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Actinin-Associated LIM Protein: Identification of a Domain Interaction Between PDZ and Spectrin-Like Repeat Motifs Houhui Xia,*‡ Sara T

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Actinin-Associated LIM Protein: Identification of a Domain Interaction Between PDZ and Spectrin-Like Repeat Motifs Houhui Xia,*‡ Sara T Actinin-associated LIM Protein: Identification of a Domain Interaction between PDZ and Spectrin-like Repeat Motifs Houhui Xia,*‡ Sara T. Winokur,i Wen-Lin Kuo,§ Michael R. Altherr,i and David S. Bredt* Departments of *Physiology, ‡Pharmaceutical Chemistry, and §Molecular Cytometry, University of California at San Francisco, San Francisco, California 94143; and iDepartment of Biological Chemistry, University of California at Irvine, Irvine, California 92697 Abstract. PDZ motifs are protein–protein interaction curs at low levels in the heart. ALP is not a component domains that often bind to COOH-terminal peptide se- of the dystrophin complex, but occurs in association quences. The two PDZ proteins characterized in skele- with a-actinin-2 at the Z lines of myofibers. Biochemi- tal muscle, syntrophin and neuronal nitric oxide syn- cal and yeast two-hybrid analyses demonstrate that the thase, occur in the dystrophin complex, suggesting a PDZ domain of ALP binds to the spectrin-like motifs role for PDZ proteins in muscular dystrophy. Here, we of a-actinin-2, defining a new mode for PDZ domain identify actinin-associated LIM protein (ALP), a novel interactions. Fine genetic mapping studies demonstrate protein in skeletal muscle that contains an NH2-termi- that ALP occurs on chromosome 4q35, near the hetero- nal PDZ domain and a COOH-terminal LIM motif. chromatic locus that is mutated in fascioscapulo- ALP is expressed at high levels only in differentiated humeral muscular dystrophy. skeletal muscle, while an alternatively spliced form oc- he cytoskeleton is a complex protein network that elled and CASK, while multiple PDZ domains occur in provides cellular structure. By partitioning the cell, PSD-95, dlg, and zona occludens (ZO)-1 and -2 proteins; T the cytoskeleton can also provide microdomains and PTP-BAS. that allow specific responses to localized stimuli. The as- Recent work indicates that PDZ domains are multifunc- sembly and maintenance of the cytoskeleton is mediated, tional protein–protein interaction motifs (Brenman and in large part, by high affinity interactions between modu- Bredt, 1997; Kornau et al., 1997; Sheng, 1996). One mode lar consensus protein-binding motifs. These sites for pro- for interaction of PDZ domains involves association with tein–protein interaction are often multifunctional, and the the COOH terminus of target proteins. Thus, the COOH specific binding partners are determined by the variations terminus of Fas binds to the third PDZ domain of PTP-BAS, in amino acid sequences between the individual domains. and this interaction participates in Fas-mediated apoptosis A recently identified motif, the PDZ domain, is an 80– of T cells (Sato et al., 1995). Similarly, the first and second 120–amino acid domain that was first identified in the PDZ domains of PSD-95 bind to the COOH termini of postsynaptic protein, PSD-95, which contains three PDZ certain ion channels in the brain, and they anchor these domains in tandem (Cho et al., 1992). Sequence analysis channels to synaptic sites at the plasma membrane (Kim et has subsequently demonstrated that PDZ domains are al., 1995; Kornau et al., 1995). PDZ–PDZ interactions have common protein motifs that occur in a variety of dissimilar also been identified. The PDZ domain of nNOS binds to proteins that interact with the cytoskeleton (Ponting and the second PDZ domain of PSD-95 (Brenman et al., 1996). Phillips, 1995). Individual PDZ domains occur in neuronal These multifunctional interactions of PDZ domains assem- nitric oxide synthase (nNOS),1 syntrophins, p55, dishev- ble a complex of nNOS / PSD-95/the N-methyl-d-aspartate receptor calcium channel at the postsynaptic density. Address all correspondence to David S. Bredt, University of California at Functional roles for PDZ domains have been demon- San Francisco School of Medicine, 513 Parnassus Avenue, San Francisco, strated in diverse tissues. Mutations in the PDZ domain of CA 94143-0444. Tel.: (415) 476-6310; Fax: (415) 476-4929; E-mail: [email protected] Drosophila inactivation no afterpotential D (INAD) alter transduction of visual signals (Shieh and Zhu, 1996), while 1. Abbreviations used in this paper: ALP, actinin-associated LIM protein; mutations in the Caenorhabditis elegans PDZ proteins Lin-2 EST, expressed sequence tag; FISH, fluorescence in situ hybridization; and Lin-7 result in abnormal vulval development (Hoskins FSHD, fascioscapulohumoral muscular dystrophy; GST, glutathione S-transferase; INAD, inactivation no afterpotential D; nNOS, neuronal et al., 1996). In all these cases, the PDZ domains are impli- nitric oxide synthase; ORF, open reading frame; RT-PCR, reverse tran- cated in targeting intracellular proteins to appropriate scription; ZO, zona occludens. multiprotein complexes at the plasma membrane. The Rockefeller University Press, 0021-9525/97/10/507/9 $2.00 The Journal of Cell Biology, Volume 139, Number 2, October 20, 1997 507–515 http://www.jcb.org 507 To understand and further define the role of PDZ do- PCR and subcloned into the GAL-4 DNA–binding domain plasmid mains in cytoskeletal assembly, we have focused on skeletal pGBT9 (Clontech Laboratories, Palo Alto, CA). This construct was cotransformed into yeast strain HF7c with a library of human skeletal muscle as a model system. The regular and defined struc- muscle cDNAs fused to the GAL-4 activation domain (Clontech). The ture of skeletal muscle makes it an ideal tissue for study. transformation mixture was plated onto a synthetic dextrose plate lacking Previous studies demonstrated that the two known PDZ tryptophan, leucine, and histidine. Growth was monitored during a 5-d in- domain proteins in skeletal muscle, the family of sytro- cubation at 308C, and color was measured by a b-galactosidase colorimet- phins and nNOS, are both components of the dystrophin ric filter assay (Fields and Song, 1989). Interacting clones were rescued, retransformed to confirm interaction, and sequenced. Deletions of inter- complex (Adams et al., 1993; Brenman et al., 1995). nNOS acting clone 9-5 were generated by digestion with XcmI (9-5X), NarI (9-5N), isoforms lacking the PDZ domain do not interact with the or BglI (9-5B). Site-directed mutagenesis of ALP L78K was performed by dystrophin complex and occur in the skeletal muscle cyto- PCR and confirmed by sequencing. sol. The PDZ domains of nNOS and syntrophin directly interact with each other, and this linkage anchors nNOS to Antibodies, Immunohistochemistry, and the dystrophin complex (Brenman et al., 1996). The absence Western Blotting of dystrophin in Duchenne muscular dystrophy results in a A glutathione S-transferase (GST) fusion protein encoding amino acids 1–207 loss of syntrophins and nNOS from the sarcolemma, and of ALP was amplified by PCR and subcloned into pGEX2T (Pharmacia these abnormalities may contribute to the disease process. Biotech, Piscataway, NJ). The fusion protein was expressed and purified We hypothesized that other PDZ proteins in muscle from Escherichia coli as described (Brenman et al., 1995). Rabbits were may also occur in the cytoskeleton. Characterization of immunized with the GST–ALP fusion protein emulsified in complete and these proteins will help better understand the function of incomplete Freund’s adjuvant. Serum bleeds were evaluated by ELISA. For affinity purification, ALP antiserum was applied to a column of GST– PDZ domains and may identify candidate genes for inher- ALP fusion protein immobilized on Reacti-Gel (Pierce Chemical Co., ited muscular dystrophies. Here, we report the cloning of a Rockford, IL). The column was washed with 20 vol of buffer containing novel cDNA encoding a protein of 39 kD that consists of 500 mM NaCl and 10 mM Tris, pH 7.5. ALP antibody was eluted with 10 an NH -terminal PDZ domain and a COOH-terminal LIM bed vol of 100 mM glycine, pH 2.5. Monoclonal antibodies to syntrophin 2 (Peters et al., 1994), nNOS (Transduction Laboratories, Lexington, KY), domain. The protein is expressed at high levels only in and a-actinin (Sigma Immunochemicals, St. Louis, MO) were also used. skeletal muscle, where it occurs at the Z lines in associa- For immunofluorescent staining, skeletal muscle samples were flash tion with a-actinin-2. We therefore name this protein acti- frozen in liquid nitrogen–cooled isobutane, sectioned on a cryostat (10 nin-associated LIM protein (ALP). Biochemical and two- mm), and melted directly onto glass slides. Sections were then postfixed in hybrid analyses indicate that the PDZ domain of ALP 2% paraformaldehyde in PBS. Tissues were blocked in PBS containing 1% normal goat serum. mAbs to a-actinin (1:100) and polyclonal ALP an- binds to the spectrin-like repeats of a-actinin-2, establish- tibody were applied to sections overnight at 48C. For indirect immunoflu- ing a novel mode of interaction for PDZ domains. Chro- orescence, secondary goat anti–mouse FITC or donkey anti–rabbit Cy-3–con- mosomal mapping indicates that the ALP gene occurs at jugated antibodies were used according to the specifications of the 4q35 within 7–10 megabase (Mb) of the heterochromatic manufacturer (Jackson ImmunoResearch Laboratories, Bar Harbor, ME). For Western blotting, protein extracts were resolved by SDS-PAGE region that is deleted in fascioscapulohumeral muscular dys- and transferred to polyvinyldifluoride membranes. Primary antibodies trophy (FSHD; Altherr et al., 1995). were diluted in block solution containing 3% BSA, and 0.1% Tween 20 in TBS, and were incubated with membranes overnight at 48C. Labeled bands were visualized using enhanced chemiluminescence (Amersham, Materials and Methods Arlington Heights, IL). mRNA Isolation and cDNA Analysis Myogenic Cell Culture RNA was isolated using the guanidine isothiocyanate/CsCl method, and C2 myogenic cell line was grown on gelatin-coated dishes in Ham’s F-10 mRNA was selected with oligo dT sepharose. For degenerate reverse nutrient mixture plus 0.8 mM extra CaCl2 supplemented with 15% horse transcriptase-PCR (RT-PCR), rat skeletal muscle mRNA was reverse serum (Life Technologies, Inc., Bethesda, MD) and 2.5 ng/ml basic fibro- transcribed with RTth polymerase using random hexamer primers.
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