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.

Show MeSH

Related in: MedlinePlus

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.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2139795&req=5

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.

Mentions: 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).


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 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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2139795&req=5

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.
Mentions: 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).

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