological processes by transferring phosphates to serine and threonine residues of substrates. Regulatory proteins such as RACK (receptor for activated PKC) and RICK (receptor for inactive PKC) have been shown to target PKC to specific subcellular compartments (1). This selective distribution of the kinase directs its catalytic activity toward restricted groups of substrates localized proximal to the enzyme. The Drosophila visual cascade is a G q -coupled phospholipase C (PLC)-mediated pathway (see Ref. 2 for review). In this cascade, an eye-specific isoform of PKC (eye-PKC) negatively regulates visual signaling (3, 4). Eye-PKC is localized to the rhabdomere (4), a densely packed microvillar structure where visual transduction takes place, by tethering to the adaptor protein INAD (inactivation-no-afterpotential D) (5).INAD contains five distinct PDZ (Postsynaptic density 95; Drosophila Discs large; and Zonula occludens 1) domains (5-8). PDZ domains are modular protein-protein interaction sequences consisting of 90 -100 amino acids. Proteins containing PDZ domains have been implicated in clustering signaling molecules, including enzymes, receptors, and channels (9). PDZ domains mediate at least three types of protein-protein associations. The most common interaction is the binding of a PDZ domain to a tetrapeptide motif, X-S/T-X--COOH (X, any amino acid; , hydrophobic residues), at the carboxyl terminus of target proteins (9, 10). The second type of interaction ...
A growing number of proteins containing PDZ 1 domains have been shown to play important roles in the organization and/or regulation of signaling events in cells. PDZ domains (or GLGF repeats) were named after three proteins identified over a decade ago: postsynaptic density-95, Drosophila Discs large, and zonula occludens-1 (3-5). These three proteins belong to the membrane-associated guanylate kinase (MAGUK) family of proteins. Most MAGUK proteins contain three PDZ domains, an Src homology 2 domain, and a guanylate kinase-like domain, each having different cellular roles. PDZ domains range from 80 to 100 amino acids in length and typically bind to the carboxyl-terminal sequence of target proteins including receptors, channels, and various signaling molecules to regulate subcellular localization, trafficking, recycling, and/or signaling (6 -10).MUPP1, a protein containing 13 putative PDZ domains, was isolated in a yeast two-hybrid screening for proteins that bound to the carboxyl-terminal tail of the 5-HT 2C R (1). MUPP1 is expressed in many tissues, whereas the 5-HT 2C R is a brainspecific protein (1, 11). The 5-HT 2C R has classically been thought to couple to G q activation; however, additional G protein families have been implicated, leading to the activation of different downstream signaling pathways including phospholipase A 2 , C, or D, and various cation channels (12-17). Since PDZ-containing proteins can scaffold many signaling molecules together into a signal transduction complex, the interaction between MUPP1 and the 5-HT 2C R was further investigated. The 5-HT 2C R contains a PDZ binding motif, Ser 458 -Ser-Val, at its extreme carboxyl terminus, which is critical for interaction with PDZ 10 of MUPP1 (18). In an alternate approach to the yeast two-hybrid system, we independently show that PDZ 10 of MUPP1 is the primary site of interaction for the 5-HT 2C R.Serotonin stimulation has previously been shown to promote phosphorylation of the two serine residues of the 5-HT 2C R PDZ binding motif, Ser 458 and Ser 459 (2). We therefore hypothesize that phosphorylation of the carboxyl-terminal serines of the 5-HT 2C R regulates receptor interaction with MUPP1. To test this hypothesis, we investigated whether a modification of Ser 458 and/or Ser 459 of the 5-HT 2C R carboxyl-terminal tail would alter PDZ 10 interaction. Ser 458 and/or Ser 459 of the receptor tail were mutated to aspartate to mimic phosphorylation (i.e. introduction of a negative charge). Next, cells expressing 5-HT 2C Rs were treated with agonist or antagonist to assess the interaction of the 5-HT 2C R with MUPP1. The results of these experiments support our hypothesis that phosphorylation is a key regulator of 5-HT 2C R interaction with MUPP1. Furthermore, the results indicated that a significant amount of basal phosphorylation of the receptor may also play a yet undetermined role in regulating PDZ-protein interactions. MATERIALS AND METHODS AntibodiesPolyclonal anti-peptide antibodies against amino acids 419 -435 (amino acids RHTNERVARKANDPE...
The remarkable hearing sensitivity and frequency selectivity in mammals is attributed to cochlear amplifier in the outer hair cells (OHCs). Prestin, a membrane protein in the lateral wall of OHC plasma membrane, is required for OHC electromotility and cochlear amplifier. In addition, GLUT5, a fructose transporter, is reported to be abundant in the plasma membrane of the OHC lateral wall and has been originally proposed as the OHC motor protein. Here we provide evidence of interactions between prestin/prestin and prestin/GLUT5 in transiently transfected HEK293T cells. We used a combination of techniques: (1) membrane colocalization by confocal microscopy, (2) fluorescence resonance energy transfer (FRET) by fluorescence activated cell sorting (FACS), (3) FRET by acceptor photobleaching, (4) FRET by fluorescence lifetime imaging (FRET-FLIM), and (5) coimmunoprecipitation. Our results suggest that homomeric and heteromeric prestin interactions occur in native OHCs to facilitate its electromotile function and that GLUT5 interacts with prestin for its elusive function.
Six protein kinase C (PKC) genes are present in Drosophila, comprising two classical PKCs (PKC53E and eye-PKC), two novel PKCs (PKC98E and PKCdelta), an atypical PKC (DaPKC), and a PKC-related kinase. Loss of function alleles affecting DaPKC and eye-PKC are available and their mutant phenotypes have been characterized. DaPKC is essential for early embryonic development because it regulates cell polarity and asymmetric cell division. Eye-PKC plays a role in the regulation of visual signaling, a G-protein coupled phospholipase Cbeta-mediated cascade. Both eye-PKC and DaPKC are differentially localized through tethering to multimolecular complexes. DaPKC interacts with partitioning-defective 3 (Par-3) and Par-6 proteins, which contain PDZ (PSD95, DLG, ZO-1) domains. Similarly, eye-PKC is anchored to a PDZ domain containing scaffolding protein INAD. Characterization of these two PKCs in Drosophila revealed a universal mechanism by which PKC is tethered to specific protein complexes for participation in distinct signal transduction processes.
Cochlear hair cells express SK2, a small-conductance Ca(2+)-activated K(+) channel thought to act in concert with Ca(2+)-permeable nicotinic acetylcholine receptors (nAChRs) alpha9 and alpha10 in mediating suppressive effects of the olivocochlear efferent innervation. To probe the in vivo role of SK2 channels in hearing, we examined gene expression, cochlear function, efferent suppression, and noise vulnerability in mice overexpressing SK2 channels. Cochlear thresholds, as measured by auditory brain stem responses and otoacoustic emissions, were normal in overexpressers as was overall cochlear morphology and the size, number, and distribution of efferent terminals on outer hair cells. Cochlear expression levels of SK2 channels were elevated eightfold without striking changes in other SK channels or in the alpha9/alpha10 nAChRs. Shock-evoked efferent suppression of cochlear responses was significantly enhanced in overexpresser mice as seen previously in alpha9 overexpresser mice; however, in contrast to alpha9 overexpressers, SK2 overexpressers were not protected from acoustic injury. Results suggest that efferent-mediated cochlear protection is mediated by other downstream effects of ACh-mediated Ca(2+) entry different from those involving SK2-mediated hyperpolarization and the associated reduction in outer hair cell electromotility.
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