Abstract
Myosin light chain phosphatase consists of three subunits, a 38-kDa catalytic subunit, a large 110-130-kDa myosin binding subunit, and a small subunit of 20-21 kDa. The catalytic subunit and the large subunit have been well characterized. The small subunit has been cloned and studied from smooth muscle, but little is known about its function and specificity in the other muscles such as cardiac muscle. In this study, cDNAs for heart-specific small subunit isoforms, hHS-M(21), were isolated and characterized. Evidence was obtained from an analysis of genome to suggest that the small subunit was the product of the same gene as the large subunit. Using permeabilized renal artery preparation and permeabilized cardiac myocytes, it was shown that the small subunit increased sensitivity to Ca(2+) in muscle contraction. It was also shown using an overlay assay that hHS-M(21) bound the large subunit. Mapping experiments demonstrated that the binding domain and the domain involved in the increasing Ca(2+) sensitivity mapped to the same N-terminal region of hHS-M(21). These observations suggest that the heart-specific small subunit hHS-M(21) plays a regulatory role in cardiac muscle contraction by its binding to the large subunit.
Highlights
Ca2ϩ plays a central role in the regulation of muscle contractile process mediated by interaction of myosin with actin
Genomic organization of the human MYPT2 gene was determined, and it was revealed that one of the large subunits (MYPT2-MBS) and the small subunit of myosin light chain phosphatase (MLCP) in the heart are the products of the same gene
We found that: 1) hHS-M21 induced an additional contraction at a constant Ca2ϩ concentration and shifted the [Ca2ϩ]i force relationship to the left; 2) the fragments containing the N-terminal half conferred this action of hHS-M21, while the deletion of the N-terminal 56 residues completely abolished the action; 3) hHS-M21 bound to the C-terminal one-third of MBS encoded by MYPT1 with high affinity, and only to a little extent, bound to the same region of MBS encoded by MYPT2; and 4) the binding domain of hHS-M21 to MYPT1-MBS was mapped to the same region as the main effective domain for the Ca2ϩ sensitivity on the Ca2ϩ-induced contraction in permeabilized porcine renal artery
Summary
PBS, phosphate-buffered saline; HPLC, high performance liquid chromatography; PAGE, polyacrylamide gel electrophoresis; TBS, Tris-buffered saline; CSS, cytoplasmic substitution solution; PIPES, piperazine1,4-bis(2-ethanesulfonic acid); aa, amino acid(s); bp, base pair(s); kbp, kilobase pair(s); PVDF, polyvinylidene difluoride. A recent report has demonstrated a possible involvement of MLC phosphorylation in the development of cardiac hypertrophy [27] Another gene for MBS, MYPT2, was isolated by Fujioka et al [25]. Because MYPT2 is expressed preferentially in the striated muscles, especially in the cardiac muscle, it was suggested that the function of MYPT2 might be related with the regulation of MLC phosphorylation in the heart [25]. We report here the isolation of cDNAs for heart-specific isoform of M20 subunit (hHS-M21), which were obtained in the process of isolating novel genes being preferentially expressed in the cardiac muscle. From an analogy of function for sm-M20 in the smooth muscle, hHS-M21 was suggested to participate in the regulation of MLC phosphorylation in the cardiac muscle. The role of hHS-M21 in the Ca2ϩ sensitivity of muscle contraction was investigated, along with its interaction with MBSs encoded by MYPT1 and MYPT2
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