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• W2015033845 abstract "INAD is a scaffolding protein containing five PSD95/dlg/zonular occludens-1 (PDZ) domains that tether NORPA (phospholipase Cβ4), the TRP calcium channel, and eye-PKC in Drosophila photoreceptors. We previously showed that eye-PKC interacted with the second PDZ domain (PDZ2) of INAD. Sequence comparison with a prototypical type I PDZ domain predicts that PDZ2 is the best candidate among the five PDZ domains to recognize eye-PKC that contains a type I PDZ ligand, Ile-Thr-Ile-Ile, at its carboxyl terminus. Replacement of Ile−3 in eye-PKC with charged residues resulted in a drastic reduction of the PDZ2 interaction. Substitution of a conserved His with Arg at the second α-helix of PDZ2 led to a reduced binding; however, a Leu replacement resulted in an enhanced eye-PKC association. We isolated and sequenced the InaD gene. The coding sequence of InaDcontains nine exons spanning 3 kilobases. Translation of coding sequences from three wild-type alleles revealed three SNPs affecting residues, 282, 319, and 333 of INAD. These polymorphisms are localized in PDZ2. Interestingly, we found two of three PDZ2 variants displayed a greater affinity for eye-PKC. In summary, we evaluated the molecular basis of the eye-PKC and PDZ2 association by mutational analysis and concluded that PDZ2 of INAD is a type I domain important for the eye-PKC interaction. INAD is a scaffolding protein containing five PSD95/dlg/zonular occludens-1 (PDZ) domains that tether NORPA (phospholipase Cβ4), the TRP calcium channel, and eye-PKC in Drosophila photoreceptors. We previously showed that eye-PKC interacted with the second PDZ domain (PDZ2) of INAD. Sequence comparison with a prototypical type I PDZ domain predicts that PDZ2 is the best candidate among the five PDZ domains to recognize eye-PKC that contains a type I PDZ ligand, Ile-Thr-Ile-Ile, at its carboxyl terminus. Replacement of Ile−3 in eye-PKC with charged residues resulted in a drastic reduction of the PDZ2 interaction. Substitution of a conserved His with Arg at the second α-helix of PDZ2 led to a reduced binding; however, a Leu replacement resulted in an enhanced eye-PKC association. We isolated and sequenced the InaD gene. The coding sequence of InaDcontains nine exons spanning 3 kilobases. Translation of coding sequences from three wild-type alleles revealed three SNPs affecting residues, 282, 319, and 333 of INAD. These polymorphisms are localized in PDZ2. Interestingly, we found two of three PDZ2 variants displayed a greater affinity for eye-PKC. In summary, we evaluated the molecular basis of the eye-PKC and PDZ2 association by mutational analysis and concluded that PDZ2 of INAD is a type I domain important for the eye-PKC interaction. PSD95/dlg/zonular occludens-1 glutathioneS-transferase polymerase chain reaction post-synaptic density protein 95 rapid amplification of cDNA ends single nucleotide polymorphisms untranslated region protein kinase C eye-specific protein kinase C polyacrylamide gel electrophoresis The InaD(inactivation-no-after potentialD) gene is preferentially expressed in the compound eye and was isolated by subtractive hybridization (1Shieh B.-H. Niemeyer B. Neuron. 1995; 14: 201-210Abstract Full Text PDF PubMed Scopus (103) Google Scholar). Molecular characterization of InaD indicates that it encodes a protein of 674 amino acid residues that contains five distinct PSD95/dlg/zonular occludens-1 (PDZ)1 domains (2Saras J. Heldin C.-H. Trends Biochem. Sci. 1996; 21: 455-458Abstract Full Text PDF PubMed Scopus (224) Google Scholar, 3Fanning A.S. Anderson J.M. Curr. Biol. 1996; 11: 1385-1388Abstract Full Text Full Text PDF Scopus (243) Google Scholar). PDZ domains are protein-protein interaction motifs of 80–100 residues and are implicated in clustering and localization of receptors and channels (4Kornau H.-C. Schenker L.T. Kennedy M.B. Seeburg P.H. Science. 1995; 269: 1737-1740Crossref PubMed Scopus (1631) Google Scholar, 5Kim E. Niethammer M. Rothschild A. Jan Y.N. Sheng M. Nature. 1995; 378: 85-88Crossref PubMed Scopus (900) Google Scholar). In Drosophila photoreceptors, INAD has been shown to interact with carboxyl-terminal sequences of three key components of the visual cascade leading to the formation of a macromolecular signaling complex (6Shieh B.-H. Zhu M.-Y. Neuron. 1996; 16: 991-998Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar, 7Shieh B.-H. Zhu M.-Y. Lee J.K. Kelly I.M. Bahiraei F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12682-12687Crossref PubMed Scopus (69) Google Scholar, 8Chevesich J. Kreuz A.J. Montell C. Neuron. 1997; 18: 95-105Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar, 9Tsunoda S. Sierralta J. Sun Y. Bodner R. Suzuki E. Becker A. Socolich M. Zuker C.S. Nature. 1997; 388: 243-249Crossref PubMed Scopus (554) Google Scholar, 10van Huizen R. Miller K. Chen D.M. Li Y. Lai Z.C. Raab R.W. Stark W.S Shortridge R.D. Li M. EMBO J. 1998; 17: 2285-2297Crossref PubMed Scopus (97) Google Scholar, 11Adamski F.M. Zhu M.-Y. Bahiraei F. Shieh B.-H. J. Biol. Chem. 1998; 273: 17713-17719Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). These INAD-interacting proteins include TRP (transient-receptor-potential), NORPA (no-receptor-potentialA), and eye-PKC. Eye-PKC is involved in a negative regulation of visual transduction (12Ranganathan R. Harris G.L. Stevens C.F. Zuker C.S. Nature. 1991; 354: 230-232Crossref PubMed Scopus (164) Google Scholar, 13Hardie R.C. Peretz A. Suss-Toby E. Rom-Glas A. Bishop S.A. Selinger Z. Minke B. Nature. 1993; 363: 634-637Crossref PubMed Scopus (135) Google Scholar), a G protein-coupled phospholipase Cβ-mediated process that converts the signal of light leading to depolarization of photoreceptors (14Scott K. Zuker C.S. Curr. Opin. Neurobiol. 1998; 3: 383-388Crossref Scopus (63) Google Scholar, 15Montell C. Annu. Rev. Cell Dev. Biol. 1999; 15: 231-268Crossref PubMed Scopus (252) Google Scholar). Negative modulation of visual signaling by eye-PKC in vivo is dependent on its interaction with INAD (11Adamski F.M. Zhu M.-Y. Bahiraei F. Shieh B.-H. J. Biol. Chem. 1998; 273: 17713-17719Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Previously we showed that eye-PKC associated with PDZ2 of INAD (11Adamski F.M. Zhu M.-Y. Bahiraei F. Shieh B.-H. J. Biol. Chem. 1998; 273: 17713-17719Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Other reports have implicated PDZ3 or PDZ4 in the eye-PKC interaction as well (9Tsunoda S. Sierralta J. Sun Y. Bodner R. Suzuki E. Becker A. Socolich M. Zuker C.S. Nature. 1997; 388: 243-249Crossref PubMed Scopus (554) Google Scholar, 16Xu X.-Z.S. Choudhury A. Li X. Montell C. J. Cell Biol. 1998; 142: 545-555Crossref PubMed Scopus (199) Google Scholar). Structural studies of PDZ domains in PSD95, human homologues ofdlg, and calcium/calmodulin-dependent serine protein kinase revealed that PDZ domains consist of six β-sheets and two α-helices forming a six-strand β-sandwich structure (17Doyle D.A. Lee A. Lewis J. Kim E. Sheng M. MacKinnon R. Cell. 1996; 85: 1067-1076Abstract Full Text Full Text PDF PubMed Scopus (976) Google Scholar, 18Morais-Cabral J.H. Petosa C. Sutcliffe M.J. Raza S. Byron O. Poy F. Marfatia S.M. Chishti A.H. Liddington R.C. Nature. 1996; 382: 649-652Crossref PubMed Scopus (292) Google Scholar, 19Daniels D.L. Cohen A.R. Anderson J.M. Brunger A.T. Nat. Struct. Biol. 1998; 5: 317-325Crossref PubMed Scopus (161) Google Scholar). With some exceptions (6Shieh B.-H. Zhu M.-Y. Neuron. 1996; 16: 991-998Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar, 20Hillier B.J. Christopherson K.S. Prehoda K.E. Bredt D.S. Lim W.A. Science. 1999; 284: 812-815Crossref PubMed Scopus (470) Google Scholar), most PDZ domains bind to the last 3–4 amino acids at the carboxyl-terminal tail of target proteins (21Songyang Z. Fanning A.S. Fu C. Xu J. Marfatia S.M. Chishti A.H. Crompton A. Chan A.C. Anderson J.M. Cantley L.C. Science. 1997; 275: 73-77Crossref PubMed Scopus (1224) Google Scholar). Based on the target or ligand sequences, PDZ domains can be subdivided into two classes: type I and type II (21Songyang Z. Fanning A.S. Fu C. Xu J. Marfatia S.M. Chishti A.H. Crompton A. Chan A.C. Anderson J.M. Cantley L.C. Science. 1997; 275: 73-77Crossref PubMed Scopus (1224) Google Scholar). Type I PDZ domains recognize ligands that contain either a Ser or Thr at the −2 position. In contrast, type II domains bind to ligands containing a bulky residue such as Phe or Tyr at the corresponding position (19Daniels D.L. Cohen A.R. Anderson J.M. Brunger A.T. Nat. Struct. Biol. 1998; 5: 317-325Crossref PubMed Scopus (161) Google Scholar,21Songyang Z. Fanning A.S. Fu C. Xu J. Marfatia S.M. Chishti A.H. Crompton A. Chan A.C. Anderson J.M. Cantley L.C. Science. 1997; 275: 73-77Crossref PubMed Scopus (1224) Google Scholar). Almost all PDZ-interacting ligands have a hydrophobic residue (Ile, Leu, or Val) at the carboxyl terminus (position 0) (2Saras J. Heldin C.-H. Trends Biochem. Sci. 1996; 21: 455-458Abstract Full Text PDF PubMed Scopus (224) Google Scholar, 3Fanning A.S. Anderson J.M. Curr. Biol. 1996; 11: 1385-1388Abstract Full Text Full Text PDF Scopus (243) Google Scholar, 4Kornau H.-C. Schenker L.T. Kennedy M.B. Seeburg P.H. Science. 1995; 269: 1737-1740Crossref PubMed Scopus (1631) Google Scholar, 5Kim E. Niethammer M. Rothschild A. Jan Y.N. Sheng M. Nature. 1995; 378: 85-88Crossref PubMed Scopus (900) Google Scholar, 8Chevesich J. Kreuz A.J. Montell C. Neuron. 1997; 18: 95-105Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar). X-ray crystallographic studies revealed that the tetrapeptide ligand anchors to a groove formed between the second β-strand and the second α-helix of the PDZ domain (17Doyle D.A. Lee A. Lewis J. Kim E. Sheng M. MacKinnon R. Cell. 1996; 85: 1067-1076Abstract Full Text Full Text PDF PubMed Scopus (976) Google Scholar, 19Daniels D.L. Cohen A.R. Anderson J.M. Brunger A.T. Nat. Struct. Biol. 1998; 5: 317-325Crossref PubMed Scopus (161) Google Scholar). Each PDZ domain appears to recognize a unique carboxyl-terminal sequence (21Songyang Z. Fanning A.S. Fu C. Xu J. Marfatia S.M. Chishti A.H. Crompton A. Chan A.C. Anderson J.M. Cantley L.C. Science. 1997; 275: 73-77Crossref PubMed Scopus (1224) Google Scholar). Because the carboxyl-terminal of eye-PKC (11Adamski F.M. Zhu M.-Y. Bahiraei F. Shieh B.-H. J. Biol. Chem. 1998; 273: 17713-17719Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar) contains a type I PDZ ligand, we sought to identify a type I domain in INAD. Sequence alignment with the third PDZ domain (PDZ3) of PSD95, a type I domain, indicates that PDZ2 is the only type I domain in INAD. PDZ3 of PSD95 interacts with a tetrapeptide, Gln-Thr-Ser-Val (17Doyle D.A. Lee A. Lewis J. Kim E. Sheng M. MacKinnon R. Cell. 1996; 85: 1067-1076Abstract Full Text Full Text PDF PubMed Scopus (976) Google Scholar), and the side chains of Gln−3 form hydrogen bonds with those of Ser and Asn in PDZ3 of PSD95. In contrast, the corresponding residue of Gln−3 in eye-PKC is Ile−3, a residue with a hydrophobic side chain. We explored how a different residue at the −3 position of the target may interact with the different type I PDZ domains. We also performed site-directed mutagenesis by modifying Ile−3 of eye-PKC and investigated the contribution of this residue in the PDZ2 recognition. To explore the basis of the type I interaction, we mutated a conserved His in the second α-helix of PDZ2. This His has been implicated in the interaction with Ser/Thr at the −2 position (17Doyle D.A. Lee A. Lewis J. Kim E. Sheng M. MacKinnon R. Cell. 1996; 85: 1067-1076Abstract Full Text Full Text PDF PubMed Scopus (976) Google Scholar). Interestingly, we found that substitution of His310 with a Leu resulted in enhanced eye-PKC interaction, whereas replacement with Arg led to a reduction of association. To gain insight into the regulation of InaD expression, we analyzed the genomic structure of InaD and mapped the transcription start site by RACE. The InaD gene contains nine exons. Interestingly, we found SNPs in the coding exons leading to substitutions in three residues of PDZ2. We analyzed these variant PDZ2 and show that two modified PDZ domains display an increase in eye-PKC binding. Ficoll, 5-bromo-4-chloro-3-indolyl phosphate, and nitroblue tetrazolium were obtained from Research Organic, Inc. (Cleveland, OH). Phenol and deoxynucleotide triphosphates were purchased from U. S. Biochemicals. Chloroform and other chemicals were from Fisher. Nitrocellulose filters were from Schleicher & Schuell. DNA size markers were purchased from Life Technologies, Inc. Restriction enzymes and modifying enzymes such as T4 DNA ligase were obtained from New England Biolabs (Beverly, MA), Stratagene (La Jolla, CA), orPromega (Madison, WI). RNasin and pGEMEX1 were obtained from Promega. [32P]dCTP was from PerkinElmer Life Sciences. A Drosophila genomic library prepared from Canton S strain in Charon 4 vector (22Maniatis T. Hardison R.C. Lacy E. Lauer J. O'Connell C. Quon D. Sim G.K. Efstratiadis A. Cell. 1978; 15: 687-701Abstract Full Text PDF PubMed Scopus (1039) Google Scholar) was plated. Filter replicas of the library were probed with radioactively labeled InaD in a hybridization solution (5× SSCP, 0.5% SDS, 10 mm EDTA, 5× Denhardt's solution) at 65 °C for 16 h. Subsequently filters were rinsed with a buffer containing 0.1× SSC and 0.1% SDS at 65 °C for 2 h (23Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989: 8.46-8.49Google Scholar). Positive plaques were identified by autoradiography and purified. The EcoRI inserts containing the InaD gene were identified and subcloned into the pBluescript KS vector (Stratagene) for sequence analysis. The nucleotide sequence was determined either by the dideoxy chain termination method (24Sanger F. Nicklen S. Coulson A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463-5467Crossref PubMed Scopus (52770) Google Scholar) using the Sequenase kit (Amersham Pharmacia Biotech) or by automatic DNA sequencing using ABI PRISM BigDyeTM Terminator Cycle Sequencing Ready reaction kit (PerkinElmer Life Sciences). For DNA obtained by PCR at least four to six independent subclones were sequenced. Total genomic DNA was isolated as described previously (1Shieh B.-H. Niemeyer B. Neuron. 1995; 14: 201-210Abstract Full Text PDF PubMed Scopus (103) Google Scholar). Briefly, 50 flies were gently homogenized in 300 μl of buffer (100 mm NaCl, 100 mmTris, pH 7.6, 100 mm EDTA, 0.5% SDS). The homogenates were incubated at 65 °C for 30 min, and 8 m potassium acetate (one sixth volume) was added. The mixture was incubated on ice for 20 min, and the supernatant was recovered following centrifugation. RNase A (50 μg) was added to the supernatant to hydrolyze RNA. The mixture was subject to phenol/chloroform extraction to remove proteins. Ethanol was added to the supernatant to precipitate genomic DNA. Fly heads from various strains were isolated, and total RNA were extracted according to Chomczynski and Sacchi (25Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63232) Google Scholar). Total RNA was precipitated by ethanol and quantified by spectrophotometry. To generate first strand cDNA, 20 μg of total RNA were added to a 25-μl reverse transcription reaction using the reverse transcription system (Promega). Following incubation at 42 °C for 1 h, the reaction was terminated by addition of 75 μl of 10 mm EDTA (pH 8.0). An aliquot of cDNA (1–2 μg of RNA equivalent) was used as templates for PCR analysis. PCR was used to amplify genomic DNA as well as cDNA using InaD-specific primers. A negative control containing no added templates was also performed to assure the specificity of amplifications. The amplified fragments were subcloned into pCR2.1 using the TOPO TA Cloning system (Invitrogen), and recombinant plasmids were purified and used for sequencing. The experimental conditions for PCR (30 cycles) were denaturation at 94 °C for 30 s and annealing at 50 °C for 30 s followed by extension at 72 °C for 2–3 min. The reaction mixture (50 μl) contained 50–100 ng of each primer, 1 μg of total genomic DNA (or first strand cDNA from 1–2 μg total RNA) as templates, 0.2 mm dNTP, and 2.5 units of Taq DNA polymerase (PerkinElmer Life Sciences), in a buffer containing 10 mm Tris (pH 8.3), 50 mm KCl, and 1.5 mm MgCl2. Primers for InaD genomic and cDNA amplification were primers b and d (see Fig.4 A). Primer sequences for the amplification of the eye-PKC carboxyl tail were TTC GAA TTC ATG GCA GGT (5′) and TAT GGA TCC TTA AAT GAT GGT TAT AAA CTC (3′). RACE was performed using the Marathon cDNA amplification kit (CLONTECH). Briefly, total RNA fromDrosophila head was primed with a modified oligo(dT)30 primer to generate first strand cDNA by avian myeloblastosis virus reverse transcriptase. The first strand cDNA was then used as templates for the second strand cDNA synthesis mediated by RNase H, Escherichia coli DNA polymerase, and E. coli DNA ligase. After ligating with an adaptor primer, the double-strand cDNA was used as templates for PCR using a gene-specific primer, b (see Fig. 4 A) and AP1 primer that anneals to the adaptor primer. A second PCR reaction using the initial PCR mixture as templates and a different set of primers, primer a (see Fig. 4 A) and AP1, was followed. Two antisense primers, a and b (see Fig. 4 A), were used for the 5′ RACE, and two sense primers, d and e (see Fig. 4 A), were used for 3′ RACE. All four InaD-specific primers have melting temperatures of 72 °C. The DNA fragments obtained from the nested PCR amplification were subcloned and sequenced. Primer sequences forInaD are CTT GTC CAG GGT CAC CAT GTG AAT (primer a), CAG CAG CAT GTC GCC CAC TTT CA (primer b), TCA AGC AGC GAG GAT GGT TCA GTT (primer d), and GGC ATG TGC GTC AAG CCC ATC AA (primer e). Site-directed mutagenesis was performed using the overlap extension method as described (26Ho S.N. Hunt H.D. Horton R.M. Pullen J.K. Pease L.R. Gene ( Amst. ). 1989; 77: 51-59Crossref PubMed Scopus (6851) Google Scholar). Carboxyl-terminal tails of wild-type eye-PKC (641) and PDZ2 (206) of INAD were expressed as fusion proteins of glutathione S-transferase (GST). Briefly, recombinant pGEX4T1 plasmids were transformed intoE. coli HMS174. Overnight cultures (1 ml) were prepared from a single colony and used to inoculate a 50-ml LB broth containing ampicillin. The cultures were grown at 37 °C for 2–3 h until the density of bacterial cultures (OD600) reached 0.6–0.7. The expression of fusion proteins was initiated by the addition of isopropyl-1-thio-β-d-galactopyranoside (final concentration, 0.1–1 mm) (27Smith D.B. Rubira M.R. Simpson R.J. Davern K.M. Tiu T.W. Board P.G. Mitchell G.F. Mol. Biochem. Parasitol. 1988; 27: 249-256Crossref PubMed Scopus (63) Google Scholar). The cultures were harvested 3 h following induction, and bacterial pellets were collected by centrifugation. Bacterial lysates containing the fusion protein were prepared by resuspending the pellets in binding buffer (50 mm K3PO4, pH 7.0, 150 mm KCl, 10 mm MgCl2, 10% glycerol, 1% Triton X-100 plus a mixture of protease inhibitors) followed by repeated sonication. Recombinant plasmids containing target cDNA in pGEMEX1 (Promega) were constructed and used as templates for T7 RNA polymerase-dependent transcription. Incorporation of [35S]methionine into T7 gene 10 fusions containing PDZ2 (206), the carboxyl-terminal tail of eye-PKC (562), and PDZ4 (485) was accomplished by in vitrotranscription and translation using the TNT-coupled reticulocyte lysate system (Promega) (6Shieh B.-H. Zhu M.-Y. Neuron. 1996; 16: 991-998Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar). Briefly, 25 μl of reticulocyte lysates were added for a 50-μl reaction that contained [35S]methionine (40 μCi), circular plasmid templates (0.2–1 μg), RNasin (40 units), and T7 RNA polymerase (1 μl).In vitro translated radiolabeled proteins were analyzed by SDS/PAGE or used directly for pull-down assays. Bacterial lysates containing similar amounts (5 μg) of GST fusion proteins or GST were incubated with radiolabeled target proteins (10–20-fold excess) in binding buffer at 4 °C for 1 h. The reaction mixture (55 μl) was transferred to an Eppendorf tube containing10 μl of glutathione-Sepharose beads (Amersham Pharmacia Biotech) prewashed with binding buffer. Incubation proceeded for 1 h at 4 °C with constant agitation for binding of GST fusion proteins to the beads. The supernatant of the mixture was removed, and an aliquot (5%) was analyzed by SDS/PAGE. The beads were rinsed with binding buffer (100 μl) three times to remove nonspecific binding. The GST fusion protein with bound radioactive proteins was eluted with SDS/PAGE loading buffer and analyzed on SDS/PAGE. Radioactivity was detected by autoradiography or with a PhosphorImager (445SI, Molecular Dynamics). Affinity of interaction was determined by the amount of bound radioactive probes. Nonspecific binding to GST was used as a negative control. Western blotting was performed using alkaline phosphatase-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories). The presence of antigens was visualized upon staining with 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium. Polyclonal anti-GST antibodies were purchased from Transduction Laboratories (San Diego, CA). Previously our laboratory reported that PDZ2 interacted with the carboxyl-terminal sequence of eye-PKC (11Adamski F.M. Zhu M.-Y. Bahiraei F. Shieh B.-H. J. Biol. Chem. 1998; 273: 17713-17719Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). However, Tsunoda et al. (9Tsunoda S. Sierralta J. Sun Y. Bodner R. Suzuki E. Becker A. Socolich M. Zuker C.S. Nature. 1997; 388: 243-249Crossref PubMed Scopus (554) Google Scholar) showed that retinal eye-PKC associated with PDZ4 by pull-down assays. Xuet al. (16Xu X.-Z.S. Choudhury A. Li X. Montell C. J. Cell Biol. 1998; 142: 545-555Crossref PubMed Scopus (199) Google Scholar) suggested the involvement of PDZ3 and PDZ4 in the eye-PKC association by heterologous expression and co-immunoprecipitation. Questions remain as to which PDZ domains are important for the eye-PKC association. Importantly, both Xu et al. (16Xu X.-Z.S. Choudhury A. Li X. Montell C. J. Cell Biol. 1998; 142: 545-555Crossref PubMed Scopus (199) Google Scholar) and Adamski et al. (11Adamski F.M. Zhu M.-Y. Bahiraei F. Shieh B.-H. J. Biol. Chem. 1998; 273: 17713-17719Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar) pointed out the involvement of the carboxyl tail of eye-PKC; a point mutation that converted the last residue of eye-PKC, Ile, to an Asp, led to a drastic reduction of the INAD interaction. To investigate which PDZ domain of INAD associates with eye-PKC, we examined the carboxyl-terminal tail of eye-PKC. Eye-PKC terminates with Ile-Thr-Ile-Ile, consistent with an interaction with a type I PDZ domain. The molecular basis of the interaction between a type I PDZ domain and a carboxyl-terminal sequence has been revealed in the x-ray crystallographic studies of PDZ3 of PSD95 and a target peptide (17Doyle D.A. Lee A. Lewis J. Kim E. Sheng M. MacKinnon R. Cell. 1996; 85: 1067-1076Abstract Full Text Full Text PDF PubMed Scopus (976) Google Scholar). We aligned PDZ3 of PSD95 with five PDZ domains of INAD and found that only PDZ2 resembles a type I domain (Fig.1 A) because several critical residues involved in target binding are conserved. In particular, the His residue located at the beginning of the second α-helix (αB) is conserved in PDZ2 of INAD, His310. The N-3 nitrogen of His310 is likely to be involved in hydrogen bonding with the hydroxyl side chain of Ser/Thr at the −2 position of the target (Fig. 1 B). The presence of (Ser/Thr)−2 is a hallmark of the type I ligand. Consistently, eye-PKC contains Thr at the −2 position for an interaction with PDZ2; none of the other PDZ domains of INAD contain a His at the corresponding position. Another conserved residue in PDZ2 includes Arg254 implicated in binding to the terminal carboxyl group of the target (Fig. 1). By analogy, the hydrophobic pocket for binding to the terminal hydrophobic residue of eye-PKC, Ile0, is contributed by Leu260, Leu262, Leu264, and Phe317 from PDZ2 (Fig. 1 B). Overall, PDZ2 of INAD and PDZ3 of PSD95 share 28% sequence identity. It is noteworthy that PDZ3 of PSD95 associates with a target sequence, Gln-Thr-Ser-Val. The polar side chain of Gln at the −3 position, specifically the free carbonyl group, forms hydrogen bonds with the polar side chains of Asn326 and Ser339 of PDZ3 (17Doyle D.A. Lee A. Lewis J. Kim E. Sheng M. MacKinnon R. Cell. 1996; 85: 1067-1076Abstract Full Text Full Text PDF PubMed Scopus (976) Google Scholar). In contrast, PDZ2 of INAD recognizes eye-PKC containing a hydrophobic residue, Ile, at the −3 position. The side chain of Ile does not participate in hydrogen bonding. Consistently, the corresponding residues in PDZ2 for bonding with Ile−3 are Ala263 and Ala278 (Fig. 1), whose side chains also do not form hydrogen bonds. The divergence of these two residues predicted to interact with Ile−3 of eye-PKC further lends support for the role of PDZ2 in interacting with the carboxyl terminus of eye-PKC. To investigate whether Ile−3 of eye-PKC is involved in the PDZ2 interaction, we generated and characterized two point mutants. The codon of Ile−3 was replaced with that of Glu (Ile−3 → Glu) or Lys (Ile−3 → Lys) by site-directed mutagenesis. Wild-type and modified eye-PKC cDNAs were used to generate radiolabeled fusion protein for PDZ2 binding. As shown in Fig. 2, we detected a drastic reduction of the PDZ2 interaction in these two mutants. In particular, the Glu substitution (Ile−3 → Glu) displayed a total loss of the association (Fig. 2 A, lane 4, and Fig. 2 B). These findings indicate that Ile−3 of eye-PKC is critically involved in the PDZ2 interaction; substitutions with charged residues almost abolished the association. As mentioned above, type I PDZ domains usually contain a basic residue, either His or Arg, at the beginning of αB (Fig. 1 A) for binding to (Ser/Thr)−2 of type I targets. In contrast, type II targets contain either a bulky hydrophobic or Tyr at the −2 position that interacts with a hydrophobic residue (e.g. Val) in the corresponding αB position of type II PDZ domains. We examined the involvement of His310 in αB of PDZ2 by amino acid replacement followed by pull-down assays. When His was substituted by Arg (H310R), the interaction with wild-type eye-PKC was reduced to about 50% (Fig. 3). Interestingly, the Leu substitution (H310L) led to a 1-fold increase in the eye-PKC interaction. We also tested the eye-PKC binding to PDZ4 of INAD and showed a much weaker binding compared with that of PDZ2 (Fig. 3). Both His and Arg have side chains capable of hydrogen bonding with (Ser/Thr)−2; however, Arg has an aliphatic side chain instead of an imidazole ring like His. It is likely that differences in the side chains at the corresponding αB position lead to a change in the eye-PKC interaction. When His310 was substituted by Leu whose side chain does not support hydrogen bonding, an increased affinity toward eye-PKC was observed. We proposed that polar side chains from a neighboring residue of His310, such as Arg308, can engage in hydrogen bonding with the hydroxyl group of Thr−2 of eye-PKC to stabilize the interaction in the H310L mutant. To investigate whether Arg308 is important for the observed eye-PKC association, we substituted Arg308 with Gly in the H310L background (R308G,H310L). We show that double mutants displayed a great reduction of the eye-PKC association (Fig. 3 B), suggesting that Arg308 is essential for the eye-PKC interaction in the H310L mutant. We identified theInaD gene and investigated its genomic organization. Genomic clones were obtained either by screening a Drosophilagenomic library or by gene amplification via PCR (Fig.4 A). The InaD gene was subjected to restriction enzyme mapping (Fig. 4 A) and nucleotide sequencing. Comparison of the genomic DNA (accession numberAF245280) with cDNA sequences provided the basis for the exon/intron organization as shown in Fig. 4 A (middle panel). The coding sequence of InaD is contained within a 3-kilobase genomic fragment that has nine exons interrupted by eight intervening sequences. All introns are rather small in size ranging from 54 to 368 nucleotides and are flanked by conserved 5′ and 3′ consensus sequences (28Padgett R.A. Grabowski P.J. Konarska M.M. Seiler S. Sharp P.A. Annu. Rev. Biochem. 1986; 55: 1119-1150Crossref PubMed Google Scholar) at each end. The InaD gene product is composed of five distinct PDZ domains (2Saras J. Heldin C.-H. Trends Biochem. Sci. 1996; 21: 455-458Abstract Full Text PDF PubMed Scopus (224) Google Scholar, 3Fanning A.S. Anderson J.M. Curr. Biol. 1996; 11: 1385-1388Abstract Full Text Full Text PDF Scopus (243) Google Scholar, 9Tsunoda S. Sierralta J. Sun Y. Bodner R. Suzuki E. Becker A. Socolich M. Zuker C.S. Nature. 1997; 388: 243-249Crossref PubMed Scopus (554) Google Scholar, 11Adamski F.M. Zhu M.-Y. Bahiraei F. Shieh B.-H. J. Biol. Chem. 1998; 273: 17713-17719Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). To reveal whether each PDZ domain is encoded within an exon, we projected the location of introns in the translation product of InaD (Fig.4 A, bottom panel). We found both PDZ1 and PDZ2 are encoded by only one exon, whereas the remaining three PDZ domains are encoded by two adjacent exons (Fig. 4 A). To determine the transcription start site of the InaD gene, we employed 5′ RACE (Fig. 4 B) followed by DNA sequencing. Similarly, the 3′-untranslated region (UTR) was also determined (Fig.4 B). The InaD cDNA has 1" @default.
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• W2015033845 title "The Second PDZ Domain of INAD Is a Type I Domain Involved in Binding to Eye Protein Kinase C" @default.
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