Limits...
Actinin-associated LIM protein: identification of a domain interaction between PDZ and spectrin-like repeat motifs.

Xia H, Winokur ST, Kuo WL, Altherr MR, Bredt DS - J. Cell Biol. (1997)

Bottom Line: 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.Biochemical and yeast two-hybrid analyses demonstrate that the PDZ domain of ALP binds to the spectrin-like motifs of alpha-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.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, University of California at San Francisco, San Francisco, California 94143, USA.

ABSTRACT
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 alpha-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 alpha-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.

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ALP protein is enriched in skeletal muscle and  colocalizes with α-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 α-actinin-2–rich (green)  Z lines.
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Figure 5: ALP protein is enriched in skeletal muscle and colocalizes with α-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 α-actinin-2–rich (green) Z lines.

Mentions: To determine whether the interaction with α-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).


Actinin-associated LIM protein: identification of a domain interaction between PDZ and spectrin-like repeat motifs.

Xia H, Winokur ST, Kuo WL, Altherr MR, Bredt DS - J. Cell Biol. (1997)

ALP protein is enriched in skeletal muscle and  colocalizes with α-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 α-actinin-2–rich (green)  Z lines.
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Related In: Results  -  Collection

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Figure 5: ALP protein is enriched in skeletal muscle and colocalizes with α-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 α-actinin-2–rich (green) Z lines.
Mentions: To determine whether the interaction with α-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).

Bottom Line: 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.Biochemical and yeast two-hybrid analyses demonstrate that the PDZ domain of ALP binds to the spectrin-like motifs of alpha-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.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, University of California at San Francisco, San Francisco, California 94143, USA.

ABSTRACT
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 alpha-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 alpha-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.

Show MeSH
Related in: MedlinePlus