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• W2000337551 abstract "The ataxic mutant mouse stargazer is a null mutant for stargazin, a protein involved in the regulation of cell surface trafficking and synaptic targeting of AMPA receptors. The extreme C terminus of stargazin (sequence, –TTPV), confers high affinity for PDZ domain-containing proteins e.g. PSD-95. Interaction with PDZ proteins enables stargazin to fulfill its role as an AMPA receptor synaptic targeting molecule but is not essential for its ability to influence AMPA receptor trafficking to the neuronal cell surface. Using the yeast-two hybrid approach we screened for proteins that interact with the intracellular C-terminal tail of stargazin. Positive interactors included PDZ domain-containing proteins e.g. SAP97, SAP102, and PIST. Interestingly, light chain 2 of microtubule-associated protein 1 (LC2), which does not contain a PDZ domain, was also a strong interactor. This was shown to be a direct interaction that occurred upstream of the -TTPV sequence of stargazin. Immunoprecipitations of Triton X-100 soluble cerebellar extracts revealed that LC2 is pulled down not only by anti-stargazin antibodies but also anti-GluR2 antibodies suggesting that stargazin and AMPA receptor subunits associate with LC2. Immunopurified full-length, native stargazin was shown to co-associate not only with GluR2 in vivo but also with full-length, native LC2. Indeed, LC2 co-associates with stargazin when part of a tripartite complex comprising LC2-stargazin-GluR2. Since this complex was extracted using Triton X-100 and was devoid of PSD95, SAP97, and actin we postulate that LC2 is involved in trafficking of AMPA receptors in cerebellar neurons before they are anchored at the synapse. The ataxic mutant mouse stargazer is a null mutant for stargazin, a protein involved in the regulation of cell surface trafficking and synaptic targeting of AMPA receptors. The extreme C terminus of stargazin (sequence, –TTPV), confers high affinity for PDZ domain-containing proteins e.g. PSD-95. Interaction with PDZ proteins enables stargazin to fulfill its role as an AMPA receptor synaptic targeting molecule but is not essential for its ability to influence AMPA receptor trafficking to the neuronal cell surface. Using the yeast-two hybrid approach we screened for proteins that interact with the intracellular C-terminal tail of stargazin. Positive interactors included PDZ domain-containing proteins e.g. SAP97, SAP102, and PIST. Interestingly, light chain 2 of microtubule-associated protein 1 (LC2), which does not contain a PDZ domain, was also a strong interactor. This was shown to be a direct interaction that occurred upstream of the -TTPV sequence of stargazin. Immunoprecipitations of Triton X-100 soluble cerebellar extracts revealed that LC2 is pulled down not only by anti-stargazin antibodies but also anti-GluR2 antibodies suggesting that stargazin and AMPA receptor subunits associate with LC2. Immunopurified full-length, native stargazin was shown to co-associate not only with GluR2 in vivo but also with full-length, native LC2. Indeed, LC2 co-associates with stargazin when part of a tripartite complex comprising LC2-stargazin-GluR2. Since this complex was extracted using Triton X-100 and was devoid of PSD95, SAP97, and actin we postulate that LC2 is involved in trafficking of AMPA receptors in cerebellar neurons before they are anchored at the synapse. The ataxic and epileptic mutant mouse, stargazer (stg), arose spontaneously as a consequence of a viral insertion of a 6-kb early transposon in intron 2 of the stargazin gene (1Noebels J.L. Qiao X. Bronson R.T. Spencer C. Davisson M.T. Epilepsy Res. 1990; 7: 129-135Google Scholar, 2Letts V.A. Felix R. Biddlecome G.H. Arikkath J. Mahaffey C.L. Valenzuela A. Bartlett II F.S. Mori Y. Campbell K.P. Frankel W.N. Nat. Genet. 1998; 19: 340-347Google Scholar). The mutation results in premature transcriptional arrest and complete ablation of stargazin expression (3Ives J.H. Drewery D.L. Tiwari P. Thompson C.L. Eur. J. Neurosci. 2000; 12: p45Google Scholar, 4Sharp III, A.H. Black J.L. Dubel S.J. Sundarraj S. Shen J.-P. Yunker A.M.R. Copeland T.D. McEnery M.W. Neuroscience. 2001; 105: 599-617Google Scholar). From P14 onwards stg display phenotypic consequences of the mutation that includes head tossing due to an inner ear defect (2Letts V.A. Felix R. Biddlecome G.H. Arikkath J. Mahaffey C.L. Valenzuela A. Bartlett II F.S. Mori Y. Campbell K.P. Frankel W.N. Nat. Genet. 1998; 19: 340-347Google Scholar), ataxia and impaired conditioned eyeblink reflex, both a consequence of cerebellar defects (5Qiao X. Chen L. Gao H. Bao S. Hefti F. Thompson R.F. Knussel B. J. Neurosci. 1998; 18: 6990-6999Google Scholar) and absence epilepsy (6Di Pasquale E. Keegan K.D. Noebels J.L. J. Neurophysiol. 1997; 77: 621-631Google Scholar). The molecular basis for these disparate defects has still to be unequivocally resolved but ultimately these must be direct or downstream consequences of ablated expression of stargazin. Based on low sequence homology to the skeletal muscle-specific L-type voltage-gated calcium channel (VGCC) 1The abbreviations used are: VGCC, voltage-gated calcium channel; TM, transmembrane; TARP, transmembrane AMPA receptor regulatory protein; MAP, microtubule-associated protein; GABAR, γ-aminobutyric acid Type AA receptors; AMPA, α-amino-3-hydroxy-5-methyl4-isoxazole propionic acid. 1The abbreviations used are: VGCC, voltage-gated calcium channel; TM, transmembrane; TARP, transmembrane AMPA receptor regulatory protein; MAP, microtubule-associated protein; GABAR, γ-aminobutyric acid Type AA receptors; AMPA, α-amino-3-hydroxy-5-methyl4-isoxazole propionic acid. γ1 subunit, stargazin was proposed to be a brain-specific γ isoform, and in this context was named CACNγ2 (2Letts V.A. Felix R. Biddlecome G.H. Arikkath J. Mahaffey C.L. Valenzuela A. Bartlett II F.S. Mori Y. Campbell K.P. Frankel W.N. Nat. Genet. 1998; 19: 340-347Google Scholar). Heterologous co-expression studies showed that stargazin had relatively minor effects on P/Q-, L- and α1L T-type VGCC kinetics and cell surface trafficking (2Letts V.A. Felix R. Biddlecome G.H. Arikkath J. Mahaffey C.L. Valenzuela A. Bartlett II F.S. Mori Y. Campbell K.P. Frankel W.N. Nat. Genet. 1998; 19: 340-347Google Scholar, 7Kang M.-G. Chen C.-C. Felix R. Letts V.A. Frankel W.N. Mori Y. Campbell K.P. J. Biol. Chem. 2001; 276: 32917-32924Google Scholar, 8Klugbauer N. Dai S. Specht V. Lacinova L. Marais E. Bohn G. Hofmann F. FEBS Lett. 2000; 470: 189-197Google Scholar, 9Rousset M. Cens T. Restituito S. Barrere C. Black III J.L. McEnery M.W. Charnet P. J. Physiol. 2001; 532.3: 583-593Google Scholar, 10Green P.J. Warre R. Hayes P.D. McNaughton N.C. Medhurst A.D. Pangalos M. Duckworth D.M. Randall A.D. J. Physiol. 2001; 533: 467-478Google Scholar). It has recently been shown that the N-type VGCC α1B subunit co-precipitates with immunoprecipitated stargazin from detergent soluble mouse brain (4Sharp III, A.H. Black J.L. Dubel S.J. Sundarraj S. Shen J.-P. Yunker A.M.R. Copeland T.D. McEnery M.W. Neuroscience. 2001; 105: 599-617Google Scholar). Cerebellar GABAA receptor expression is also severely compromised in stargazer mice (11Thompson C.L. Jalilian Tehrani M.H. Barnes Jr., E.M. Stephenson F.A. Mol. Brain Res. 1998; 60: 282-290Google Scholar, 12Chen L. Bao S. Qiao X. Thompson R.F. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12132-12137Google Scholar). The apparent fickle nature of stargazin associations was compounded when it was shown that it also played an intimate role in the trafficking and synaptic targeting of AMPA receptors to cerebellar granule cell synapses through a VGCC-activity-independent mechanism (13Chen L. Chetkovich D.M. Petralia R.S. Sweeney N.T. Kawasaki Y. Wenthold R.J. Bredt D.S. Nicoll R.A. Nature. 2000; 408: 936-943Google Scholar). Heterologous expression studies revealed that stargazin co-associates with AMPAR GluR1, -2, and -4 (but not NMDA receptor subunit, NR1) and PDZ domains of postsynaptic density proteins, e.g. PSD-95 (13Chen L. Chetkovich D.M. Petralia R.S. Sweeney N.T. Kawasaki Y. Wenthold R.J. Bredt D.S. Nicoll R.A. Nature. 2000; 408: 936-943Google Scholar, 14Schnell E. Sizemore M. Karimzadegan S. Chen L. Bredt D.S. Nicoll R.A. Proc. Natl. Acad. Sci. 2002; 99: 13902-13907Google Scholar). Sharp et al. (4Sharp III, A.H. Black J.L. Dubel S.J. Sundarraj S. Shen J.-P. Yunker A.M.R. Copeland T.D. McEnery M.W. Neuroscience. 2001; 105: 599-617Google Scholar) have since provided evidence that stargazin complexes with GluR1 in vivo, this has been confirmed and expanded upon by Tomita et al. (15Tomita S. Chen L. Kawasaki Y. Petralia R.S. Wenthold R.J. Nicoll R.A. Bredt D.S. J. Cell Biol. 2003; 161: 805-816Google Scholar) to include GluR2 and GluR4. The stargazin ability to influence recruitment of AMPA receptors to the mossy fiber-cerebellar granule cell synapse is, at least in part, dictated by its C-terminal RRTTPV sequence that confers interaction with PDZ domain-containing proteins such as PSD-95. The extent to which AMPA receptor subunits are targeted/clustered at synapses in a stargazin-mediated manner appears to be limited by the number of available PSD-95 molecules with which stargazin-AMPAR complexes can interact while the availability of stargazin seems to dictate the extent of surface-trafficked AMPA receptors (14Schnell E. Sizemore M. Karimzadegan S. Chen L. Bredt D.S. Nicoll R.A. Proc. Natl. Acad. Sci. 2002; 99: 13902-13907Google Scholar). It is not yet known, however how stargazin regulates AMPA receptor trafficking, nor whether other stargazin-protein interactions are required for synaptic targeting or synaptic stability of receptors already docked at the synapse. The work described herein shows that stargazin interacts with light chain 2 (LC2) of microtubule-associated protein 1A (MAP1A) in vivo and that this complex includes GluR2 but not PSD-95, SAP97, nor actin. LC2 is synthesized from MAP1A mRNA, which is translated to give a precursor protein that is subsequently cleaved into the N-terminal heavy chain (∼270 kDa) and the C-terminal LC2 (∼30 kDa, Ref. 16Langkopf A. Hammarback J.A. Muller R. Vallee R.B. Garner C.C. J. Biol. Chem. 1992; 267: 16561-16566Google Scholar). The light and heavy chains can associate non-covalently, though it is now considered that the light chain is the biochemically active entity responsible for regulating neuronal differentiation and microtubule dynamics. The heavy chain acts as a light chain regulatory subunit (17Tögel M. Wiche G. Propst F. J. Cell Biol. 1998; 143: 695-707Google Scholar, 18Mei X. Sweatt A.J. Hammarback J.A. Brain Res. Bulletin. 2000; 53: 801-806Google Scholar). Thus, we propose that stargazin is involved in a tripartite complex with LC2 and AMPA receptors after exit from the endoplasmic reticulum but prior to, or following incorporation into a synapse. Animals—Wild-type (C3B6Fe+; +/+), heterozygous (C3B6Fe+; +/stg) and homozygous stargazer mutant mice (C3B6Fe+; stg/stg) were obtained from heterozygous breeding pairs originally obtained from The Jackson Laboratory (Bar Harbor, ME) and maintained in the University of Durham vivarium on a 12 h light/dark cycle with food and water available ad libitum. Animal husbandry, breeding, and procedures performed during these experiments were conducted according to the Scientific Procedures Act 1986. We, in accordance with others (19Qiao X. Hefti F. Knussel B. Noebels J.L. J. Neurosci. 1998; 16: 640-648Google Scholar, 20Hashimoto K. Fukaya M. Qiao X. Sakimura K. Watanabe M. Kano M. J. Neurosci. 1999; 19: 6027-6036Google Scholar), have found no differences between wild-type (+/+) and heterozygous (+/stg) mice in terms of their phenotype, behavior or any of the molecular entities we have studied. We routinely use, therefore, a mixture of +/+ and +/stg mice brains in our control experiments. New-Zealand White (NZW) rabbits used for polyclonal antibody production were obtained from B & K Universal Ltd. (Hull). Yeast Two-hybrid Screen—The C-terminal stargazin bait representing amino acid residues 202–323 was constructed by PCR amplification and cloned into pGBKT7 (Clontech). DNA was sequenced from clones to confirm the correct orientation and sequence accuracy. A T7 transcription reaction followed by translation in a rabbit reticulocyte system (TnT, Promega) was performed from the pGBKT7 clone, which has a c-Myc tag inserted upstream of the bait. The expected size protein product was detected by immunoblotting, performed as previously described by Thompson and Stephenson (21Thompson C.L. Stephenson F.A. J. Neurochem. 1994; 62: 2037-2044Google Scholar). The bait plasmid was introduced into yeast strain AH109 by the lithium acetate-mediated method, a protocol provided by Clontech, which was adapted from Gietz et al. (22Gietz D. St Jean A. Woods R.A. Schiestl R.H. Nucleic Acids Res. 1992; 20: 1425Google Scholar). The bait was used to screen an adult mouse brain MATCHMAKER cDNA library (Clontech), constructed in pACT2 and pre-transformed into yeast strain Y187, in a mating reaction, which was plated onto S.D. agar deficient in leucine, tryptophan, and histidine (a low stringency screen). Colonies grew in 3–6 days and were picked and re-screened on high stringency media (S.D. –leucine, tryptophan, histidine, adenine) with the addition of X-α-gal, a chromogenic substrate used to detect α-galactosidase, which is the secreted reporter expressed from the MEL1 gene. Blue colonies from this screen were re-screened on high stringency media to confirm the phenotype. Clone Analysis—DNA was extracted from cultures of each clone by standard methods and then PCR-amplified with pACT2-specific primers (AD-LD Insert Screening Amplimer Set, Clontech). Identical clones were identified by restriction enzyme digest analysis, and hybridization of probes to DNA dot blots (standard methods). To identify PDZ proteins a 2125 bp PSD-95 hybridization probe (encompassing all three PDZ domains the SH3 domain and the guanylate kinase-like domain) was generated by reverse transcriptase-PCR using the primers F (5′-TGTCTCTGTATAGTGACAACCA-3′) and R (5′-AGGTCTTCGATGACACGTTTCA-3′) and applied to DNA dot blots. Identities of clones were established by direct sequencing. Yeast mating assays were performed using constructs and plasmids supplied with the Matchmaker 2 kit (Clontech, methods according to the manufacturer's instructions) to eliminate false positive interacting clones. Antibodies—Mouse monoclonal anti-c-Myc antibody (Clontech) was used at 2 μg/ml. Goat anti-LC2 and anti-GluR2 (Santa Cruz Biotechnology) antibodies were employed at 0.2 μg/ml. Anti-stargazin extreme C terminus-targeted polyclonal antibodies were raised against customsynthesized peptide Cys-DSLHANTANRRTTPV (Stg 309–323; Immune Systems Ltd., Paignton, UK) coupled to thyroglobulin in NZW rabbits. A second anti-stargazin specific antibody was raised to a custom-synthesized peptide sequence Cys-RATDYLQASAITRIPS (Stg 213–228; Immune Systems Ltd.) corresponding to the sequence found in the intracellular C-terminal tail but more proximal to the fourth putative transmembrane domain (TM4). This antibody is subsequently referred to as the TM4 proximal C terminus anti-stargazin antibody. Antibodies were purified on peptide-coupled affinity columns by standard methods (23Ives J.H. Drewery D.L. Thompson C.L. J. Neurochem. 2002; 80: 317-327Google Scholar) and used at 1–4 μg/ml. Cloning and Expression of Protein Constructs—LC2 residues 2554–2774, stargazin residues 202–323 (stargazin-full), and stargazin residues 202–317 (stargazin-truncated) were cloned into pENTR/D then recombined into pDEST17 (Invitrogen), which has an N-terminal His6 tag. Constructs were sequenced to confirm their open reading frames, transformed, and then expressed in Escherichia coli strain BL21-AI. Proteins were purified on Ni-nitrilotriacetic acid columns (Invitrogen), and competent expression was verified by immunoblot analysis. Gel Overlay Analysis—Essentially as Wu et al. (24Wu L. Davies S.L. North P.S. Goulaouic H. J. Biol. Chem. 2000; 275: 9636-9644Google Scholar) with the following modifications: Proteins (1 μg) were subjected to SDS-PAGE and transferred to nitrocellulose filters. Filters were then initially immersed in 6 m guanidine-HCl for 10 min at 4 °C followed by six sequential 10 min, 4 °C incubations in 1:1 serially diluted guanidine-HCl (final incubation in 0.09375 m). Filters were blocked in Tris-buffered saline (TBS; 25 mm Tris, 137 mm NaCl, 2.7 mm KCl, pH 7.4) supplemented with 10% (w/v) fat-free skimmed dry milk and 0.3% (v/v) Tween-20 for 30 min. The block was discarded and the filters rinsed twice with incubation buffer (TBS with 0.25% (w/v) milk, 0.3% (v/v) Tween-20, 1 mm dithiothreitol, 1 mm phenylmethylsulfonyl fluoride) before overnight incubation in the overlay protein at 17–30 μg/ml, at 4 °C in incubation buffer. Filters were washed four times, 10 min each in TBS with 0.3% (v/v) Tween-20, 0.25% (w/v) milk with the second wash also containing 0.0001% (v/v) glutaraldehyde. The presence of overlaid proteins on the filter was detected by immunoblotting. SDS-PAGE Gel Silver Staining—Proteins were separated by SDS-PAGE, gels were fixed by 2× immersion for 15 min in fixative (10% (v/v) acetic acid: 40% (v/v) methanol) followed by 30 min in 250 ml of sensitizer (30% methanol, 13 mm sodium thiosulfate, 83 mm sodium acetate). Gels were then washed 3× 5 min in H2O and then exposed to 15 mm silver nitrate for 20 min. Following 2× 1-min washes in H2O, gels were exposed to developer (24 mm sodium carbonate, 0.15% (v/v) formaldehyde in 250 ml H2O) until silver-stained protein bands emerged. Development was terminated by addition of a saturated aqueous solution of EDTA. Immunoaffinity Purification of Stargazin—Adult mouse cerebella from control (+/+ and +/stg) and stargazer mice were rapidly dissected and flash frozen in liquid nitrogen and stored at –70 C until required. Cerebellar membrane homogenates of both sets of tissue were simultaneously prepared according to Kannenberg et al. (25Kannenberg K. Baur R. Sigel E. J. Neurochem. 1997; 68: 1352-1360Google Scholar). Briefly, cerebella were rapidly thawed and homogenized in buffer A comprising 10 mm HEPES, pH 7.1, 100 mm KCl, 2 mm MgCl2, 1 mm EDTA, 2 mm benzamidine, 1 mm phenylmethylsulfonyl fluoride, and protease inhibitors leupeptin (1 μg/ml), aprotinin (2 μg/ml) and pepstatin A (1 μg/ml), all at 4 °C. The homogenates were centrifuged at 45,000 × g, 30 min, 4 °C. The pellets were rehomogenized in buffer A supplemented with 1% (v/v) Triton X-100, stirred for 30 min at 4 °C before ultracentrifugation at 147,000 × g for 75 min, 4 °C. The Triton X-100 soluble material was passed through glass wool before being applied to separate, but identical, anti-stargazin antibody-coupled agarose gels (1.5-ml bed volume, hereafter termed the immunoaffinity columns) in 15-ml conical tubes and incubated with agitation, overnight at 4 °C. The gels were poured into 15-ml Bio-Rad columns. Unbound material was allowed to elute off the column and was collected for analysis (flow-through). The immunoaffinity columns were subsequently washed with 50-column volumes of buffer A supplemented with 1.0% (v/v) Triton X-100. Antibody-bound proteins were eluted from the respective columns by one of the following methods. Acid Elution—Proteins were eluted with acid elution buffer comprising 50 mm glycine pH 2.3, 500 mm KCl, 10% (w/v) sucrose, 1% (v/v) Triton X-100. 1-ml fractions were collected in Eppendorf tubes and immediately neutralized with 1 m Tris. Peptide Elution—To test for the specificity of association of proteins with the immunoaffinity columns and specificity of elution, peptide whose sequence matched part of the extracellular domain of stargazin (CKTKSVSENETSK) and thus not competitive for the stargazin-anti-stargazin antibody binding sequences on the two different anti-stargazin antibody immunoaffinity columns was applied (0.5 mm, 4 ml), with circulation, to the immunoaffinity columns in buffer A supplemented with protease inhibitors, 10% sucrose (w/v), and 1% (v/v) Triton X-100 for 12 h at 4 °C. The column eluate was collected, and the columns subsequently washed with 10 ml of buffer A as above and then the respective peptide, used as antigen to raise the anti-stargazin antibodies coupled to the immunoaffinity columns, was applied as for the nonspecific peptide above. After 12 h of circulation through the column, the eluate was collected. The column was subsequently washed with 10 ml of Buffer A supplemented with sucrose (10%, w/v) and Triton X-100 (1%, v/v) before being subjected to the acid-elution protocol above to elucidate whether all proteins had been displaced from the column and to displace peptide so the columns could be used again. Immunoprecipitations—Stargazin and associated proteins purified by immunoaffinity column chromatography and acid eluted as above was prepared for immunoprecipitation studies by first incubating with the stargazin C-terminal peptide (0.5 mm) to compete out the interaction of stargazin with the anti-stargazin antibody that leached off the immunoaffinity column by the acid elution protocol. This preparation was precleared with EZview Red protein G affinity gel (Sigma) and then dialyzed against IP buffer (10 mm HEPES pH 7.5, 100 mm KCl, 2 mm MgCl2, 1 mm EGTA, 1% (v/v) Triton X-100) plus 10% (w/v) sucrose). In the immunoprecipitation reaction, purified stargazin or Triton X-100 solubilized cerebellar membranes were combined with 10–20 μl of anti-LC2, anti-stargazin or anti-GluR2 antibodies, in 1-ml reactions in IP buffer with protease inhibitors (aprotinin, 2 μg/ml; pepstatin A, 1 μg/ml; leupeptin 1 μg/ml, Sigma) overnight at 4 °C. Samples were incubated with EZview Red protein G affinity gel, pelleted, washed, and the protein complexes eluted with 50 mm glycine pH 2.5. Neutralized samples were prepared for immunoblotting. Protein Determination—Protein concentrations were determined according to the method of Lowry et al. (26Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Google Scholar) employing bovine serum albumin as standard for calibration. Yeast Two-hybrid Screen: the C Terminus Stargazin Bait Interacts with Both PDZ and Non-PDZ Domain-containing Proteins—The fidelity of PCR amplification of our stargazin bait was confirmed by DNA sequencing. We confirmed that we had subcloned the bait sequence in-frame by in vitro translating the construct and analyzing the product by immunoblotting using antibodies recognizing the N terminus c-Myc sequence and the extreme C terminus of stargazin. A unique immunopositive band was identified with both antibodies that had the identical, predicted molecular size (21 kDa). These bands were not detected when empty vector was analyzed in the same way (Fig. 1). The bait sequence is depicted in Fig. 2A.Fig. 2Schematic representations of stargazin and MAPIA proteins. A, schematic representation of the stargazin protein. The position of the four predicted transmembrane domains of stargazin are indicated (I, residues 11–29; II, residues 106–124; III, residues 134–155; IV, residues 180–202: Ref. 2Letts V.A. Felix R. Biddlecome G.H. Arikkath J. Mahaffey C.L. Valenzuela A. Bartlett II F.S. Mori Y. Campbell K.P. Frankel W.N. Nat. Genet. 1998; 19: 340-347Google Scholar). The domain of stargazin that confers its ability to interact with PDZ domain-containing proteins is shown as a shaded box. Below is the region used as the stargazin bait in the yeast two-hybrid screen. Also shown are the two protein expression constructs used in the gel overlay assays. The region of interaction of stargazin is narrowed to residues 202–317. B, schematic of the MAP1A polyprotein. The positions of the self-similarity 1 and 2 domains (SS1, SS2), and the guanylate kinase binding site (GK) are indicated (31Brenman J.E. Topinka J.R. Cooper E.C. McGee A.W. Rosen J. Milroy T. Ralston H.J. Bredt D.S. J. Neurosci. 1998; 18: 8805-8813Google Scholar). Light chain 2 (LC2) spans residues 2465–2774. The shortest LC2-like clone found by yeast two-hybrid is indicated as is the region used in the protein expression construct; from this analysis the region of interaction with stargazin is narrowed to residues 2554–2774.View Large Image Figure ViewerDownload (PPT) To identify stargazin C terminus interacting proteins we screened a mouse brain cDNA library with the C-terminal bait. A total of 5.5 × 106 clones were screened. By low stringency auxotrophic challenge, 600 interacting clones were identified. Following re-screening on high stringency media, the number of positive interacting clones was reduced to 129. Identical clones were identified by restriction enzyme digest analysis and some overlapping clones were identified by dot blot hybridizations (data not shown). Since the extreme C-terminal amino acid sequence of stargazin confers a consensus high affinity binding site for PDZ domain-containing proteins we used a PSD-95 hybridization probe in dot blot screens to identify the presence of PDZ domain containing clones. PDZ domain-containing proteins were indeed identified among the group of strong interactors. Sequence analysis revealed the identity of the clones, and a list of proteins represented by 34 independent clones is shown in Table I. Following yeast mating analyses (see “Experimental Procedures” Clone Analysis), three of these strong positive interacting clones, representing Na+/K+ ATPase β-polypeptide, axonal-associated cell adhesion molecule (BIG-2) and protein phosphatase-3, were eliminated from further studies as these failed this secondary round of screening. Those strong interactors that passed the yeast mating screens comprised the PDZ domain-containing proteins SAP97, SAP102, and PIST. Two proteins that do not contain PDZ domains were also recognized as stargazin interactors. Three clones of microtubule-associated protein 1A light chain 2 (LC2) were identified as strong interactors. A single clone of microtubule-associated protein 1B light chain 1 (LC1) was also identified and also survived the yeast mating secondary screening analyses, however, based on the time this clone took to grow and express α-galactosidase activity we considered this to be a weaker interaction.Table IStargazin C terminus bait interacting proteins detected by yeast two-hybrid assaysProteinNumber of independent clonesOutcome of yeast matingMAP1A-LC23PassedMAP1B-LC11PassedSAP971PassedSAP1021PassedPIST1PassedNa+/K+ ATPase β-polypeptide18FailedAxonal-associated cell adhesion molecule (BIG-2)4FailedProtein phosphatase 31Failed Open table in a new tab Stargazin Interacts with the Light Chains of Microtubule-associated Proteins—Sequence analysis of the three stargazin-interacting LC2 clones and the single LC1 clone revealed that association was through a common conserved sequence of their light chain (LC) regions. The shortest LC2 clone identified from the screen extended from amino acid residue 2550 to 2774 (Fig. 2B), and the only LC1 clone to be pulled out of the library screen extended from residue 2233, equivalent to residue 2570 of LC2 when their sequences are aligned (16Langkopf A. Hammarback J.A. Muller R. Vallee R.B. Garner C.C. J. Biol. Chem. 1992; 267: 16561-16566Google Scholar), to residue 2774. As the LC1 clone was twenty residues shorter than the shortest LC2 domain, we assumed these residues were unlikely to contribute to the interacting domain and therefore felt justified in omitting the 12 base pairs coding for the first four amino acids of LC2, thus making it easier to create the LC construct for in vitro translation. Since the yeast two-hybrid screen detected only one LC1 clone but three LC2 clones as stargazin interactors (Table I) and that the LC1 clone-stargazin bait yeast hybrid grew more slowly on high stringency media than the LC2 clones (data not shown) implying a weaker interaction in the former case, we subsequently used the LC2 sequence for further investigation. The LC2 Domain and the Stargazin Bait Interaction Is Direct, Is Preserved Outside of the Yeast System, and It Is Independent of the PDZ Domain-interacting Sequence of Stargazin—We next investigated whether the stargazin C-terminal domain and LC2 interacted directly with each other, and whether this had some significance outside of the yeast two-hybrid system. The interacting domains were expressed in E. coli, purified, and then analyzed for their ability to interact using the gel overlay approach. Fig. 3A confirms the strict specificity of the anti-stargazin and anti-LC2 antibodies for their intended targets. Our extreme C terminus anti-stargazin antibody recognizes both the full-length and truncated versions of stargazin (Fig. 3A, lanes 2 and 3, respectively) but does not cross-react with the LC2 domain (Fig. 3A, lane 1). Likewise, the anti-LC2 antibody recognizes the LC2 construct (Fig. 3A, lane 4) and does not cross-react with the full-length or truncated version of stargazin devoid of the PDZ-interacting domain (Fig. 3A, lanes 5 and 6). When the in vitro translated stargazin bait protein was used to overlay the LC2 construct the anti-stargazin antibody recognized its substrate at a molecular weight corresponding to that of the LC2 construct, i.e. the stargazin C terminus construct interacted with the LC2 domain (Fig. 3B, lane 1). Likewise, when this was performed in reverse, i.e. the LC2 protein was used to overlay both the full-length and truncated stargazin bait proteins, anti-LC2 antibody immunoreactivity was identified at the mass of the stargazin constructs (Fig 3B, lanes 2 and 3), confirming that the stargazin C terminus protein and the LC2 domain specifically and directly interact with each other outside of the yeast system and do so independently of the PDZ domain" @default.
• W2000337551 created "2016-06-24" @default.
• W2000337551 creator A5014692946 @default.
• W2000337551 creator A5019616733 @default.
• W2000337551 creator A5021253585 @default.
• W2000337551 creator A5037061644 @default.
• W2000337551 creator A5047590016 @default.
• W2000337551 date "2004-07-01" @default.
• W2000337551 modified "2023-09-29" @default.
• W2000337551 title "Microtubule-associated Protein Light Chain 2 Is a Stargazin-AMPA Receptor Complex-interacting Protein in Vivo" @default.
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