: Smooth muscles, which surround the periphery of hollow organs, contract to change organ shape or maintain tension to fix the shape and thereby control the flow of vital fluids, which are essential to the normal functioning of the cardiovascular, respiratory, digestive, and reproductive systems. If the regulation of smooth muscle contraction does not function properly, it could contribute to such diseases as high blood pressure, asthma, and premature birth. The goal of our work is to understand the molecular basis of the normal regulation of contraction. Smooth muscle contraction is primarily regulated by the Ca2+ controlled phosphorylation of myosin in the thick filament. However there is not a strict coupling between phosphorylation levels and the level of the resulting contractile force. Evidence indicates that there is additional regulation in the actin thin filament possibly involving tropomyosin (Tm). However the mechanism of this function is poorly understood. The long-range goal of this project is to uncover the molecular mechanisms whereby Tm, in concert with other thin filament proteins, regulates smooth muscle contraction. The main hypothesis of this proposal is that thin filament regulation occurs mainly by controlling the movement of Tm on the thin filament by myosin in the thick filament and by the other thin filament proteins, caldesmon and calponin, which are in turn regulated by phosphorylation and Ca2+binding proteins. This will be tested by monitoring Tm's position, and movement by measuring the Tm-actin distances as a function of myosin, caldesmon and calponin by fluorescence resonance energy transfer and correlated with actomyosin ATPase activity, an in vitro analogue of contraction. The results of these studies, which will be conducted on reconstituted thick and thin filaments, will help to further our understanding of the switching on/off of smooth muscle contraction and of smooth muscle's unique ability, especially vascular muscle, to maintain tension, and thus organ shape, at the cost of very little energy. These studies will compare myosin from vascular and gastrointestinal smooth muscles in order to better understand the ability of vascular muscle to maintain this tension.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL066219-03
Application #
6607294
Study Section
Experimental Cardiovascular Sciences Study Section (ECS)
Program Officer
Lin, Michael
Project Start
2001-08-20
Project End
2005-07-31
Budget Start
2003-08-01
Budget End
2004-07-31
Support Year
3
Fiscal Year
2003
Total Cost
$472,950
Indirect Cost
Name
Boston Biomedical Research Institute
Department
Type
DUNS #
058893371
City
Watertown
State
MA
Country
United States
Zip Code
02472
Graceffa, Philip; Lee, Eunhee; Stafford, Walter F (2013) Disulfide cross-linked antiparallel actin dimer. Biochemistry 52:1082-8
Hayes, David; Napoli, Vanessa; Mazurkie, Andrew et al. (2009) Phosphorylation dependence of hsp27 multimeric size and molecular chaperone function. J Biol Chem 284:18801-7
Graceffa, Philip; Mazurkie, Andrew (2005) Effect of caldesmon on the position and myosin-induced movement of smooth muscle tropomyosin bound to actin. J Biol Chem 280:4135-43
Graceffa, Philip; Dominguez, Roberto (2003) Crystal structure of monomeric actin in the ATP state. Structural basis of nucleotide-dependent actin dynamics. J Biol Chem 278:34172-80
Otterbein, Ludovic R; Cosio, Christophe; Graceffa, Philip et al. (2002) Crystal structures of the vitamin D-binding protein and its complex with actin: structural basis of the actin-scavenger system. Proc Natl Acad Sci U S A 99:8003-8