Smooth muscle, which lines the periphery of hollow organs, contracts to change the shape of the organ or maintains tension to fix the shape. An understanding of the regulation of smooth muscle contraction could help to form the basis for understanding a number of diseases, including hypertension in the vascular system and asthma in the airways of the lung. Smooth muscle contraction is primarily regulated by Ca2+-calmodulin dependent phosphorylation of myosin in the thick filament. Evidence is accumulating for an additional regulatory mechanism associated with the actin thin filament involving caldesmon, a long, thin, actin-binding protein, and tropomyosin. A much shorter isoform of caldesmon is also found in a wide range of non-muscle tissue including brain, liver, kidney, and platelets. Non-muscle caldesmon is thought to play a role in regulating the shape and motility of cells. However very little is known about its function. Thus the long range goal of this project is to uncover the molecular mechanisms whereby caldesmon, in concert with tropomyosin, regulates smooth muscle contraction and non-muscle motility and shape. The immediate objective is to map the arrangement of smooth muscle and non-muscle caldesmon, and tropomyosin, on the actin filament. The results of such studies will be correlated with actomyosin ATPase activity, the test tube analogue of contraction, in order to connect structure with function.
These aims will be pursued with reconstituted and native thin filaments and intact myofibrils. The project will make use of spectroscopic and cross-linking probes specifically attached to particular domains of caldesmon and tropomyosin in order to probe these regions of the proteins in their interaction with each other and other contractile proteins. In particular, spin labels, fluorescence labels, photocross-linkers, and disulfide cross-linkers will be used. Electron microscopy and the hydrodynamic techniques of viscosity and analytical ultracentrifugation will also be employed.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Research Project (R01)
Project #
5R01AR030917-10
Application #
2078732
Study Section
Molecular Cytology Study Section (CTY)
Project Start
1982-04-01
Project End
1997-04-30
Budget Start
1994-05-01
Budget End
1995-04-30
Support Year
10
Fiscal Year
1994
Total Cost
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
Mabuchi, K; Li, Y; Carlos, A et al. (2001) Caldesmon exhibits a clustered distribution along individual chicken gizzard native thin filaments. J Muscle Res Cell Motil 22:77-90
Graceffa, P (2000) Phosphorylation of smooth muscle myosin heads regulates the head-induced movement of tropomyosin. J Biol Chem 275:17143-8
Graceffa, P (1999) Movement of smooth muscle tropomyosin by myosin heads. Biochemistry 38:11984-92
D'Angelo, G; Graceffa, P; Wang, C A et al. (1999) Mammal-specific, ERK-dependent, caldesmon phosphorylation in smooth muscle. Quantitation using novel anti-phosphopeptide antibodies. J Biol Chem 274:30115-21
Graceffa, P (1997) Arrangement of the COOH-terminal and NH2-terminal domains of caldesmon bound to actin. Biochemistry 36:3792-801
Graceffa, P; Adam, L P; Morgan, K G (1996) Strong interaction between caldesmon and calponin. J Biol Chem 271:30336-9
Graceffa, P (1995) Cross-linking and fluorescence study of the COOH- and NH2-terminal domains of intact caldesmon bound to actin. J Biol Chem 270:30187-93
Szczesna, D; Graceffa, P; Wang, C L et al. (1994) Myosin S1 changes the orientation of caldesmon on actin. Biochemistry 33:6716-20
Stafford, W F; Chalovich, J M; Graceffa, P (1994) Turkey gizzard caldesmon molecular weight and shape. Arch Biochem Biophys 313:47-9

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