Mammalian Nonsarcomeric Myosin Regulatory Light Chains Are Encoded by Two Differentially Regulated and Linked Genes

The myosin 20,000-D regulatory light chain (RLC) has a central role in smooth muscle contraction. Previous work has suggested either the presence of two RLC isoforms, one specific for nonmuscle and one specific for smooth muscle, or the absence of a true smooth muscle-specific isoform, in which instance smooth muscle cells would use nonmuscle isoforms. To address this issue directly, we have isolated rat RLC cDNAs and corresponding genomic sequences of two smooth muscle RLC based on homology to the amino acid sequence of the chicken gizzard RLC. These cDNAs are highly homologous in their amino acid coding regions and contain unique 3'-untranslated regions. RNA analyses of rat tissue using these unique 3'-untranslated regions revealed that their expression is differentially regulated. However, one eDNA (RLC-B), predominantly a nonmuscle isoform, based on abundant expression in nonmuscle tissues including brain, spleen, and lung, is easily detected in smooth muscle tissues. The other cDNA (RLC-A; see Taubman, M., J. W. Grant, and B. Nadal-Ginard. 1987. J. Cell Biol. 104:1505-1513) was detected in a variety of nonmuscle, smooth muscle, and sarcomeric tissues. RNA analyses comparing expression of both RLC genes with the actin gene family and smooth muscle specific o~-tropomyosin demonstrated that neither RLC gene was strictly smooth muscle specific. RNA analyses of cell lines demonstrated that both of the RLC genes are expressed in a variety of cell types. The complete genomic structure of RLC-A and close linkage to RLC-B is described. M YOSIN, the major contractile element of the thick filament, is composed of two identical 200-kD heavy chains and two sets of light chains, the 20kD regulatory and the 17and 23-kD alkalai light chains. The 20-kD regulatory light chain (RLC) ~ of smooth muscle contributes significantly to the functional characteristics of smooth muscle contraction and myosin structure (2, 17). In vertebrates, phosphorylation of RLC by myosin light chain kinase increases active tension in intact smooth muscle tissue and in actin-activated myosin ATPase activity in vitro (2). Phosphorylation of the RLC occurs at several sites including serine-19, the adjacent threonine-20, threonine-9, and serine-2 (4, 10, 18, 19). The RLC is involved in filament structure and conformation by interactions with the globular Portions of this work were presented at the Scientific Sessions of the American Heart Association in November 1988. J. W. Grant's present address is the Brookhaven Center for Molecular Biology, Department of Medicine, Mt. Sinai School of Medicine, New York. 1. Abbreviations used in this paper: RASM, rat aortic smooth muscle; RLC, regulatory light chain. head of the myosin heavy chain and the alkalai light chains (17). In vitro filament assembly, conformation of the myosin, stability and the solubility characteristics of nonmuscle and smooth muscle myosin are directly influenced by phosphorylation of the RLC (8, 9, 23, 49, 50). Recent data suggest a direct contribution of RLC pbosphorylation on both myosin conformation and its ATP activity (51). Protein sequence data has demonstrated distinct isoforms of the RLC for smooth muscle and sarcomeric tissues (27, 29). A separate isoform for a nonmuscle RLC has not been identified, although a 20-kD phosphorylated myosin light chain exists in a variety of tissues including fibroblasts, platelets, intestinal brush border, and pancreatic acinar cells (1, 3, 5, 6, 8, 14, 53). The smooth muscle and fibroblast 20-kD RLC co-migrate on isoelectric focusing gels (6). The presence of at least two different isoforms has been reported for vascular, uterine smooth muscles (11, 14) and pancreatic acinar cells (5), though, precise identification of isoforms is complicated by the potential for shifts caused by multiple phosphorylations. The sequence analyses of cDNAs and the corresponding genes isolated for mammalian skeletal, cardiac, and smooth © The Rockefeller University Press, 0021-9525/90/09/1127/9 $2.00 The Journal of Cell Biology, Volume 111, September 199