All eukaryotic cells contain one or more isozymes of nonmuscle myosin. Two forms of the myosin heavy chain (MHC-A and MHC-B) have been cloned in this laboratory and are the products of two different genes in humans. One of the isozymes, the B form, is also subject to alternative splicing of messenger RNA to yield four possible variants. This is due to the existence of combinations of two possible inserted nucleotide sequences in the head region of myosin. Levels of messenger RNA and distribution of the isoforms differ depending on the source of tissue. We would like to understand the function of the A and B isoforms and the function of the insertions in the head region of the B isoform. To do this, we wish to overexpress the full-length human MHC isoforms in mammalian cells and in transgenic mice. CDNA clones for MHCs, which have been previously isolated, will be spliced together to give CDNAS coding for the entire heavy chain. These clones will be introduced into a mammalian expression vector (Prc/RSV) under the constitutive control of the Rous Sarcoma virus promoter. Constructs which contain the 20,000 dalton myosin light chain (LC20) and the 17,000 dalton light chain (LC17) have been made in this same expression vector. The light chain constructs will be co-transfected with the MHC by electroporation into various cell lines. Both stable and transient transfectants will be assayed for overexpression of the encoded proteins and for functional and morphological changes in the cells. Studies are also being carried out in collaboration with Dr. Oksoon Choi (LCP, NHLBI) to overexpress the myosin LC20 in RBL-2H3 cells using the LC20/Prc/RSV construct. Experiments have shown that when these cells are triggered to secrete by aggregation of IgE receptors on the cell surface, phosphorylation of LC20 at the myosin light chain kinase and protein kinase C sites occurs and precedes cell secretion. We wish to test the effect of overexpression of the LC20 and to then introduce mutant forms of the LC20 which cannot be phosphorylated. It may then be possible to assess the importance of phosphorylation during secretion.
