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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Association of  ALP and α-actinin-2 and  specificity of the PDZ–spectrin-like repeat interaction.  (A) Affinity chromatography  demonstrates that α-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  α-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 α-actinin, ALP, nNOS, and syntrophin. Load is 10% of the input used for the immunoprecipitation.
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Figure 4: Association of ALP and α-actinin-2 and specificity of the PDZ–spectrin-like repeat interaction. (A) Affinity chromatography demonstrates that α-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 α-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 α-actinin, ALP, nNOS, and syntrophin. Load is 10% of the input used for the immunoprecipitation.

Mentions: 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 α-actinin-2 from solubilized skeletal muscle cytoskeleton (Fig. 4) but did not retain α1-syntrophin. By contrast, an analogous column containing the PDZ domain of nNOS “pulled-down” α1-syntrophin but not α-actinin-2.


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)

Association of  ALP and α-actinin-2 and  specificity of the PDZ–spectrin-like repeat interaction.  (A) Affinity chromatography  demonstrates that α-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  α-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 α-actinin, ALP, nNOS, and syntrophin. Load is 10% of the input used for the immunoprecipitation.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2139795&req=5

Figure 4: Association of ALP and α-actinin-2 and specificity of the PDZ–spectrin-like repeat interaction. (A) Affinity chromatography demonstrates that α-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 α-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 α-actinin, ALP, nNOS, and syntrophin. Load is 10% of the input used for the immunoprecipitation.
Mentions: 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 α-actinin-2 from solubilized skeletal muscle cytoskeleton (Fig. 4) but did not retain α1-syntrophin. By contrast, an analogous column containing the PDZ domain of nNOS “pulled-down” α1-syntrophin but not α-actinin-2.

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