Actinin-associated LIM protein: identification of a domain interaction between PDZ and spectrin-like repeat motifs - PubMed (original) (raw)

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

H Xia et al. J Cell Biol. 1997.

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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Figures

Figure 1

Figure 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.

Figure 1

Figure 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.

Figure 2

Figure 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, α1-syntrophin, (α1syn), 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.

Figure 2

Figure 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, α1-syntrophin, (α1syn), 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.

Figure 2

Figure 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, α1-syntrophin, (α1syn), 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.

Figure 3

Figure 3

The PDZ domain of ALP binds to the spectrin repeats of α-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 α-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 α1-syntrophin did not interact with α-actinin-2. Mutation of ALP leucine 78 to lysine abolished interaction with α-actinin-2.

Figure 4

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.

Figure 5

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.

Figure 6

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).

Figure 6

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).

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