Departments of * Physiology, Pharmaceutical Chemistry, and § Molecular Cytometry, University of California at San Francisco,
San Francisco, California 94143; and
Department of Biological Chemistry, University of California at Irvine, Irvine, California
92697
PDZ motifs are protein-protein interaction
domains that often bind to COOH-terminal peptide sequences. The two PDZ proteins characterized in skeletal muscle, syntrophin and neuronal nitric oxide synthase, occur in the dystrophin complex, suggesting a role for PDZ proteins in muscular dystrophy. Here, we
identify actinin-associated LIM protein (ALP), a novel
protein in skeletal muscle that contains an NH2-terminal PDZ domain and a COOH-terminal LIM motif.
ALP is expressed at high levels only in differentiated skeletal muscle, while an alternatively spliced form occurs at low levels in the heart. ALP is not a component
of the dystrophin complex, but occurs in association
with -actinin-2 at the Z lines of myofibers. Biochemical and yeast two-hybrid analyses demonstrate that the
PDZ domain of ALP binds to the spectrin-like motifs
of
-actinin-2, defining a new mode for PDZ domain
interactions. Fine genetic mapping studies demonstrate
that ALP occurs on chromosome 4q35, near the heterochromatic locus that is mutated in fascioscapulohumeral muscular dystrophy.
THE cytoskeleton is a complex protein network that
provides cellular structure. By partitioning the cell,
the cytoskeleton can also provide microdomains
that allow specific responses to localized stimuli. The assembly and maintenance of the cytoskeleton is mediated,
in large part, by high affinity interactions between modular consensus protein-binding motifs. These sites for protein-protein interaction are often multifunctional, and the
specific binding partners are determined by the variations
in amino acid sequences between the individual domains.
A recently identified motif, the PDZ domain, is an 80-
120-amino acid domain that was first identified in the
postsynaptic protein, PSD-95, which contains three PDZ domains in tandem (Cho et al., 1992 Recent work indicates that PDZ domains are multifunctional protein-protein interaction motifs (Brenman and
Bredt, 1997 Functional roles for PDZ domains have been demonstrated in diverse tissues. Mutations in the PDZ domain of
Drosophila inactivation no afterpotential D (INAD) alter
transduction of visual signals (Shieh and Zhu, 1996 To understand and further define the role of PDZ domains in cytoskeletal assembly, we have focused on skeletal
muscle as a model system. The regular and defined structure of skeletal muscle makes it an ideal tissue for study.
Previous studies demonstrated that the two known PDZ
domain proteins in skeletal muscle, the family of sytrophins and nNOS, are both components of the dystrophin complex (Adams et al., 1993 We hypothesized that other PDZ proteins in muscle
may also occur in the cytoskeleton. Characterization of
these proteins will help better understand the function of
PDZ domains and may identify candidate genes for inherited muscular dystrophies. Here, we report the cloning of a
novel cDNA encoding a protein of 39 kD that consists of
an NH2-terminal PDZ domain and a COOH-terminal LIM
domain. The protein is expressed at high levels only in
skeletal muscle, where it occurs at the Z lines in association with mRNA Isolation and cDNA Analysis
RNA was isolated using the guanidine isothiocyanate/CsCl method, and
mRNA was selected with oligo dT sepharose. For degenerate reverse
transcriptase-PCR (RT-PCR), rat skeletal muscle mRNA was reverse
transcribed with RTth polymerase using random hexamer primers. PCR,
using a 54°C annealing temperature, was then performed using the following primer pair: GGGGGATCCGGXGGXCTXGGXHT and GCGGAATTCAGDARXATRTCXCC. Amplified products were resolved on agarose
gels. Bands of 120-160 bp were isolated and subcloned into pBluescript
vectors (Stratagene, La Jolla, CA) for sequence analysis. Before transformation, ligation products were digested with BglII to remove syntrophin
cDNAs. Clones encoding ALP were isolated from a rat skeletal muscle
cDNA library (catalog no. 937510; Stratagene) by plaque hybridization with the 32P-labeled DNA fragment obtained above by RT-PCR. One
clone of 1,586 nucleotides was sequenced on both strands and contained an open reading frame (ORF) of 362 amino acids. The initiation site for
translation was assigned to the first methionine codon in the ORF at nucleotide 100. This ATG occurs in a consensus Kozak sequence for initiation (Kozak, 1991 For Northern blotting, RNA was separated on a formaldehyde agarose gel
and transferred to a nylon membrane. A random-primed 32P probe was generated using the 160-bp ALP cDNA obtained from RT-PCR as a template.
The filter was washed at high stringency, 63°C, 0.1% sodium chloride/sodium citrate and 0.1% SDS, and was exposed to x-ray film for 2 h at Yeast Two-Hybrid Analysis
The nucleotides encoding amino acids 1-128 of ALP were amplified by
PCR and subcloned into the GAL-4 DNA-binding domain plasmid pGBT9 (Clontech Laboratories, Palo Alto, CA). This construct was cotransformed into yeast strain HF7c with a library of human skeletal muscle cDNAs fused to the GAL-4 activation domain (Clontech). The
transformation mixture was plated onto a synthetic dextrose plate lacking
tryptophan, leucine, and histidine. Growth was monitored during a 5-d incubation at 30°C, and color was measured by a Antibodies, Immunohistochemistry, and
Western Blotting
A glutathione S-transferase (GST) fusion protein encoding amino acids 1-207
of ALP was amplified by PCR and subcloned into pGEX2T (Pharmacia
Biotech, Piscataway, NJ). The fusion protein was expressed and purified
from Escherichia coli as described (Brenman et al., 1995 For immunofluorescent staining, skeletal muscle samples were flash
frozen in liquid nitrogen-cooled isobutane, sectioned on a cryostat (10 µm), and melted directly onto glass slides. Sections were then postfixed in
2% paraformaldehyde in PBS. Tissues were blocked in PBS containing
1% normal goat serum. mAbs to For Western blotting, protein extracts were resolved by SDS-PAGE
and transferred to polyvinyldifluoride membranes. Primary antibodies
were diluted in block solution containing 3% BSA, and 0.1% Tween 20 in
TBS, and were incubated with membranes overnight at 4°C. Labeled
bands were visualized using enhanced chemiluminescence (Amersham,
Arlington Heights, IL).
Myogenic Cell Culture
C2 myogenic cell line was grown on gelatin-coated dishes in Ham's F-10
nutrient mixture plus 0.8 mM extra CaCl2 supplemented with 15% horse
serum (Life Technologies, Inc., Bethesda, MD) and 2.5 ng/ml basic fibroblast growth factor (Promega, Madison, WI). Myoblasts were fused in media containing DME (GIBCO BRL, Gaithersburg, MD) supplemented
with 2% horse serum. Protein was extracted from cultured cells in buffer
containing 25 mM Tris-HCl, pH 7.4, containing 1% Triton X-100, 1 mM
EDTA, and 100 µM PMSF. RNA was purified from cultured cells as described above.
GST Fusion Protein Affinity Chromatography
and Immunoprecipitation
Skeletal muscle tissue was homogenized in 10 vol of cold water containing
1 mM PMSF and centrifuged at 15,000 g for 10 min. To extract cytoskeletal proteins, the pellet was treated with 10 vol of buffer containing 2 mM
Tris-HCl, pH 9, and 1 mM EGTA at 37°C for 30 min with gentle agitation.
After centrifugation at 15,000 g for 30 min, the supernatant was titrated to
pH 7.5. For "pull-down" assays, the solubilized tissue samples were incubated with control or GST fusion proteins linked to glutathione Sepharose beads for 1 h. Beads were washed three times with buffer containing 0.5%
Triton X-100 and 350 mM NaCl, and proteins were eluted with SDS loading buffer. The GST-nNOS fusion protein was purified as described previously (Brenman et al., 1995 For immunoprecipitation, polyclonal antibodies (1 µg) to ALP or preimmune serum were added to 0.5-ml aliquots of solubilized skeletal muscle extract, and samples were incubated on ice for 1 h. Protein A Sepharose
(50 µl) was used to precipitate antibodies. Protein A pellets were washed
three times with buffer containing 100 mM NaCl and 1% Triton X-100.
Immunoprecipitated proteins were denatured with loading buffer and resolved by SDS-PAGE.
In Situ Hybridization
In situ hybridization used 35S-labeled RNA probes exactly as described
(Sassoon and Rosenthal, 1993 Human Chromosome Mapping
Two P1 clones corresponding to human ALP (Genome Systems, St.
Louis, MO) were used to determine the location of ALP on human chromosomes by fluorescence in situ hybridization (FISH). The hybridized signal was detected by antidigoxigenin conjugated with FITC, as described
(Sakamoto et al., 1995 Fine mapping of ALP was performed by PCR amplification of human
hamster somatic cell hybrids and radiation-derived hybrids containing all
or portions of human chromosome 4q35. The somatic cell hybrids included HHW986, containing intact human chromosome 4 (Carlock et al.,
1986 Hybrids were screened by PCR for the presence of the ALP gene. The
oligonucleotides (sense: GGGAGCTGTACTGCGAAA) and (antisense: CGATTGTTTTCTCGTGTA) amplify a 150-bp product within the 3 Molecular Cloning of ALP and Characterization of
mRNA Expression
To identify novel PDZ proteins expressed in skeletal muscle, we performed RT-PCR with degenerate primers that
amplify amino acids GLGF (sense) and GDXIL (antisense; see Materials and Methods for experimental details). These sequences are conserved among many PDZ
proteins. A library of these cDNA fragments was constructed and processed to remove the syntrophins leading to the
identification of novel cDNAs encoding PDZ sequences.
Northern analysis was then used to identify gene products
that were enriched in skeletal muscle. The mRNA for one
of the products, SK-2 (hereafter called ALP), migrated at
1.6 kb and occurred at high levels in skeletal muscle tissue,
at low levels in the heart, and was undetectable in other
tissues examined (Fig. 1 A). Human ALP showed a similar tissue-specific expression pattern (Fig. 1 B). To better understand mechanisms that regulate ALP during muscle development, we evaluated expression in myogenic cultures.
ALP expression was dramatically induced after myotube
fusion in culture (Fig. 1 C).
In situ hybridization of embryonic day (E15) mouse embryo with antisense ALP probe demonstrated the highest
levels of ALP expression in developing skeletal muscle
and heart (Fig. 1 D). ALP mRNA also occurred in embryonic intestine. No specific hybridization was observed using sense RNA control (Fig. 1 E).
Structural Features of ALP and Comparison with
Its Homologues
cDNA clones encoding the full 1.6-kb mRNA were obtained from a rat skeletal muscle cDNA library. The mRNA
contains a single ORF of 1,086 bp encoding a protein of 39 kD. Sequence analysis shows that ALP has a homology at
the NH2 terminus to PDZ domains. Alignment of ALP
with other PDZ domains is shown (Fig. 2 A). The presence
of histidine at amino acid 62 suggests that the PDZ domain may be in group I (Brenman and Bredt, 1997
The central region of ALP showed no homology to any
other cloned gene while the COOH terminus encodes a
LIM domain. Though ALP has not previously been reported, other proteins with a similar domain structure have
been described. A database homology search with BLAST
indicated that ALP shares high homology to a number of
newly identified transcripts including CLP-36, RIL, and
enigma (Fig. 2 B). CLP-36 was identified as a cDNA whose
expression in the heart is downregulated by hypoxia (Wang
et al., 1995 Our analysis of the expressed sequence tag (EST) database showed that overlapping cDNAs corresponding to
human ALP have been deposited. The human ALP is
91% identical to the rat sequence. We noted that EST
clones from human heart libraries were consistently different in the central region from those in human skeletal
muscle libraries (Fig. 2 C). Exons encoding the central 112 amino acids of skeletal muscle ALP are likely to be spliced
out in the heart and replaced by exons encoding 64 different amino acids. To confirm this differential expression,
we amplified the region that was unique to heart transcripts and reprobed the Northern blot. As expected, we
found heart-specific expression of this region of ALP (data
not shown). We therefore define two subtypes ALPSK and ALPH for the alternative transcripts that occur in skeletal
muscle and heart, respectively.
The PDZ Domain of ALP Binds Previous studies have shown that PDZ domains participate in protein-protein interactions. To determine potential targets for the PDZ domain of ALP, we used the yeast
two-hybrid system. We screened 106 clones from an adult
skeletal muscle library (Clontech) and obtained 120 positive clones, 35 of which were recovered and analyzed. All
positive clones encoded fragments of
Interaction of a PDZ domain with spectrin-like repeats
is unprecedented. We therefore asked whether this interaction was specific. We found that the PDZ domains of
nNOS, To further confirm this interaction, we expressed a bacterial fusion protein linking GST to the PDZ domain of
ALP. We found that this fusion protein specifically retained
ALP Colocalizes with To determine whether the interaction with
To determine whether ALP and The ALP Gene Maps to Human Chromosome 4q35
To determine whether ALP might map close to any
known genetic mutations that are associated with muscle
disease, we determined the chromosomal location of human ALP. We performed FISH with two independent
digoxigenin-labeled genomic ALP probes on normal human metaphase chromosomes. Hybridization with these
probes resulted in specific labeling of only chromosome 4. The location of the probes was determined by digital image microscopy after FISH and was localized by the fractional length from the p terminus (FLpter) as described
previously (Sakamoto et al., 1995 Interestingly, FSHD, the most common autosomal dominant muscular dystrophy, maps to the telomeric region of
chromosome 4, at 4q35. Since FSHD is associated with deletions of a subtelomeric repeat sequence (van Deutekom
et al., 1993
The primary finding in this study is the identification of a
functional interaction between a PDZ domain and the
spectrin-like repeats of Interaction with the spectrin-like repeat represents a
new mode of binding for a PDZ domain. Previous work
has shown that PDZ domains of the postsynaptic density
protein, PSD-95, bind to certain glutamate receptors and
K+ channels in the brain that terminate with a consensus
of E-T/S-X-V/I (Cohen et al., 1996 The binding interface between the PDZ domain of ALP
and the spectrin-like repeats of We find that ALP expression is normal in Duchenne and
Becker muscular dystrophies (Xia, H., and D.S. Bredt, unpublished data). On the other hand, certain inherited muscular
dystrophies result from mutations in cytoskeletal proteins
that do not interact with the dystrophin complex (Hoffman
et al., 1995 Our chromosomal mapping studies indicate that ALP
occurs on human chromosome 4q 35. Interestingly, the location is ~7-10 Mb from the subtelomeric region that is
mutated in FSHD, an autosomal dominant disease (Wijmenga et al., 1992 What might be the normal function of ALP? Determining the role of the LIM motif in ALP remains a critical
question. LIM motifs were first identified in protein products from three different genes, lin11 (Freyd et al., 1990). Sequence analysis
has subsequently demonstrated that PDZ domains are
common protein motifs that occur in a variety of dissimilar
proteins that interact with the cytoskeleton (Ponting and
Phillips, 1995
). Individual PDZ domains occur in neuronal
nitric oxide synthase (nNOS),1 syntrophins, p55, dishevelled and CASK, while multiple PDZ domains occur in
PSD-95, dlg, and zona occludens (ZO)-1 and -2 proteins; and PTP-BAS.
; Kornau et al., 1997
; Sheng, 1996
). One mode
for interaction of PDZ domains involves association with
the COOH terminus of target proteins. Thus, the COOH
terminus of Fas binds to the third PDZ domain of PTP-BAS,
and this interaction participates in Fas-mediated apoptosis of T cells (Sato et al., 1995
). Similarly, the first and second PDZ domains of PSD-95 bind to the COOH termini of
certain ion channels in the brain, and they anchor these
channels to synaptic sites at the plasma membrane (Kim et
al., 1995
; Kornau et al., 1995
). PDZ-PDZ interactions have
also been identified. The PDZ domain of nNOS binds to
the second PDZ domain of PSD-95 (Brenman et al., 1996
).
These multifunctional interactions of PDZ domains assemble a complex of nNOS / PSD-95/the N-methyl-D-aspartate
receptor calcium channel at the postsynaptic density.
), while
mutations in the Caenorhabditis elegans PDZ proteins Lin-2
and Lin-7 result in abnormal vulval development (Hoskins
et al., 1996
). In all these cases, the PDZ domains are implicated in targeting intracellular proteins to appropriate multiprotein complexes at the plasma membrane.
; Brenman et al., 1995
). nNOS
isoforms lacking the PDZ domain do not interact with the
dystrophin complex and occur in the skeletal muscle cytosol. The PDZ domains of nNOS and syntrophin directly
interact with each other, and this linkage anchors nNOS to
the dystrophin complex (Brenman et al., 1996
). The absence
of dystrophin in Duchenne muscular dystrophy results in a
loss of syntrophins and nNOS from the sarcolemma, and
these abnormalities may contribute to the disease process.
-actinin-2. We therefore name this protein actinin-associated LIM protein (ALP). Biochemical and two-hybrid analyses indicate that the PDZ domain of ALP
binds to the spectrin-like repeats of
-actinin-2, establishing a novel mode of interaction for PDZ domains. Chromosomal mapping indicates that the ALP gene occurs at
4q35 within 7-10 megabase (Mb) of the heterochromatic
region that is deleted in fascioscapulohumeral muscular dystrophy (FSHD; Altherr et al., 1995
).
Materials and Methods
) and is preceded by an in-frame stop codon. A polyadenylation sequence occurs ~400 bp downstream of the stop codon.
70°C.
-galactosidase colorimetric filter assay (Fields and Song, 1989
). Interacting clones were rescued,
retransformed to confirm interaction, and sequenced. Deletions of interacting clone 9-5 were generated by digestion with XcmI (9-5X), NarI (9-5N),
or BglI (9-5B). Site-directed mutagenesis of ALP L78K was performed by
PCR and confirmed by sequencing.
). Rabbits were
immunized with the GST-ALP fusion protein emulsified in complete and
incomplete Freund's adjuvant. Serum bleeds were evaluated by ELISA.
For affinity purification, ALP antiserum was applied to a column of GST-
ALP fusion protein immobilized on Reacti-Gel (Pierce Chemical Co.,
Rockford, IL). The column was washed with 20 vol of buffer containing
500 mM NaCl and 10 mM Tris, pH 7.5. ALP antibody was eluted with 10 bed vol of 100 mM glycine, pH 2.5. Monoclonal antibodies to syntrophin (Peters et al., 1994
), nNOS (Transduction Laboratories, Lexington, KY),
and
-actinin (Sigma Immunochemicals, St. Louis, MO) were also used.
-actinin (1:100) and polyclonal ALP antibody were applied to sections overnight at 4°C. For indirect immunofluorescence, secondary goat anti-mouse FITC or donkey anti-rabbit Cy-3-conjugated antibodies were used according to the specifications of the
manufacturer (Jackson ImmunoResearch Laboratories, Bar Harbor, ME).
).
). Sense and antisense probes to ALP (full
length) were synthesized from a pBluescript vector using T3 and T7 polymerases.
; Stokke et al., 1995
).
), HHW986, retaining only 4q35 translocated to a derivative 5p, and
HHW1372, in which only the telomeric region of 4q35 (distal to D4S187)
is retained on a derivative X (Bodrug et al., 1990
). The radiation hybrids
were derived from HHW416 and retain varying fragments of 4q35 (Winokur et al., 1993
). Negative controls included a human lymphoblastoid
cell line GM7057 (NIGMS) and a Chinese hamster fibroblast cell line
UCW 104.
untranslated region of ALP. Amplifications with a 51°C annealing temperature were done in 25 µl containing 250 ng genomic DNA, 50 µl each oligonucleotide, 1.25 mM each dNTP, 16.6 mM ammonium sulfate, 67 mM
Tris-HCl, pH 8, 6.7 mM MgCl2, 10 mM
-mercaptoethanol, and 2 U Taq
polymerase.
Results
Fig. 1.
ALP mRNA expression in skeletal muscle, heart, and
other tissues. (A) Northern blot of poly(A+) RNA (10 µg/lane)
from rat kidney (K), spleen (Sp), liver (L), heart (H), skeletal
muscle (M), and brain (B) was probed with 32P-labeled ALP. (B)
Human multiple-tissue Northern blot purchased from Clontech
(7760-1) was probed with hALP. Each lane contains ~2 µg of
poly(A+) RNA from human heart (H), brain (B), placenta (Pl),
lung (Lu), liver (Li), skeletal muscle (M), kidney (K), and pancreas (Pa). (C) ALP expression is induced after the fusion of C2
myotubes. Each lane in c contains 2 µg of total RNA. (D) In situ
hybridization of E15 rat embryo shows highest levels of ALP in
developing skeletal muscles, including the tongue (To), sternocephalic (St), and tail (Ta). Hybridizing signals are also seen in
the heart atrium (At) and ventricle (Ve) and in a circular pattern
in the intestine (In). (E) No specific hybridization is seen using
sense control probe. Hybridization in the liver (Li) was considered nonspecific since it was detected with sense and antisense
probes.
[View Larger Versions of these Images (32 + 82K GIF file)]
; Songyang et
al., 1997
).
Fig. 2.
Sequence analysis of ALP isoforms. (A) Amino acids 5-80 of ALP encode a consensus PDZ domain. Alignment of ALP with
PDZ domains from CLP-36, PSD95, 1-syntrophin, (
1syn), nNOS, and INAD. Histidine 62 of ALP is marked with an asterisk and leucine 78 with a pound sign. (B) Predicted sequences of rat ALP (GenBank/EMBL/DDBJ accession no. AF002281) and human ALP
(hALP) are aligned with two homologous proteins, CLP-36 and RIL. (C) An alternative ALP isoform is expressed in the heart. Schematic model shows the domain structure of ALP and the divergence of ALP between skeletal muscle (hALPSK; accession no.
AF002280) and heart (hALPH; accession no. AF002282). The alignment shows that the central region of ALP is different between skeletal muscle and heart. The accession numbers for ESTs used to construct hALPH are F12229, R20192, AA147575, AA211287, and
D56502. The accession numbers for ESTs encoding the skeletal muscle-specific splice for hALPsk are Z28845, Z19288, and Z28703.
[View Larger Versions of these Images (27 + 44 + 12K GIF file)]
). RIL, short for reversion-induced LIM protein,
is downregulated in H-ras-transformed cells (Kiess et al.,
1995
). Enigma was identified as an insulin receptor-interacting protein (Wu et al., 1996
). These investigators, however, did not recognize the homology of the NH2-terminal
regions of CLP-36, RIL, or enigma with the PDZ domain.
The PDZ domain of ALP shares 55, 48, and 45% amino
acid identity with the PDZ domains of CLP36, RIL, and
enigma, respectively. The LIM domain of ALP shares
even stronger homology (67% identity) with CLP36 and
RIL. While ALP, CLP36, and RIL all only have one LIM
domain, enigma has three LIM domains. The sequence
homology indicates that ALP, CLP36, and RIL constitute
a new family of proteins containing an NH2-terminal PDZ
domain and a COOH-terminal LIM domain.
-Actinin-2
-actinin-2, a muscle- specific cytoskeletal protein that contains an NH2-terminal
actin-binding domain, four central spectrin-like repeats,
and a COOH-terminal region homologous to calcium-binding EF hands (Beggs et al., 1992
). ALP interacts only with
the spectrin-like repeat region of
-actinin-2, and all interacting clones encode spectrin repeat three (Fig. 3). However, a deletion construct (9-5N) containing repeat three did not interact with ALP, indicating that repeat three is
necessary but not alone sufficient for binding.
Fig. 3.
The PDZ domain of ALP
binds to the spectrin repeats of
-actinin-2. The sequence encoding amino acids 1-128 of ALP was
fused to the GAL4 DNA-binding
domain. Clones 9-2, 4, 5, 6, 7, and
12, which were rescued from a yeast
two-hybrid screen of a human skeletal muscle library, encode different
fragments of
-actinin-2. Clone 9-5 was truncated with restriction enzymes to yield clones 9-5X, N, and
B. All ALP-interacting clones encoded at least two complete spectrin-like repeats, one of which was
the third repeat. nNOS, PSD-95,
and
1-syntrophin did not interact
with
-actinin-2. Mutation of ALP
leucine 78 to lysine abolished interaction with
-actinin-2.
[View Larger Version of this Image (27K GIF file)]
1-syntrophin, and the three PDZ domains of PSD-95
(Brenman et al., 1996
) did not interact with
-actinin-2 in
the yeast two-hybrid system. We previously identified a
point mutation that universally disrupts all known types of
PDZ interactions (Christopherson, K., W. Lim, and D.S. Bredt, manuscript submitted for publication). This change
corresponds to a mutation found in INAD (Shieh and Niemeyer, 1995
), a PDZ protein in Drosophila that binds to
the transient receptor potential (TRP) calcium channel (Shieh
and Zhu, 1996
). This mutation corresponds to position
Leu78 in ALP. Mutating Leu78 of ALP to Lys abolished the interaction with
-actinin-2 (Fig. 3), demonstrating
specificity of the PDZ domain interaction with
-actinin-2.
-actinin-2 from solubilized skeletal muscle cytoskeleton
(Fig. 4) but did not retain
1-syntrophin. By contrast, an
analogous column containing the PDZ domain of nNOS
"pulled-down"
1-syntrophin but not
-actinin-2.
Fig. 4.
Association of
ALP and -actinin-2 and
specificity of the PDZ-spectrin-like repeat interaction.
(A) Affinity chromatography demonstrates that
-actinin-2
is selectively retained by an
immobilized ALP fragment
(amino acids 1-128) fused to
GST, not by GST-NOS (amino acids 1-299) fusion
protein, which selectively
brings down syntrophin. The
load is 20% of the input used
for affinity chromatography experiment. (B) Immunoprecipitation of skeletal muscle
extracts shows selective
coimmunoprecipitation of
-actinin-2 with ALP antiserum but not with preimmune serum. By contrast, two control proteins, nNOS and syntrophin, were not coimmunoprecipitated. Immunoprecipitated proteins were resolved on four replicate
gels and probed with antisera to
-actinin, ALP, nNOS, and syntrophin. Load is 10% of the input used for the immunoprecipitation.
[View Larger Version of this Image (30K GIF file)]
-Actinin-2 at the Z Lines in
Skeletal Muscle
-actinin-2 is
physiologically important in localizing ALP to specific cytoskeletal domains, we developed an affinity-purified antiserum (see Materials and Methods). We evaluated the
specificity of the serum by Western blot analysis of crude
tissue extracts. As expected, the antiserum recognized a
prominent band of 39 kD in skeletal muscle and a much
less intense band of 35 kD in the heart (Fig. 5 A). No immunoreactive bands were noted in the spleen, kidney,
brain, or liver. Western blotting of myogenic cell extracts
showed that ALP is absent from myoblasts but is induced
within 3 d after myotube fusion (Fig. 5 B).
Fig. 5.
ALP protein is enriched in skeletal muscle and
colocalizes with -actinin-2
at the Z lines. (A) Rat tissue
extracts (100 µg/lane) from
rat kidney (K), spleen (S),
liver (L), heart (H), skeletal muscle (M), and brain (B)
was run on SDS-PAGE gel,
transferred to a polyvinyldifluoride membrane, and then
probed with a polyclonal antibody against GST-ALP fusion protein. (B) Western
blotting of protein extracts
from C2 myogenic cultures
shows that ALP is absent
from myoblasts and is
present in myotubes 3 and 5 d
after fusion. (C) Immunofluorescent staining of rat skeletal
muscle longitudinal sections
shows that ALP (red) occurs
at the
-actinin-2-rich (green)
Z lines.
[View Larger Version of this Image (72K GIF file)]
-actinin-2 occur together in a protein complex in skeletal muscle, we performed immunoprecipitation studies (Fig. 4 B). We found
that the antiserum to ALP specifically coimmunoprecipitated
-actinin-2 from solubilized cytoskeletal extracts
from skeletal muscle. By contrast, neither nNOS nor syntrophin, cytoskeletal components of the dystrophin complex, were coimmunoprecipitated with ALP. We next compared the cellular distribution of ALP with
-actinin-2 in
skeletal muscle. Immunofluorescent staining of longitudinal sections of adult skeletal muscle showed that ALP colocalized with
-actinin-2 on the Z lines (Fig. 5 C). No
ALP immunoreactivity was found at the sarcolemma, nucleus, or other structures of the myofiber.
; Stokke et al., 1995
).
Both clones mapped to the most telomeric region of chromosome 4, at 4q34-qter with FLpter values of 0.962 ± 0.004 and 0.959 ± 0.005.
), the localization of ALP relative to the 4q telomere was of interest. We therefore used somatic cell and
radiation hybrid panels to map ALP within chromosomal
band 4q35. PCR analysis of the somatic cell hybrids revealed that ALP maps within the proximal portion of this
band (Fig. 6 B). ALP is not present in HHW1372, which
contains only the telomeric 2 Mb of chromosome 4q distal
to the locus D4S187. Analysis of the radiation hybrid panel
DNAs, which contain independent fragments of 4q35, localizes ALP to the interval between D4S171 and the Factor XI gene (Fig. 6 C). Thus, the approximate distance of
ALP from the telomere is 7-10 Mb.
Fig. 6.
Human ALP maps to chromosome 4q35. (A) FISH of P1 clones to human metaphase chromosomes. Hybridizing signals (arrows) were detected by FITC (green), and the chromosomes were counterstained with propidium iodide and DAPI (purple, combined
color). Inset on the lower left corner shows chromosome 4 with P1 hybridization aligned with a black and white image of DAPI-stained
chromosome 4. (B) PCR analysis of human hamster somatic cell and radiation hybrids containing various portions of chromosomal
band 4q35. The 150-bp amplification product from the ALP gene is present only in somatic cell hybrids containing the portion of 4q35
proximal to D4S187. Only those radiation hybrids that contain a portion of the interval between D4S171 and FXI were positive for
ALP. (C) Schematic of the 4q35 locus contained within each somatic cell and radiation hybrid. The order and retention of the 12 loci between IRF2 (centromeric) and D4S809 (telomeric) in the radiation hybrids were determined previously (Winokur et al., 1993).
[View Larger Versions of these Images (18 + 26K GIF file)]
Discussion
-actinin-2. This association targets a novel LIM protein, ALP, to the actinin-rich Z lines
of skeletal muscle fibers. PDZ domains are recently recognized protein-protein interaction motifs that are implicated in protein association with the cytoskeleton (Marfatia et al., 1996
) and in signal transduction (Brenman and Bredt, 1997
; Sheng, 1996
). Previous studies demonstrated that the two PDZ proteins in skeletal muscle, nNOS and the
syntrophins, are constituents of the dystrophin complex (Adams et al., 1993
; Brenman et al., 1995
). Our work here shows
that the PDZ protein ALP does not associate with the dystrophin complex, but instead binds to
-actinin-2, which is
in the dystrophin superfamily of cytoskeletal proteins.
; Kim et al., 1995
; Kornau et al., 1995
). These interactions appear to anchor ion
channels to synaptic sites in neurons. Interaction with specific COOH-terminal peptides may be a general property
of PDZ domains, and two recent studies demonstrate that
distinct PDZ domains, bind to specific COOH-terminal
peptide sequences (Songyang et al., 1997
; Stricker et al.,
1997
). Certain PDZ domains can also associate with each
other in a homotypic-type interaction (Brenman et al., 1996
).
The PDZ domain of nNOS binds to the second PDZ domain of PSD-95 in the brain and to the PDZ domain of
1-syntrophin in skeletal muscle.
-actinin-2 represents a
third mode for protein interactions mediated by PDZ domains. We suspect that this type of interaction is not
unique to ALP and may explain cytoskeletal interactions
of diverse PDZ proteins. Z-1 protein of epithelial tight
junctions contains three PDZ domains and associates with
spectrin in the cell cortex (Willott et al., 1993
). Electron micrographic studies indicate that ZO-1 forms a complex
with the central rodlike repeat domains of spectrin. It is
not yet clear whether the PDZ domains of ZO-1 mediate
this interaction. A complex ternary interaction between
the spectrin-like repeats of dystrophin and the PDZ domains of nNOS/syntrophin may occur at the skeletal muscle sarcolemma (Chao et al., 1996
). Thus, in vitro assays
demonstrate that nNOS binds directly to syntrophin, but
not to dystrophin. However, the nNOS/syntrophin interaction in skeletal muscle requires that certain spectrin-like
repeats of dystrophin be intact. Also, nNOS is selectively
absent from skeletal muscle sarcolemma in patients with
Becker muscular dystrophy who have mutations within the spectrin-like repeats of dystrophin (Chao et al., 1996
).
). Plectin, a cytoskeleton-membrane anchorage
protein of hemidesmosomes, links intermediate filaments to the sarcolemma and also occurs at the Z lines in skeletal
muscle (Wiche et al., 1983
). Mutations in plectin do not effect the dystrophin complex, but they cause an autosomal recessive muscular dystrophy associated with skin blistering
(Smith et al., 1996
). It will be important to assess ALP expression in a variety of inherited muscular dystrophies to determine whether it may play a role in any of these diseases.
). The specific genetic defect in FSHD
disease appears to be a deletion of heterochromatin (Lyle
et al., 1995
; Winokur et al., 1994
). It is not clear how this
mutation results in muscular dystrophy. It is postulated that the telomeric mutation mediates a "position effect"
that alters the expression of a nearby muscle-specific gene
(Altherr et al., 1995
). Genes separated by genomic distances >2 Mb from heterochromatin have been reported
to be affected by position effect variegation in Drosophila
(Bedell et al., 1996
). Therefore, ALP should be considered
a candidate gene for FSHD. In preliminary studies, we
have not detected consistent changes in ALP expression in
muscle biopsies from FSHD tissues. However, the muscle
samples from FSHD patients analyzed for ALP expression
may not have been from the critically affected muscle
groups or from appropriate developmental stages.
),
isl1 (Karlsson et al., 1990
), and mec3 (Way and Chalfie,
1988
), which all contain two LIM domains in association
with a homeodomain DNA binding motif. These transcription factor LIM proteins participate in cell fate determination. Many distinct classes of LIM proteins have now been
identified that do not have a homeodomain but still participate in cell fate determination (Sanchez-Garcia and Rabbitts,
1994
). At the biochemical level, LIM motifs are implicated
in protein-protein interactions. LIM motifs have been
shown to interact with basic helix-loop-helix transcription
factors (Wadman et al., 1994
), protein kinase C (Kuroda et
al., 1996
), receptor tyrosine kinase tight turn structure (Wu et al., 1996
), and other LIM motifs (Schmeichel and
Beckerle, 1994
). In striated muscle, the LIM-only protein,
MLP, occurs at the Z lines and is an essential regulator of
myogenic differentiation (Arber et al., 1994
). Targeted disruption of MLP results in a disorganization of the myocyte
cytoarchitecture (Arber et al., 1997
). It will be interesting
to determine whether the LIM domain of ALP interacts
with MLP or other LIM proteins at the Z lines. To decisively
determine the role of ALP in cellular physiology and muscular dystrophy, it will be important to delete the function of ALP by knockout and dominant negative approaches.
Received for publication 6 May 1997 and in revised form 22 July 1997.
Address all correspondence to David S. Bredt, University of California at San Francisco School of Medicine, 513 Parnassus Avenue, San Francisco, CA 94143-0444. Tel.: (415) 476-6310; Fax: (415) 476-4929; E-mail: bredt{at}itsa.ucsf.eduThe authors thank Jay Brenman for performing in situ hybridization and Rick Topinka for excellent technical assistance.
This work was supported by grants (to D.S. Bredt) from the National Institutes of Health, the National Science Foundation, the Council for Tobacco Research, the Searle Scholars Program, and the Lucille P. Markey Charitable Trust, as well as by grants (to D.S. Bredt, S.T. Winokur, and M.R. Altherr) from the Muscular Dystrophy Association.
ALP, actinin-associated LIM protein; EST, expressed sequence tag; FISH, fluorescence in situ hybridization; FSHD, fascioscapulohumoral muscular dystrophy; GST, glutathione S-transferase; INAD, inactivation no afterpotential D; nNOS, neuronal nitric oxide synthase; ORF, open reading frame; RT-PCR, reverse transcription; ZO, zona occludens.
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