Liprins, a Family of LAR Transmembrane Protein-tyrosine Phosphatase-interacting Proteins*

Carles Serra-PagèsDagger §, Quintus G. MedleyDagger , May TangDagger , Anne Hartparallel , and Michel StreuliDagger **

From the Dagger  Division of Tumor Immunology, Dana-Farber Cancer Institute, Boston, Massachusetts 02115, the Departments of § Medicine and  Pathology, Harvard Medical School, Boston, Massachusetts 02115, and the parallel  Massachusetts General Hospital Cancer Center, Charlestown, Massachusetts 02129

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

LAR family transmembrane protein-tyrosine phosphatases function in axon guidance and mammary gland development. In cultured cells, LAR binds to the intracellular, coiled coil LAR-interacting protein at discrete ends of focal adhesions, implicating these proteins in the regulation of cell-matrix interactions. We describe seven LAR-interacting protein-like genes in humans and Caenorhabditis elegans that form the liprin gene family. Based on sequence similarities and binding characteristics, liprins are subdivided into alpha -type and beta -type liprins. The C-terminal, non-coiled coil regions of alpha -liprins bind to the membrane-distal phosphatase domains of LAR family members, as well as to the C-terminal, non-coiled coil region of beta -liprins. Both alpha - and beta -liprins homodimerize via their N-terminal, coiled coil regions. Liprins are thus multivalent proteins that potentially form complex structures. Some liprins have broad mRNA tissue distributions, whereas others are predominately expressed in the brain. Co-expression studies indicate that liprin-alpha 2 alters LAR cellular localization and induces LAR clustering. We propose that liprins function to localize LAR family tyrosine phosphatases at specific sites on the plasma membrane, possibly regulating their interaction with the extracellular environment and their association with substrates.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

The highly organized and coordinated response of cells to diverse extracellular stimuli is partially mediated by tyrosine phosphorylation of proteins, some of which relay information from the cell surface to the nucleus and others of which control cytoskeletal organization. The degree of tyrosine phosphorylation of such signaling proteins, including enzymes, adapter proteins, and structural proteins, is regulated by the concerted activities of protein-tyrosine kinases and protein-tyrosine phosphatases (PTPases).1 Both protein-tyrosine kinases and PTPases comprise large gene families encoding transmembrane-type and intracellular-type enzymes (1, 2). The physiological role of many protein-tyrosine kinases, and some PTPases, is well documented (3-6). Recent genetic analysis of LAR-like transmembrane PTPases indicates that members of this subfamily play a role in Drosophila axon guidance (7, 8) and murine mammary gland development and function (9). About half of the embryos with Drosophila LAR (DLAR)-inactivating mutations die as late instar larvae, and the other half die before or at eclosion (7). Examination of the nervous systems of Dlar-/- mutant embryos revealed specific defects in motor axon guidance and, to a lesser degree, in the formation of certain CNS axon pathways. Female mice with a targeted disruption of the Lar gene are incapable of delivering milk due to impaired terminal differentiation of alveoli at late pregnancy (9). Consequently, the glands fail to switch to a lactational state and rapidly involute postpartum. The molecular basis for the axon guidance defect in the Dlar-/- mutants and the impaired development of mammary alveoli in the Lar-/- mutant mice is unknown, but given that axon guidance, as well as mammary epithelial differentiation and lactation, is regulated by soluble factors, cell-cell interactions, and cell-matrix interactions, it is likely that DLAR and LAR function in one or more of these signaling pathways (10, 11).

LAR and DLAR are members of the LAR subfamily of transmembrane PTPases, which consists of the highly related vertebrate LAR (12, 13), PTPdelta (14-16), and PTPsigma (17-20), and Drosophila DLAR (21, 22). These PTPases contain extracellular regions comprised of three N-terminal Ig-like domains and a variable number of fibronectin type III-like domains connected via a transmembrane segment to an intracellular region containing two PTPase domains. The overall architecture of the LAR family extracellular regions is similar to several cell or matrix adhesion molecules, indicating that these PTPases function as receptors for cell surface molecules and/or extracellular matrix molecules (3, 23). Furthermore, human LAR localizes to focal adhesions (FAs) (24), which are sites of cell-extracellular matrix interactions, and to sites of cell-cell contact (25, 26). Because FAs are assembled by a tyrosine phosphorylation-dependent process following integrin ligation (27), LAR may play a role in FA disassembly.

Two proteins were identified that bind the LAR membrane-distal D2 PTPase domain. One of these, Trio, contains a rac1 guanine nucleotide exchange factor domain, a rhoA guanine nucleotide exchange factor domain, a protein kinase domain, and several auxiliary domains (28). Because rac and rho are regulators of actin reorganization and cell growth (29), a LAR/Trio complex may integrate multiple signals and determine the response of cells to diverse extracellular stimuli. The second protein, LAR-interacting protein 1 (LIP.1), is a coiled coil protein that colocalizes with LAR at FAs (24). LIP.1 may form rod-like dimers or higher order structures similar to other proteins that contain coiled coil alpha -helical domains, such as the myosin II heavy chain and intermediate filaments (30). The APC colorectal tumor suppressor gene product also contains coiled coil domains, and APC is believed to mediate the attachment of cadherin/catenin complexes to the cytoskeleton (31). LIP.1 does not appear to be tyrosine-phosphorylated and hence is unlikely to be a PTPase substrate (24). Instead, LIP.1 likely anchors LAR at FAs where LAR may dephosphorylate FA-associated protein(s) to alter FA assembly and/or signaling. Thus, the LAR/LIP.1 complex may represent a matrix/cytoskeletal linkage that augments the actin/integrin and cadherin/catenin linkages by its intrinsic PTPase activity.

Herein we describe the identification and characterization of human and Caenorhabditis elegans LIP.1-related genes, which we have designated the liprin (derived from "LIP-related protein") gene family. Based on sequence homology and binding properties, liprins are divided into alpha -liprins and beta -liprins. alpha -Liprins, including LIP.1 (renamed liprin-alpha 1) bind to the membrane-distal D2 PTPase domains of the LAR family PTPases, LAR, PTPdelta , and PTPsigma , whereas beta -liprins bind to alpha -liprins but not to LAR family PTPases. Furthermore, both alpha - and beta -liprins homodimerize via their N-terminal, coiled coil regions. Whereas some of the liprins have a broad mRNA tissue distribution, others are highly restricted, particularly to brain. Co-expression studies indicate that liprin-alpha 2 alters the cellular distribution of LAR, supporting a role for liprins in localizing LAR family members to specific sites within the cell and creating specific linkages between the extracellular environment and the cytoskeleton.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Interaction Trap Assay-- Plasmid DNAs and yeast strains used for the interaction trap assay were provided by Dr. R. Brent and colleagues (32) and used as described (24, 32). The human fibroblast cell WI-38 (ATCC CCL 75) cDNA library used for the interaction trap assay was kindly provided by Dr. C. Sardet. The various liprin and LAR-like PTPase regions fused to LexA or the B42 transcription activation domain are given in Tables I and II.

cDNA Cloning and Plasmid Constructions-- Liprin cDNA clones were isolated from lambda gt11 human fetal brain, adult brain, heart, and kidney cDNA libraries (CLONTECH, Palo Alto, CA), as well as from the interaction trap fibroblast cDNA library, essentially as described (33). In the human expression sequence tag data base, two sequences were identified (accession nos. H08934 and H11896) that encode peptides with high sequence identity to liprin-alpha 1 (83 and 73% identity over 77-102 amino acids, respectively). The initial ~300-bp liprin-alpha 2 and liprin-alpha 3 cDNAs probes used for library screenings were generated by reverse transcription polymerase chain reaction using kidney poly(A)+ mRNA and oligonucleotides derived from the human expression sequence tag sequences H08934 and H11896, respectively. Liprin cDNAs were sequenced by the dideoxy method. For COS cell transient transfections, liprin cDNAs were cloned into pMT.2 or pMT.HAtag, which encodes a hemagglutinin (HA) epitope tag sequence immediately upstream of the cloning site (24). pMT.Liprin-alpha 1 encodes liprin-alpha 1 amino acids 1-1202 (24); pMT.Liprin-alpha 2 encodes liprin-alpha 2 amino acids 1-1257; pMT.Liprin-beta 1 encodes liprin-beta 1 amino acids 1-1005; pMT.HA-Liprin-alpha 1Delta C (HA-Lalpha 1Delta C) encodes HA-tagged liprin-alpha 1 amino acids 3-670; pMT.HA-Liprin-alpha 2Delta C (HA-Lalpha 2Delta C) encodes HA-tagged liprin-alpha 2 amino acids 3-701; pMT.HA-Liprin-alpha 2Delta N encodes HA-tagged liprin-alpha 2 amino acids 821-1257; pMT.HA-Liprin-beta 1Delta C (HA-Lbeta 1Delta C) encodes HA-tagged liprin-beta 1 amino acids 1-227; pMT.HA-Liprin-beta 1Delta N encodes HA-tagged liprin-beta 1 amino acids 678-1005; pMT.HA-Liprin-beta 2Delta N encodes HA-tagged liprin-beta 2 amino acids 257'-783'; pMT.LAR encodes amino acids 1-1881 (12); pMT.LAR-D1 encodes amino acids 1-1615; and pGST.LAR encodes LAR amino acids 1275-1881 fused to GST (20). The celiprin-alpha and celiprin-beta coding sequences were obtained by others using Genefinder (GenBankTM accession nos. Z50794 and Z78546, respectively). The 5' promoter regions of celiprin-alpha and celiprin-beta used to construct the green fluorescent protein (GFP) reporter plasmids pPD.celiprin-alpha -GFP and pPD.celiprin-beta -GFP were generated by polymerase chain reaction using C. elegans cosmid DNA (clones F59F5 and T21H8, respectively, obtained from the Sanger Centre, Cambridge, United Kingdom) and inserted into the pPD95.67 plasmid, which encodes GFP and the SV40 nuclear localization signal sequence (kindly provided by Dr. Andrew Fire). The 5' promoter region isolated for celiprin-alpha spans bp 29331-24665 of the C. elegans cosmid clone F59F5 (34), with the predicted initiation methionine codon located at bp 24762-24760. The 5' promoter region isolated for celiprin-beta spans bp 11771-6365 of the C. elegans cosmid clone T21H8 (34), with the predicted initiation methionine codon located at bp 8760-8758. Sequence comparisons were done using the Wisconsin Package (version 8) software from Genetics Computer Group (Madison, WI).

The GenBankTM accession numbers for the liprins are as follows: human liprin-alpha 2, AFO34799; human liprin-alpha 3, AFO34800; human liprin-alpha 4, AFO34801; human liprin-beta 1, AFO34802; and human liprin-beta 2, AFO34803. The accession numbers for the two liprin-alpha 1 splice variants, liprin-alpha 1a and liprin-alpha 1b (previously designated LIP.1a and LIP.1b) are U22815 and U22816, respectively. The C. elegans celiprin-alpha and celiprin-beta structures were derived from GenBankTM accession numbers Z50794 (F59F5.6) and Z78546 (T21H8.1), respectively.

Northern Blot Analysis-- Northern blot analysis was done using a human multiple tissue Northern blot (CLONTECH) that contains 2 µg of poly(A)+ selected RNA from different human tissues per lane and was sequentially hybridized with the following random primed [alpha -32P]dCTP labeled liprin cDNA probes: liprin-alpha 1, nucleotides 236-1514 (encoding aa 3-429); liprin-alpha 2, nucleotides 3447-3722 (encoding aa 1093-1184); liprin-alpha 3, nucleotides 877'-1191' (encoding aa 293'-397'); liprin-alpha 4, nucleotides 1'-489' (encoding aa 1'-163'); liprin-beta 1, nucleotides 2274-2739 (encoding aa 679-833); and liprin-beta 2, nucleotides 1978'-2226' (encoding aa 660'-741'); it was also hybridized with a beta -actin cDNA probe.

C. elegans Expression Analysis-- Animals were injected with plasmid pPD.celiprin-alpha -GFP and pPD.celiprin-beta -GFP DNAs at 50 ng/µl using lin-15 as a marker (35, 36). At least two independent extrachromosomal array lines were examined for each construct. GFP was observed in both the cytoplasm and nucleus for both constructs, despite the SV40 nuclear localization signal in the GFP vector. Adobe Photoshop was used to create "negatives" of black and white images originally captured using a Sensys camera and ImagePro Plus software.

Monoclonal Antibodies-- The anti-liprin-alpha 1(LIP.1) mAb anti-LIP.1.77 was described previously (24), as were the anti-LAR mAbs 11.1A, 75.3A, and 128.4A (12). The anti-HA mAbs 11A and 12CA5 were from Berkeley Antibody Co. (Richmond, CA) and the Harvard University mAb facility (Cambridge, MA), respectively. To generate anti-liprin-alpha 2 and anti-liprin-beta 1 Abs, mice were immunized with purified, Escherichia coli-derived GST-Liprin-alpha 2 (amino acids 3-470) and GST-Liprin-beta 1 (amino acids 63-446) fusion proteins (33). Hypoxanthine/aminopterin/thymidine-resistant hybridomas derived from GST-Liprin-beta 1 immunized mice were initially selected using enzyme-linked immunosorbent assay and then by immunoprecipitation studies. The anti-liprin mAb thus obtained was termed anti-liprin-beta 1.68.1 (IgG1). The anti-liprin-alpha 2 polyserum was obtained from mice immunized with the GST-Liprin-alpha 2 fusion protein.

Cell Labeling and Protein Analysis-- Cell proteins were metabolically labeled with [35S]methionine as described previously (24). Following labeling, cells were washed in PBS, lysed in Nonidet P-40 lysis buffer (1% Nonidet P-40, 150 mM NaCl, 50 mM Tris-HCl (pH 8.0), 1 mM EDTA) containing 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 10 mM sodium fluoride. Insoluble material was removed from the lysates by centrifugation in a microcentrifuge. Cell lysates were then precleared once with 25 µl of protein A-Sepharose slurry (Amersham Pharmacia Biotech) for 1-2 h. For immunoprecipitations, ~2 µg of anti-LIP.1.77 mAb (anti-liprin-alpha 1), 3 µl of mouse anti-liprin-alpha 2 serum, 100 µl of anti-liprin-beta 1.68.1 hybridoma supernatant, 2 µg of control isotype-matched mAb, or 1 µl of ascites fluid of anti-HA mAb 11A and 25 µl of protein A-Sepharose slurry were added per ml of precleared lysate for 2 h. Immunoprecipitates were then washed with buffer containing 0.1% Nonidet P-40, 0.05% SDS, 150 mM NaCl, and 50 mM Tris-HCl (pH 8.0). Immunoprecipitated proteins were analyzed using SDS-PAGE with reducing conditions followed by autoradiography (16-40 h).

Cell Transfections-- COS-7 cell transient transfections were done by the DEAE-dextran/Me2SO method using 2 µg of plasmid DNA per 2 × 105 cells per 9-cm2 dish, and cells were harvested ~20 h after transfection (33). Proteins were metabolically labeled with [35S]methionine during the final 4 h prior to harvesting of cells.

Immunofluorescence-- COS-7 cells were plated on glass coverslips 5 h following transfection with pMT.2-based expression plasmids and grown for ~20 h prior to staining. Cells were rinsed in PBS, fixed in 2% paraformaldehyde/PBS for 15 min, and then permeabilized for 10 min in 0.1% Triton X-100/PBS-containing 2% horse serum. Nonspecific antibody binding sites were blocked by a 30-min incubation in blocking buffer (10% normal goat serum in PBS). To detect liprin-alpha 1 and liprin-beta 1, permeabilized cells were exposed to the anti-liprin-alpha 1 mAb anti-LIP.1.77 mAb and the anti-liprin-beta 1.68.1 mAb at a concentration of 2 µg of alpha -LIP.1.77/ml blocking buffer and a 1:3 dilution of anti-liprin-beta 1.68.1 hybridoma supernatant in blocking buffer for 1 h and washed, and the primary antibody was detected with a 30-min treatment of 1:1000 goat anti-mouse IgG2a-Texas red (Southern Biotechnology, Birmingham, AL) and 1:1000 goat anti-mouse IgG1-fluorescein isothiocyanate (Southern Biotechnology). To detect LAR and HA-tagged liprin-alpha 2, permeabilized COS cells were exposed to a 1:1:1 mixture of the anti-LAR mAbs, 75.3A, 11.1A, and 128.4A at 2 µg/ml blocking buffer, and/or the anti-HA mAb 12CA5 at 0.5 µg/ml blocking buffer for 1 h and washed, and the primary antibody detected as above with a 30-min treatment of 1:1000 goat anti-mouse IgG2b-Texas red and 1:1000 goat anti-mouse IgG1-fluorescein isothiocyanate. Slides were mounted in a polyvinyl alcohol medium and viewed on a Olympus BX60 microscope equipped for epifluorescence. Photographs were taken on Kodax Ektachrome film.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

LIP.1 Is the Prototype Member of the Evolutionarily Conserved Liprin Family-- LIP.1 is a coiled coil protein originally isolated by virtue of its binding to the intracellular region of the LAR transmembrane PTPase and was shown to co-localize with LAR at FAs (24). To learn more about LIP.1 function, we performed an interaction trap screen to identify additional LIP.1-binding proteins (32). Using as bait the non-coiled coil, C-terminal region of LIP.1 (aa 794-1202), we isolated two novel proteins we have named liprin-beta 1 (aa 678-1005; Fig. 1A) and liprin-beta 2 (aa 257'-783'; Fig. 1A), in addition to the expected LAR and PTPsigma . Sequence determination of liprin-beta 1 cDNA clones predict that liprin-beta 1 is a 1005-amino acid long protein, the primary sequence of which is similar to LIP.1 (25% sequence identity; Fig. 1A). Liprin-beta 1 was named LIP.1-related protein; beta 1 indicates that liprin-beta 1 is the first member of the beta  subfamily of liprins (see below). Consequently, LIP.1 was renamed liprin-alpha 1, because it is the first member of the liprin-alpha subfamily.


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Fig. 1.   Predicted structures of liprins. A, deduced amino acid sequence of human liprin-beta 1, human liprin-beta 2, and C. elegans celiprin-beta . B, deduced amino acid sequence of human liprin-alpha 2, human liprin-alpha 3, human liprin-alpha 4, and C. elegans celiprin-alpha . Sequences are aligned with the liprin-alpha 1(LIP.1) sequence; residues conserved in three out of the four sequences are shown with a black background. Numbers followed by a prime symbol (') indicate provisional amino acid numbering because the structures are incomplete. Underlined sequences are the LH domain sequences. C, the overall structures of alpha - and beta -liprins, with their N-terminal, coiled coil regions and their C-terminal LH domains are schematically depicted for liprin-alpha 1 and -beta 1. D, phylogenetic tree for the liprins. The tree was obtained using the PIMA-PAUP program of the Genetics Computer Group package using the entire cloned amino acid and the coding nucleic acid sequence of each liprin. Numbers represent relative distances between the indicated liprins.

Characterization of liprin-beta 2 cDNA clones indicated that the partial 783-amino acid liprin-beta 2 sequence is most similar to liprin-beta 1 (51% identity; the liprin-beta 2 sequence is provisionally numbered 1'-783'; Fig. 1A). Similar to liprin-alpha 1, the liprin-beta 1 and liprin-beta 2 N-terminal regions (aa 97-330 and 1'-223', respectively) are predicted to form alpha -helical coiled coils (37). A murine cDNA sequence encoding a 882-amino acid protein is likely to be mouse liprin-beta 2 because it shares 91% sequence identity with human liprin-beta 2 (38). The highest degree of sequence similarity between liprin-alpha 1 and -beta 1 or -beta 2 resides within the C-terminal, non-coiled coil region (25-26% identity, compared with 17-20% identity of the N-terminal, coiled coil regions). The liprin-alpha 1, -beta 1, and -beta 2 C-terminal, non-coiled coil regions are unrelated to any other proteins (with the exception of liprin family members; see below), whereas the N-terminal, coiled coil regions of liprin-alpha 1, -beta 1, and -beta 2 are weakly similar to coiled coil regions of other proteins, such as the myosin heavy chain (18% identity). Thus, the screening for liprin-alpha 1(LIP.1)-binding proteins identified two novel proteins, liprin-beta 1 and liprin-beta 2, that are similar to each other, as well as to liprin-alpha 1 itself.

A search of the GenBankTM expression sequence tag data base for liprin-related sequences revealed the existence of two additional mammalian liprin-like genes. One of these genes, termed liprin-alpha 2, was isolated in its entirety and encodes a 1257-amino acid protein (Fig. 1B). Liprin-alpha 2 has 72% sequence identity with liprin-alpha 1 and 26% with liprin-beta 1. Screening of cDNA libraries yielded clones that encode only a partial 474-amino acid liprin-alpha 3 structure, which has 72% identity with liprin-alpha 1 and 29% identity with liprin-beta 1 (Fig. 1B). During colony hybridization screening for liprin cDNAs, a fourth human alpha -liprin gene was isolated. The partial liprin-alpha 4 cDNA clones encode a 500-amino acid peptide possessing 75% identity with liprin-alpha 1 (Fig. 1B). Like liprin-alpha 3 and -beta 2, the -alpha 4 sequence is likely to be missing N-terminal sequences, and in the case of liprin-alpha 4, C-terminal sequences are also absent (Fig. 1B). Thus, the amino acid numbering of liprin-alpha 3, -alpha 4, and -beta 2 is provisional. A search of a C. elegans data base revealed the existence of two liprin-like genes that were designated celiprin-alpha (for C. elegans liprin) and celiprin-beta , encoding putative proteins of 1139 amino acids (Fig. 1B) and 1036 amino acids (Fig. 1A), respectively. Celiprin-alpha has 50% identity with liprin-alpha 1 and 22% identity with liprin-beta 1, whereas celiprin-beta has 29% identity with liprin-beta 1 and 22% identity with liprin-alpha 1. The N-terminal regions of liprin-alpha 2, celiprin-alpha , and celiprin-beta are predicted to form coiled coils (aa 29-696, 28-702, and 211-411, respectively) (37).

Based on sequence comparisons, the six mammalian liprins and two C. elegans liprins are subdivided into the alpha -liprin subfamily, which contains liprin-alpha 1, -alpha 2, -alpha 3, -alpha 4, and the C. elegans celiprin-alpha ; and the beta -liprin family, which contains liprin-beta 1, -beta 2, and the C. elegans celiprin-beta (see Fig. 1D). Overall, the human liprin-alpha subfamily members are ~69-76% identical to each other, ~52% identical to celiprin-alpha , ~28% to either liprin-beta 1 or liprin-beta 2, and ~22% identical to celiprin-beta . Liprin-beta 1 and liprin-beta 2 are 51% identical to each other and ~30% identical to celiprin-beta . These findings demonstrate the existence of a family of structurally related proteins that contains at least six mammalian members and two C. elegans members.

A notable feature of liprins is the high degree of sequence conservation within a ~250-amino acid domain located within the C-terminal, non-coiled coil region (underlined sequences in Fig. 1, A and B). Within this domain, which we designate the liprin homology (LH) domain, 34 amino acids are absolutely conserved in all six human liprins and the two C. elegans liprins, and 95 amino acids are conserved in at least six out of the eight members. The extensive sequence conservation of LH domains suggests that these domains are functionally important. One possibility is that LH domains are protein-protein interaction domains, because the LH domain in liprin-alpha 1 binds to LAR (24) and to liprin-beta 1 (see below, and data not shown). Thus, LH domains may be novel protein-protein interaction domains. The overall structures of alpha - and beta -liprins, with their N-terminal, coiled coil regions and their C-terminal LH domains are schematically depicted for liprin-alpha 1 and liprin-beta 1 in Fig. 1C.

The relatively high degree of sequence conservation between the mammalian alpha -liprins and C. elegans celiprin-alpha (50-58% identity), as well as the high degree of conservation between the mammalian beta -liprins and C. elegans liprin-beta (29-31% identity), indicates that liprins are evolutionarily conserved. Based on the predicted phylogenetic tree (Fig. 1D), it is likely that alpha - and beta -liprins were derived from an ancestral gene by gene duplication prior to the divergence of chordates and nematodes.

Tissue-specific Expression of Human Liprins-- The human LAR, PTPdelta , and PTPsigma PTPases have distinct, but overlapping, mRNA tissue distributions (20). For instance, all three mRNAs are expressed in brain, whereas only LAR and PTPsigma mRNA are expressed in kidney and pancreas. To determine the tissue distribution of alpha - and beta -liprin mRNAs, Northern blot analysis was performed using poly(A)+ RNA isolated from various human tissues and gene-specific cDNA probes (Fig. 2). The 5.3-kb liprin-alpha 1 mRNA and the less abundant 6.8- and 4.0-kb liprin-alpha 1 mRNAs are expressed in all eight samples (heart, lung, placenta, lung, liver, skeletal muscle, kidney, and pancreas), whereas the 6.5-kb liprin-alpha 2 mRNA and the 5.0-kb liprin-alpha 3 mRNA are present only in the brain sample. The 7.5-kb liprin-alpha 4 mRNA is present only in the heart, brain, and skeletal muscle samples. Both the 7.0-kb liprin-beta 1 and the 3.9-kb liprin-beta 2 are broadly expressed, being present either in all eight samples (liprin-beta 2) or in seven of the eight samples (liprin-beta 1 is absent in the liver sample). In addition to the 3.9-kb liprin-beta 2 mRNA species, there is also a 5.1-kb species present in the heart and skeletal muscle samples. These results indicate that liprin mRNAs, like LAR family PTPase mRNAs, have distinct, but overlapping, expression patterns. The tissue restriction of liprin-alpha 2 and liprin-alpha 3 mRNA to brain suggests that these liprins may play specific roles in brain, perhaps in localizing PTPdelta and PTPsigma , which are both predominately expressed in the brain.


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Fig. 2.   Liprin mRNA expression. Northern blot analysis of poly(A)+ RNA isolated from different human tissues (indicated at the top of the figure) using radiolabeled cDNA probes specific for liprin-alpha 1 (A), liprin-alpha 2 (B), liprin-alpha 3 (C), liprin-alpha 4 (D), liprin-beta 1 (E), liprin-beta 2 (F), and beta -actin (G). A single RNA blot was sequentially probed with the indicated cDNAs. Autoradiography at -80 °C with an intensifying screen was performed for 16-74 h.

Expression of C. elegans Liprins-- The expression of the C. elegans liprins celiprin-alpha and celiprin-beta was examined using celiprin promoter driven expression of the GFP in transgenic nematodes (35). Two extrachromosomal array lines were characterized for each GFP reporter construct (Fig. 3). Celiprin-alpha -GFP expression is detected in vulval muscle and other cells near the vulva; in neurons located in the lateral ganglion, posterior ganglion, ventral cord, and lateral body; and in pharyngeal and body wall muscle cells (Fig. 3, A and B). Celiprin-beta 1-GFP expression is seen in pharyngeal muscle, particularly posterior bulb, adjacent to the dorsal and ventral cord (but not in ventral cord neurons), and in body wall muscles (Fig. 3, C and D). Overall, celiprin-alpha and celiprin-beta expression appears predominately in neurons and muscle cells, with celiprin-alpha and celiprin-beta being co-expressed in pharyngeal and body wall muscle but not in any other obvious regions.


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Fig. 3.   Celiprin-alpha and celiprin-beta expression in C. elegans. Nematodes express the GFP reporter under the control of either the celiprin-alpha promoter (A and B) or the celiprin-beta promoter (C and D). GFP expression in these images is shown in black. Each panel corresponds to an independent extrachromosomal array line. Celiprin-alpha -GFP reporter construct expression (A and B) is detected in pharyngeal muscle (p), vulval muscle (v), body wall muscle (m), neurons of the lateral and posterior ganglion (g), and neurons (n) of the ventral cord and lateral body. Vulval muscle expression is visible in a ventral view in B. Celiprin-beta -GFP reporter construct expression (C and D) is detected in pharyngeal muscle (p), particularly posterior bulb, body wall muscle (m), and surrounding the dorsal and ventral cords. Scale bar represents 50 µm.

Liprins Homodimerize and Heterodimerize via Their N-terminal, Coiled Coil Regions-- Previously, co-immunoprecipitation studies demonstrated that the N-terminal region of liprin-alpha 1(LIP.1) dimerizes, consistent with the predicted coiled coil structure of this region (24). To determine whether the N-terminal regions of liprin-alpha 2 and -beta 1 homodimerize or heterodimerize, full-length liprin-alpha 1, -alpha 2, or -beta 2 was co-transfected into COS cells together with HA-tagged liprin C-terminal truncation (Delta C) constructs encoding only the N-terminal, coiled coil regions of liprin-alpha 1, -alpha 2, or -beta 2. The ability of one liprin to bind another liprin was then assessed by co-immunoprecipitation experiments (Fig. 4). Liprin-alpha 1, -alpha 2, and -beta 1 were efficiently and comparably expressed in all of the co-transfection experiments (data not shown). HA-liprin-alpha 1Delta C (aa 3-670) and HA-liprin-alpha 2Delta C (aa 1-701), which both contain only the N-terminal, coiled coil region, but not HA-liprin-beta 1Delta C (aa 1-227) co-precipitated full-length liprin-alpha 1 (aa 1-1202) (Fig. 4, lanes 1-3). Similarly, HA-liprin-alpha 2Delta C co-precipitated full-length liprin-alpha 1 and -alpha 2 but not -beta 1 (Fig. 4, lanes 4-6). Thus, the N-terminal regions of liprin-alpha 1 and -alpha 2 interact to form liprin-alpha 1/liprin-alpha 1 and liprin-alpha 2/liprin-alpha 2 homodimers, as well as liprin-alpha 1/liprin-alpha 2 heterodimers, although it appears that alpha -liprins preferentially homodimerize (Fig. 4; homodimerization is seen in lanes 1 and 5, whereas liprin-alpha 1/liprin-alpha 2 heterodimerization is seen in lanes 2 and 4). HA-liprin-beta 1Delta C co-precipitated full-length liprin-beta 1 but not liprin-alpha 1 or -alpha 2, demonstrating that the liprin-beta 1 N-terminal region homodimerizes (Fig. 4, lanes 7-9). These binding studies indicate that the liprin-alpha 2 and liprin-beta 1 N-terminal regions, like liprin-alpha 1, form coiled coil structures. Furthermore, the ability of the coiled coil region of liprin-alpha 1 to heterodimerize with liprin-alpha 2 but not with liprin-beta 1 indicates that liprin coiled coil regions within a subfamily may form homodimers, heterodimers, and/or higher order structures.


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Fig. 4.   Homo- and heterodimerization of liprins via their N-terminal, coiled coil regions. Shown is an SDS-PAGE analysis of [35S]methionine-labeled proteins isolated by co-immunoprecipitation with HA-tagged liprin-alpha 1, liprin-alpha 2, and liprin-beta 1 C-terminal truncation (Delta C) mutants containing only the N-terminal, coiled coil regions. COS-7 cells were transiently transfected with expression vectors encoding the HA-tagged, C-terminal truncated forms of liprin-alpha 1 (HA-Lalpha 1Delta C (aa 3-670), lanes 1-3), liprin-alpha 2 (HA-Lalpha 2Delta C (aa 3-701), lanes 4-6), and liprin-beta 1 (HA-Lbeta 1Delta C (aa 1-227), lanes 7-9) together with either full-length, non-HA-tagged liprin-alpha 1 (lanes 1, 4, and 7), liprin-alpha 2 (lanes 2, 5, and 8), or liprin-beta 1 (lanes 3, 6, and 9). 16 h after transfection, cells were metabolically labeled with [35S]methionine for 4 h. Following labeling, cell extracts were prepared, and immunoprecipitation analysis was performed using the anti-HA antibody 11A. Control precipitations using the anti-liprin-alpha 1 mAb, anti-LIP.1.77, an anti-liprin-alpha 2 mouse polyserum, or anti-liprin-beta 1.68.1 hybridoma supernatant demonstrated that all three of the full-length liprins were about equally well expressed in all of the co-transfections (data not shown). Immunoprecipitated proteins were resolved by 10% SDS-PAGE and visualized by autoradiography after 16 h (lanes 1-6) or 40 h (lanes 7-9). Liprin-beta 1 (lanes 3 and 6) was not observed even in the 40 h exposure (data not shown). Molecular mass standards in kDa are shown at the right of the figure.

Interaction between the C-terminal Regions of alpha -Liprins and beta -Liprins-- Liprin-beta 1 and liprin-beta 2 were identified in the interaction trap screen for liprin-alpha 1-binding proteins. Both the liprin-alpha 1 bait and the original liprin-beta 1 and liprin-beta 2 interactors isolated contain only the C-terminal, non-coiled coil regions, demonstrating that liprin-alpha 1/liprin-beta 1 and liprin-alpha 1/liprin-beta 2 binding occurs via the C-terminal, non-coiled coil regions. To determine whether the C-terminal regions of liprin-alpha 2 and -alpha 3 also bind beta -liprins, interaction trap assays were performed. Both the C-terminal, non-coiled coil region of liprin-alpha 2 (aa 821-1257) and liprin-alpha 3 (aa 3'-443') bound the C-terminal region of liprin-beta 1 and -beta 2, indicating that a general property of alpha -liprins is the ability to bind beta -liprins through their C-terminal regions, which contain the LH domains (Table I). The binding of alpha - and beta -liprins was not observed in co-precipitation experiments using mammalian cells (data not shown). The reason for this lack of binding is unknown, but possibly the alpha - and beta -liprin interaction is sensitive to the detergents present in the lysis buffer. Interaction of liprins via their C-terminal regions was restricted to alpha /beta interactions because the C-terminal regions of alpha -liprin subfamily members did not bind other alpha -liprins, and the C-terminal region of liprin-beta 1 did not bind liprin-beta 2 (Table I). Taken together, these data indicate that the C-terminal, non-coiled coil regions of alpha -liprins bind to the C-terminal regions of beta -liprins.

                              
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Table I
Protein-protein interaction between the C-terminal, non-coiled coil regions of alpha -liprins and beta -liprins using the interaction-trap assay
Shown are beta -galactosidase levels in liquid cultures for various pairs of liprin interaction-trap baits and interactors. Numbers in parentheses are the amino acid residues of the liprins (see Fig. 1) fused to the LexA DNA binding domain (the baits) or to the B42 transcription activation domain (the interactors). Control indicates the interactor expression plasmid pJG45 only. For each transfection five clones were analyzed for growth on Leu-deficient medium, as well as beta -galactosidase activity on X-galactosidase-containing medium. Measurements of beta -galactosidase levels in liquid cultures were done in duplicate, and the average values of beta -galactosidase units are shown.

To determine whether alpha - and beta -liprins interact in vivo, COS cells were co-transfected with liprin-alpha 1 and -beta 1 expression vectors, and co-localization was assessed by immunofluorescence (Fig. 5). Liprin-alpha 1 (Fig. 5B, red) and liprin-beta 1 (Fig. 5A, green) co-localize predominantly to the plasma membrane of COS cells (Fig. 5C, double exposure of co-localized green and red results in yellow/orange). The localization of either liprin-alpha 1 or -beta 1 in singly transfected COS cells is similar to their localization in co-transfected cells (data not shown), indicating that localization of liprin-alpha 1 or -beta 1 to the membrane in COS cells is independent of the expression of the second liprin. The co-localization of liprin-alpha 1 and -beta 1 in COS cells supports the possibility that alpha - and beta -liprins interact in vivo. The ability of liprins to dimerize via their N-terminal regions to form alpha /alpha and beta /beta dimers, as well as their ability to form alpha /beta heterodimers via their C-terminal regions, suggests that liprins are multivalent proteins that form complex structures.


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Fig. 5.   Co-localization of liprin-alpha 1 and liprin-beta 1 in COS cells. Immunofluorescence images of cells transiently transfected with expression vectors encoding liprin-alpha 1 and -beta 1, stained for liprin-alpha 1 and -beta 1, and photographed for liprin-beta 1 (green) (A), liprin-alpha 1 (red) (B), or liprin-alpha 1 and liprin-beta 1 with antigen colocalization seen as orange-yellow (e.g. see arrows) (C). COS-7 cells were plated onto coverslips 5 h after being transiently transfected with the expression vectors pMT.Liprin-alpha 1 (aa 1-1202) and/or pMT.Liprin-beta 1 (aa 1-1005). 16 h following plating onto coverslips, cells were fixed, permeabilized, and stained simultaneously with the anti-liprin-alpha 1 mAb anti-LIP.1.77 and the anti-liprin-beta 1.68.1 mAb, followed by isotype-specific, goat anti-mouse fluorescein isothiocyanate or Texas red-conjugated Abs as described under "Experimental Procedures." Scale bar represents 25 µm.

alpha -Liprins, but Not beta -Liprins, Bind LAR Family PTPases-- Previously, we demonstrated that liprin-alpha 1(LIP.1) binds LAR, PTPdelta , and PTPsigma (20, 24). To assess whether other alpha -liprins also bind LAR, PTPdelta , PTPsigma , or the cytoplasmic regions of the transmembrane PTPases PTPµ and CD45, interaction trap assays were performed. In this assay system, the C-terminal regions of LAR, PTPdelta , and PTPsigma but not of PTPµ or CD45 bound liprin-alpha 1, -alpha 2, and -alpha 3 but not liprin-beta 1 or -beta 2 (Table II). These results indicate that the C-terminal, non-coiled coil region of alpha -liprins but not beta -liprins binds the C-terminal region of LAR family PTPases (Table II). The binding of liprin-alpha 1 and -alpha 2 to LAR, as well as the lack of binding of liprin-beta 1 or -beta 2 to LAR, was also observed in co-precipitation experiments using cell lysates prepared from [35S]methionine-labeled COS cells transiently expressing the liprin-alpha 1, -alpha 2, -beta 1, or -beta 2 C-terminal regions and GST-LAR fusion protein or control GST protein (Fig. 6). These results demonstrate that in addition to binding the C-terminal, non-coiled coil regions of beta -liprins, the alpha -liprin C-terminal regions also bind the three mammalian LAR family PTPases (Table II). Furthermore, because beta -liprins do not bind LAR family PTPases, the binding of LAR family PTPases by liprins is restricted to members of the alpha -liprin subfamily.

                              
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Table II
Protein-protein interaction between LAR family PTPases and alpha -liprins using the interaction trap assay
Shown are beta -galactosidase levels in liquid cultures for various pairs of PTPase interaction trap baits and liprin interactors. Numbers in parentheses are the amino acid residues of the PTPases LAR (aa 1275-1881 (39)), PTPdelta (aa 1624-1892 (16)), PTPµ (aa 1189-1452 (46)), and CD45 (aa 584-1281 (47)) fused to the LexA DNA binding domain (the baits) or liprins (see Fig. 1) fused to the B42 transcription activation domain (the interactors). Control indicates the interactor expression plasmid pJG45 only.


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Fig. 6.   LAR co-precipitates alpha -type liprins. SDS-PAGE analysis of [35S]methionine-labeled proteins isolated by co-precipitation using GST-LAR fusion protein. COS-7 cells were transiently transfected with expression vectors encoding HA-tagged, N-terminal truncated (Delta N) forms of liprin-alpha 1 (Liprin-alpha 1Delta N, aa 794-1202; lanes 1, 2, and 8), liprin-alpha 2 (Liprin-alpha 2Delta N, aa 821-1257; lanes 3, 4, and 10), liprin-beta 1 (Liprin-beta 1Delta N, aa 678-1005; lanes 5, 6, and 11), and liprin-beta 2 (Liprin-beta 2Delta N, aa 678-1005; lanes 7, 8, and 12). 16 h after transfection, cells were metabolically labeled with [35S]methionine for 4 h. Following labeling, cell extracts were prepared, and co-precipitation analysis was performed using control GST protein (lanes 1, 3, 5, and 7) or LAR-GST fusion protein (aa 1275-1881; lanes 2, 4, 6, and 8). Control anti-HA 11A mAb immunoprecipitation (lanes 9-12) was done using 0.2 volumes of extract compared with the GST precipitations. Precipitated proteins were resolved by 10% SDS-PAGE and visualized by autoradiography after 16 h.

Liprin-alpha 2 Expression Affects LAR Cellular Localization-- To determine whether liprin-alpha 2, like -alpha 1, colocalizes with LAR in cells, transiently transfected COS cells were analyzed by immunofluorescence (Fig. 7). COS cells were transfected with various combinations of expression vectors encoding LAR, a LAR truncation mutant (LAR-D1) that lacks the alpha -liprin interacting D2 PTPase domain, and HA-liprin-alpha 2. In cells transfected with LAR only, LAR (green) was uniformly distributed throughout the plasma membrane and Golgi (Fig. 7A), whereas in liprin-alpha 2-only transfected cells, liprin-alpha 2 (red) was observed in large plaque-like structures at the cell surface (Fig. 7B). In cells co-expressing LAR (green) and liprin-alpha 2 (red) both proteins co-localized at the cell surface in plaque-like structures and to a lesser extent at ruffling edges (single exposures of the same field for LAR (Fig. 7C) and liprin-alpha 2 (Fig. 7D). The specificity of the LAR-liprin-alpha 2 association is supported by the lack of significant co-localization of LAR-D1 with liprin-alpha 2 (Fig. 7, single exposures of the same field for LAR-D1 (Fig. 7E) and liprin-alpha 2 (Fig. 7F)). In contrast to the punctate expression pattern of LAR in the LAR/liprin-alpha 2 co-expressing cells (Fig. 7C), the LAR-D1 expression pattern in the LAR-D1/liprin-alpha 2 co-expressing cells (Fig. 7E) is similar to the expression pattern of LAR in the LAR-only transfected cells (Fig. 7A). Furthermore, the liprin-alpha 2 expression pattern is similar in the liprin-alpha 2-only transfected cells and in the LAR/liprin-alpha 2 or LAR-D1/liprin-alpha 2 doubly transfected cells (Fig. 7, B, D, and F), indicating that LAR expression does not alter liprin-alpha 2 localization. Taken together, these results demonstrate that LAR and liprin-alpha 2 co-localize in COS cells, and that the LAR membrane-distal D2 PTPase domain is required for co-localization and LAR clustering. Thus, liprin expression modifies LAR distribution.


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Fig. 7.   Liprin-alpha 2 expression alters LAR cellular distribution. Immunofluorescence images of cells transiently transfected with expression vectors encoding LAR and stained for LAR (green) (A); encoding liprin-alpha 2 and stained for liprin-alpha 2 (red) (B); encoding LAR and liprin-alpha 2 and stained for LAR and liprin-alpha 2, photographed for LAR (green) (C) and photographed for liprin-alpha 2 (red) (D); encoding LAR-D1 and liprin-alpha 2 and stained for LAR and liprin-alpha 2, photographed for LAR (green) (E) and photographed for liprin-alpha 2 (red) (F). Examples of LAR and liprin-alpha 2 co-localization are indicated by arrows. COS-7 cells were plated onto coverslips 5 h after being transiently transfected with the pMT.HA-Liprin-alpha 2 (aa 3-1257, HA-tagged), pMT.LAR (aa 1-1881), and pMT.LAR-D1 (aa 1-1615, which lacks the alpha -liprin binding PTPase D2 domain) expression vectors. 16 h following plating onto coverslips, cells were fixed, permeabilized, and stained individually or simultaneously with the anti-LAR mAb 11.1A/75.3A/128.4A mixture and/or the anti-HA 12CA5 mAb, followed by isotype-specific, goat anti-mouse fluorescein isothiocyanate or Texas red-conjugated Abs as described under "Experimental Procedures." Scale bar represents 25 µm.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

We describe the liprins, a novel gene family that contains at least six mammalian and two C. elegans members. The overall predicted structure of alpha -liprins and beta -liprins is an N-terminal, coiled coil region and a C-terminal, non-coiled coil region. This structure suggests that the liprin N-terminal regions intertwine to form rod-like structures, similar to those seen in intermediate filaments and myosin II heavy chains (30). The prototype member of this family, LIP.1 (renamed liprin-alpha 1), was previously identified as a LAR PTPase-binding protein and is thought to function in anchoring LAR at FAs (24). Based on sequence homology, as well as their binding to LAR family PTPases or to themselves, liprins are subdivided into alpha -liprins and beta -liprins.

The characterization of the liprins genes suggests several general properties of liprins: 1) the N-terminal, coiled coil region of alpha -liprins mediates homodimerization, as well as heterodimerization with other alpha -liprin subfamily members. The coiled coil region of beta -liprins also allows for homodimerization and possibly for heterodimerization with other beta -liprins. The coiled coil regions of alpha - and beta -liprins do not heterodimerize. 2) alpha -Liprins and beta -liprins interact via their C-terminal LH domains. However, alpha -LH domains do not bind other alpha -LH domains, and beta -LH domains do not bind other beta -LH domains. 3) Only alpha -LH domains, not beta -LH domains, bind to the membrane-distal D2 domain of LAR family PTPases.

The ability of the N-terminal, coiled coil regions to form alpha /alpha or beta /beta dimers and the C-terminal LH domains to form alpha /beta dimers suggests that liprins are multivalent proteins that form complex structures. Such structures could function as scaffolds for the recruitment and anchoring of LAR family PTPases. For instance, in tissues such as brain, in which all the known alpha - and beta -liprins are expressed, as well as LAR, PTPdelta , and PTPsigma , the potential complexities of the interaction between liprins and PTPases are substantial. Distinct combinations of alpha - and beta -liprins may determine where in the plasma membrane particular PTPases are located and determine the protein composition of the liprin/PTPase complexes. It is unknown whether alpha -liprins can simultaneously bind LAR family PTPases and beta -liprins or whether the PTPases and beta -liprins compete for alpha -liprin binding. LAR family PTPases bound to alpha -liprins may also be brought together with other liprin-binding proteins by liprin dimerization.

C. elegans liprins may function in a manner similar to mammalian liprins in LAR family PTPase signaling. Indeed, there exists a LAR-like gene in C. elegans (CECO9D8-1/2; GenBankTM accession number Z46811) that is predicted to contain an extracellular region composed of Ig-like and fibronectin III-like domains, connected via a transmembrane peptide to an intracellular region with two PTPase domains. However, because the expression pattern of celiprin-alpha and -beta is only partially overlapping, it is unclear whether celiprin-alpha and -beta interaction is essential for liprin function in C. elegans. If liprin function, at least in part, depends on the interaction of alpha - and beta -liprins, then one would assume that there are additional C. elegans liprins or proteins that functionally substitute for liprins. Alternatively, the function of C. elegans liprins does not require alpha /beta association.

Based on the binding properties of liprins, we postulate that liprins recruit LAR family PTPases to specific areas within the plasma membrane, as well as facilitating the recruitment/anchoring of other signaling proteins. A role for alpha -liprins in localizing LAR family PTPases within the cell was initially indicated by the co-localization of LAR and liprin-alpha 1 to discrete ends of FAs (24). Such a role for other liprins is supported by the altered cellular distribution of LAR in LAR/liprin-alpha 2 co-expressing COS cells. In singly transfected cells, LAR is expressed homogeneously throughout the plasma membrane, whereas liprin-alpha 2 is distributed into large plaque-like structures and to the cell edges. In doubly transfected cells, LAR is redistributed to the liprin-alpha 2 aggregates, indicating that liprin-alpha 2 recruits and clusters LAR. Controlling LAR family PTPase localization is likely to be a key feature in determining the substrates that these PTPases dephosphorylate because there is currently little evidence to support regulated catalytic activity for LAR family PTPases. Further insight into the physiological function of liprins and LAR family PTPases should be greatly aided by genetic analyses of the C. elegans liprins and LAR-like PTPase(s).

    ACKNOWLEDGEMENTS

We thank Drs. N. Kedersha and H. Saito for critical review of the manuscript; Dr. R. Brent and colleagues for plasmid DNAs and yeast strains used for the interaction trap assay; Dr. C. Sardet for the fibroblast cDNA library; J. Shapiro for expert technical assistance; and Dr. S. F. Schlossman for encouragement and support.

    FOOTNOTES

* This work was supported by Grant CA55547 from the National Institutes of Health, a Barr Investigator Award, and a Medical Research Council of Canada Fellowship (to Q. G. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

** Leukemia Society of America Scholar. To whom correspondence should be addressed: Division of Tumor Immunology, Dana Farber Cancer Institute, 44 Binney St., Boston, MA 02115. Tel.: 617-632-3526; Fax: 617-632-4569; E-mail: Michel_Streuli{at}dfci.harvard.edu.

1 The abbreviations used are: PTPase, protein-tyrosine phosphatase; DLAR, Drosophila LAR; FA, focal adhesion; LIP.1, LAR-interacting protein 1; LH, liprin homology; kb, kilobase(s); HA, hemagglutinin; GFP, green fluorescent protein; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; mAb, monoclonal antibody; aa, amino acid(s).

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Top
Abstract
Introduction
Procedures
Results
Discussion
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