Smooth muscle exhibits significant phenotypic and functional diversity. The overall goal of these studies is to understand the molecular mechanisms for the generation of this diversity. We have selected the smooth muscle myosin phosphatase (SMMP), the primary mediator of smooth muscle relaxation, as the focus for understanding the diversity in smooth muscle relaxant properties. The myosin targeting subunit (MYPT1) of SMMP is a critical substrate for signaling pathways that regulate smooth muscle tone. Signaling molecules such as nitric oxide (NO) that relax smooth muscle enhance SMMP activity via the dimerization of the cGMP-dependent protein kinase with the C-terminal leucine zipper of MYPT1. Since MYPT1 isoforms have been described with variable presence o the C-terminal leucine zipper, we postulated that MYPT1 isoforms might determine the differential sensitivity of distinct smooth muscle phenotypes to NO/cGMP-mediated relaxation. To examine this, we characterized the SMMP subunit isoforms and correlated their expression with relaxant properties of smooth muscle tissues. Chicken and rat MYPT1 isoforms are generated by tissue- specific and developmentally regulated cassette-type alternative splicing of exons at the center and C-terminus. Preliminary experiments suggest that the expression of the MYPT1 isoforms correlate with cGMP relaxant response of mature smooth muscle tissues, as well as during a phenotypic switch of developing smooth muscle. Tissues that express the MYPT1 isoform that contains the C-terminal leucine zipper relax in response to cGMP in the presence of activating calcium, while those that express the isoform lacking the C-terminal leucine zipper do not. Given the role of the MYPT1 C-terminal leucine zipper in the transmission of the cGMP relaxant signal, and the correlation of the presence of this motif with cGMP sensitivity, we hypothesize that tissue-specific expression of MYPT1 isoforms determines the differential sensitivity of smooth muscle tissues to NO/cGMP-mediated relaxation. To test this hypothesis we propose to: 1) Define the relationship between SMMP subunit isoform expression and smooth muscle relaxant properties. The ability of NO donors and cGMP analogues to activate SMMP and cause smooth muscle relaxation will be measured in developing and mature smooth muscle tissues that express distinct MYPT1 isoforms. 2) Demonstrate the role of the SMMP subunit isoforms in determining sensitivity to cGMP-mediated relaxation by forcing their expression in vitro or in vivo. 3) Identify the mechanisms for the tissue-specific expression of the MYPT1 isoforms. Preliminary results using an in vivo gene delivery system have identified a putative intronic enhancer and a tissue-specific repressor of splicing of a MYPT1 alternative exon. These experiments will advance our understanding of the molecular mechanisms by which alternative splicing of exons leads to SMMP subunit isoforms and smooth muscle phenotypic diversity, and the role of the SMMP subunit isoforms in determining the diversity in smooth muscle relaxant properties.
Showing the most recent 10 out of 25 publications
