STRUCTURAL AND FUNCTIONAL-RELATIONSHIPS BETWEEN H-CALDESMONS AND L-CALDESMONS

Two different M(r) forms of caldesmon as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (M(r) values in the range of 120,000-150,000, h-caldesmon and 70,000-80,000, l-caldesmon) have been already identified. h-Caldesmon is predominantly expressed in smooth muscle cells, whereas l-caldesmon widely distributes in non-muscle cells. Most recently, the molecular cloning of h-caldesmon has been reported (Hayashi,K., Kanda, K., Kimizuka, F., kato, I., and Sobue, K. (1989) Biochem. Biophys. Res. Commun. 164, 503-511; Bryan, J., Imai, M., Lee, R., Moore, P., Cook, R.G., and Lin, W-G. (1989) J. Biol. Chem. 264, 13873-13879). The calculated M(r) of this protein from its primary structure is 88,743. Here, the nucleotide and deduced amino acid sequences of l-caldesmon have been determined by cloning and sequencing the cDNA from chick brain and compared with those of h-caldesmon. The l-caldesmon cDNA encodes a sequence of 517 amino acids with the calculated M(r) of 58,844. Two isoforms of caldesmon conserve the completely identical sequences in the NH2- and COOH-terminal domains except for the insertion of Ala-508 in l-caldesmon. Interestingly, the central repeating sequence of h-caldesmon (residues 201-477) is deleted in the l-caldesmon molecule. The short NH2-terminals of two caldesmons individually show the unique sequences. The results of Northern and Southern blot analyses suggest that two mRNAs (4.8 and 4.1 kilobases) coding for caldesmon isoforms may be generated from a single gene by alternative splicing. Using a series of truncated caldesmons expressed in Escherichia coli, the common calmodulin-, tropomyosin-, and acting-binding sites and the minimum regulatory domains, which are involved in the Ca2+-dependent regulation of actin-myosin interaction, have been identified within the limited consensus sequences (residues 381-433 for l-caldesmon and residues 636-688 for h-caldesmon).