From the 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
Massachusetts General
Hospital Cancer Center, Charlestown, Massachusetts 02129
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ABSTRACT |
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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 -type and
-type liprins. The
C-terminal, non-coiled coil regions of
-liprins bind to the
membrane-distal phosphatase domains of LAR family members, as well as
to the C-terminal, non-coiled coil region of
-liprins. Both
- and
-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-
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.
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INTRODUCTION |
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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), PTP
(14-16), and PTP
(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 -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 -liprins and
-liprins.
-Liprins, including LIP.1
(renamed liprin-
1) bind to the membrane-distal D2 PTPase domains of
the LAR family PTPases, LAR, PTP
, and PTP
, whereas
-liprins
bind to
-liprins but not to LAR family PTPases. Furthermore, both
- and
-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-
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.
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EXPERIMENTAL PROCEDURES |
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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 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-
1 (83 and 73% identity over 77-102
amino acids, respectively). The initial ~300-bp liprin-
2 and
liprin-
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-
1 encodes
liprin-
1 amino acids 1-1202 (24); pMT.Liprin-
2 encodes
liprin-
2 amino acids 1-1257; pMT.Liprin-
1 encodes liprin-
1
amino acids 1-1005; pMT.HA-Liprin-
1
C (HA-L
1
C) encodes
HA-tagged liprin-
1 amino acids 3-670; pMT.HA-Liprin-
2
C (HA-L
2
C) encodes HA-tagged liprin-
2 amino acids 3-701;
pMT.HA-Liprin-
2
N encodes HA-tagged liprin-
2 amino acids
821-1257; pMT.HA-Liprin-
1
C (HA-L
1
C) encodes HA-tagged
liprin-
1 amino acids 1-227; pMT.HA-Liprin-
1
N encodes
HA-tagged liprin-
1 amino acids 678-1005; pMT.HA-Liprin-
2
N encodes HA-tagged liprin-
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-
and celiprin-
coding sequences were obtained by others
using Genefinder (GenBankTM accession nos. Z50794 and
Z78546, respectively). The 5' promoter regions of celiprin-
and
celiprin-
used to construct the green fluorescent protein (GFP)
reporter plasmids pPD.celiprin-
-GFP and pPD.celiprin-
-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-
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-
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).
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 [-32P]dCTP
labeled liprin cDNA probes: liprin-
1, nucleotides 236-1514 (encoding aa 3-429); liprin-
2, nucleotides 3447-3722 (encoding aa
1093-1184); liprin-
3, nucleotides 877'-1191' (encoding aa 293'-397'); liprin-
4, nucleotides 1'-489' (encoding aa 1'-163'); liprin-
1, nucleotides 2274-2739 (encoding aa 679-833); and
liprin-
2, nucleotides 1978'-2226' (encoding aa 660'-741'); it was
also hybridized with a
-actin cDNA probe.
C. elegans Expression Analysis--
Animals were injected with
plasmid pPD.celiprin--GFP and pPD.celiprin-
-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-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-
2
and anti-liprin-
1 Abs, mice were immunized with purified,
Escherichia coli-derived GST-Liprin-
2 (amino acids
3-470) and GST-Liprin-
1 (amino acids 63-446) fusion proteins (33).
Hypoxanthine/aminopterin/thymidine-resistant hybridomas derived
from GST-Liprin-
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-
1.68.1 (IgG1). The anti-liprin-
2 polyserum was
obtained from mice immunized with the GST-Liprin-
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-1), 3 µl of mouse anti-liprin-
2 serum, 100 µl of
anti-liprin-
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-1 and liprin-
1, permeabilized cells were exposed to
the anti-liprin-
1 mAb anti-LIP.1.77 mAb and the
anti-liprin-
1.68.1 mAb at a concentration of 2 µg of
-LIP.1.77/ml blocking buffer and a 1:3 dilution of anti-liprin-
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-
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.
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RESULTS |
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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-1 (aa 678-1005; Fig. 1A) and liprin-
2 (aa
257'-783'; Fig. 1A), in addition to the expected LAR and
PTP
. Sequence determination of liprin-
1 cDNA clones predict
that liprin-
1 is a 1005-amino acid long protein, the primary
sequence of which is similar to LIP.1 (25% sequence identity; Fig.
1A). Liprin-
1 was named LIP.1-related protein;
1
indicates that liprin-
1 is the first member of the
subfamily of
liprins (see below). Consequently, LIP.1 was renamed liprin-
1, because it is the first member of the liprin-
subfamily.
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Tissue-specific Expression of Human Liprins--
The human LAR,
PTP, and PTP
PTPases have distinct, but overlapping, mRNA
tissue distributions (20). For instance, all three mRNAs are
expressed in brain, whereas only LAR and PTP
mRNA are expressed
in kidney and pancreas. To determine the tissue distribution of
-
and
-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-
1 mRNA and the less abundant 6.8- and 4.0-kb liprin-
1
mRNAs are expressed in all eight samples (heart, lung, placenta,
lung, liver, skeletal muscle, kidney, and pancreas), whereas the 6.5-kb
liprin-
2 mRNA and the 5.0-kb liprin-
3 mRNA are present
only in the brain sample. The 7.5-kb liprin-
4 mRNA is present
only in the heart, brain, and skeletal muscle samples. Both the 7.0-kb
liprin-
1 and the 3.9-kb liprin-
2 are broadly expressed, being
present either in all eight samples (liprin-
2) or in seven of the
eight samples (liprin-
1 is absent in the liver sample). In addition
to the 3.9-kb liprin-
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-
2 and liprin-
3 mRNA to brain suggests that these
liprins may play specific roles in brain, perhaps in localizing PTP
and PTP
, which are both predominately expressed in the brain.
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Expression of C. elegans Liprins--
The expression of the
C. elegans liprins celiprin- and celiprin-
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-
-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-
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-
and celiprin-
expression appears predominately in neurons and muscle
cells, with celiprin-
and celiprin-
being co-expressed in
pharyngeal and body wall muscle but not in any other obvious
regions.
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Liprins Homodimerize and Heterodimerize via Their N-terminal,
Coiled Coil Regions--
Previously, co-immunoprecipitation studies
demonstrated that the N-terminal region of liprin-1(LIP.1)
dimerizes, consistent with the predicted coiled coil structure of this
region (24). To determine whether the N-terminal regions of liprin-
2
and -
1 homodimerize or heterodimerize, full-length liprin-
1,
-
2, or -
2 was co-transfected into COS cells together with
HA-tagged liprin C-terminal truncation (
C) constructs encoding only
the N-terminal, coiled coil regions of liprin-
1, -
2, or -
2.
The ability of one liprin to bind another liprin was then assessed by
co-immunoprecipitation experiments (Fig.
4). Liprin-
1, -
2, and -
1 were
efficiently and comparably expressed in all of the co-transfection
experiments (data not shown). HA-liprin-
1
C (aa 3-670) and
HA-liprin-
2
C (aa 1-701), which both contain only the N-terminal,
coiled coil region, but not HA-liprin-
1
C (aa 1-227)
co-precipitated full-length liprin-
1 (aa 1-1202) (Fig. 4,
lanes 1-3). Similarly, HA-liprin-
2
C co-precipitated
full-length liprin-
1 and -
2 but not -
1 (Fig. 4, lanes
4-6). Thus, the N-terminal regions of liprin-
1 and -
2
interact to form liprin-
1/liprin-
1 and liprin-
2/liprin-
2
homodimers, as well as liprin-
1/liprin-
2 heterodimers, although
it appears that
-liprins preferentially homodimerize (Fig. 4;
homodimerization is seen in lanes 1 and 5,
whereas liprin-
1/liprin-
2 heterodimerization is seen in
lanes 2 and 4). HA-liprin-
1
C
co-precipitated full-length liprin-
1 but not liprin-
1 or -
2,
demonstrating that the liprin-
1 N-terminal region homodimerizes
(Fig. 4, lanes 7-9). These binding studies indicate that
the liprin-
2 and liprin-
1 N-terminal regions, like liprin-
1,
form coiled coil structures. Furthermore, the ability of the coiled
coil region of liprin-
1 to heterodimerize with liprin-
2 but not
with liprin-
1 indicates that liprin coiled coil regions within a
subfamily may form homodimers, heterodimers, and/or higher order
structures.
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Interaction between the C-terminal Regions of -Liprins and
-Liprins--
Liprin-
1 and liprin-
2 were identified in the
interaction trap screen for liprin-
1-binding proteins. Both the
liprin-
1 bait and the original liprin-
1 and liprin-
2
interactors isolated contain only the C-terminal, non-coiled coil
regions, demonstrating that liprin-
1/liprin-
1 and
liprin-
1/liprin-
2 binding occurs via the C-terminal, non-coiled
coil regions. To determine whether the C-terminal regions of
liprin-
2 and -
3 also bind
-liprins, interaction trap assays
were performed. Both the C-terminal, non-coiled coil region of
liprin-
2 (aa 821-1257) and liprin-
3 (aa 3'-443') bound the
C-terminal region of liprin-
1 and -
2, indicating that a general
property of
-liprins is the ability to bind
-liprins through
their C-terminal regions, which contain the LH domains (Table
I). The binding of
- and
-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
- and
-liprin interaction is sensitive to the
detergents present in the lysis buffer. Interaction of liprins via
their C-terminal regions was restricted to
/
interactions because
the C-terminal regions of
-liprin subfamily members did not bind
other
-liprins, and the C-terminal region of liprin-
1 did not
bind liprin-
2 (Table I). Taken together, these data indicate that
the C-terminal, non-coiled coil regions of
-liprins bind to the
C-terminal regions of
-liprins.
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|
-Liprins, but Not
-Liprins, Bind LAR Family
PTPases--
Previously, we demonstrated that liprin-
1(LIP.1) binds
LAR, PTP
, and PTP
(20, 24). To assess whether other
-liprins also bind LAR, PTP
, PTP
, 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, PTP
,
and PTP
but not of PTPµ or CD45 bound liprin-
1, -
2, and
-
3 but not liprin-
1 or -
2 (Table
II). These results indicate that the C-terminal, non-coiled coil region of
-liprins but not
-liprins binds the C-terminal region of LAR family PTPases (Table II). The
binding of liprin-
1 and -
2 to LAR, as well as the lack of binding
of liprin-
1 or -
2 to LAR, was also observed in co-precipitation experiments using cell lysates prepared from
[35S]methionine-labeled COS cells transiently expressing
the liprin-
1, -
2, -
1, or -
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
-liprins, the
-liprin C-terminal regions also bind the three mammalian LAR family PTPases (Table II). Furthermore, because
-liprins do not bind LAR family PTPases, the binding of LAR family PTPases by liprins is restricted to members of the
-liprin
subfamily.
|
|
Liprin-2 Expression Affects LAR Cellular Localization--
To
determine whether liprin-
2, like -
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
-liprin interacting D2 PTPase domain, and HA-liprin-
2. In cells
transfected with LAR only, LAR (green) was uniformly
distributed throughout the plasma membrane and Golgi (Fig.
7A), whereas in liprin-
2-only transfected cells, liprin-
2 (red) was observed in large plaque-like
structures at the cell surface (Fig. 7B). In cells
co-expressing LAR (green) and liprin-
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-
2 (Fig.
7D). The specificity of the LAR-liprin-
2 association is supported by the lack of significant co-localization of LAR-D1 with
liprin-
2 (Fig. 7, single exposures of the same field for LAR-D1
(Fig. 7E) and liprin-
2 (Fig. 7F)). In contrast
to the punctate expression pattern of LAR in the LAR/liprin-
2
co-expressing cells (Fig. 7C), the LAR-D1 expression pattern
in the LAR-D1/liprin-
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-
2 expression
pattern is similar in the liprin-
2-only transfected cells and in the
LAR/liprin-
2 or LAR-D1/liprin-
2 doubly transfected cells (Fig. 7,
B, D, and F), indicating that LAR
expression does not alter liprin-
2 localization. Taken together, these results demonstrate that LAR and liprin-
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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DISCUSSION |
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We describe the liprins, a novel gene family that contains at
least six mammalian and two C. elegans members. The overall predicted structure of -liprins and
-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-
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
-liprins and
-liprins.
The characterization of the liprins genes suggests several general
properties of liprins: 1) the N-terminal, coiled coil region of
-liprins mediates homodimerization, as well as heterodimerization with other
-liprin subfamily members. The coiled coil region of
-liprins also allows for homodimerization and possibly for heterodimerization with other
-liprins. The coiled coil regions of
- and
-liprins do not heterodimerize. 2)
-Liprins and
-liprins interact via their C-terminal LH domains. However,
-LH
domains do not bind other
-LH domains, and
-LH domains do not
bind other
-LH domains. 3) Only
-LH domains, not
-LH domains,
bind to the membrane-distal D2 domain of LAR family PTPases.
The ability of the N-terminal, coiled coil regions to form /
or
/
dimers and the C-terminal LH domains to form
/
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
- and
-liprins are
expressed, as well as LAR, PTP
, and PTP
, the potential
complexities of the interaction between liprins and PTPases are
substantial. Distinct combinations of
- and
-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
-liprins can simultaneously bind LAR family
PTPases and
-liprins or whether the PTPases and
-liprins compete
for
-liprin binding. LAR family PTPases bound to
-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- and -
is only partially
overlapping, it is unclear whether celiprin-
and -
interaction is
essential for liprin function in C. elegans. If liprin
function, at least in part, depends on the interaction of
- and
-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
/
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 -liprins in localizing LAR family
PTPases within the cell was initially indicated by the co-localization
of LAR and liprin-
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-
2 co-expressing COS cells. In singly transfected
cells, LAR is expressed homogeneously throughout the plasma membrane,
whereas liprin-
2 is distributed into large plaque-like structures
and to the cell edges. In doubly transfected cells, LAR is
redistributed to the liprin-
2 aggregates, indicating that
liprin-
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).
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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* 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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REFERENCES |
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