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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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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).
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Figure 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).

Mentions: 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 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.


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)

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).
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Figure 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).
Mentions: 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 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.

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