alpha2Macroglobulin is an abundant blood protein, that may play a critical role in cell growth and regulation through its ability to interact with many proteases and growth factors. The long term goals of this project are to elucidate the mechanism of action of alpha2-macroglobulin and to understand the basis for its physiological importance in interacting with proteases and growth factors. The experiments detailed in the present proposal are aimed at testing two hypotheses:- 1. That the specificity and efficiency of trapping of proteases by alpha2- macroglobulin can be understood in terms of the structure and location of the bait region (the site of limited proteolysis) and the internal thiol ester. 2. That growth factors interact only with an activated form of alpha2- macroglobulin which results either from initial reaction with limiting amounts of protease or else is present at a small equilibrium concentration, which is normally overlooked in examination of stoichiometric reactions. To test these hypotheses it is proposed to pursue the following specific aims:- 1. To further localize the functionally important bait region an thiol ester groups within the alpha2-macroglobulin tetramer using spectroscopic methods. 2. To obtain as much structural information as possible about the bait region from human alpha2-macroglobulin before and after reaction with various proteases using 1H NMR spectroscopic methods. 3. To continue attempts to obtain crystals of native and modified forms of alpha2-macroglobulin suitable for X-ray crystallographic analysis. 4. To determine the conditions necessary for interaction of the growth factors bFGF, EGF, PDGF, and TGF-beta with alpha2-macroglobulin, and to characterize the stoichiometry of interaction, the nature of the interaction, and the state of the alpha2-macroglobulin.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Research Project (R01)
Project #
5R01HD028187-02
Application #
3329805
Study Section
Pathobiochemistry Study Section (PBC)
Project Start
1991-05-01
Project End
1996-04-30
Budget Start
1992-05-01
Budget End
1993-04-30
Support Year
2
Fiscal Year
1992
Total Cost
Indirect Cost
Name
Vanderbilt University Medical Center
Department
Type
Schools of Medicine
DUNS #
004413456
City
Nashville
State
TN
Country
United States
Zip Code
37212
Gettins, P G (1995) Thiol ester cleavage-dependent conformational change in human alpha 2-macroglobulin. Influence of attacking nucleophile and of Cys949 modification. Biochemistry 34:12233-40
Gettins, P G; Hahn, K H; Crews, B C (1995) Alpha 2-macroglobulin bait region variants. A role for the bait region in tetramer formation. J Biol Chem 270:14160-7
Gettins, P G; Crews, B; Beth, A H et al. (1995) Bait region-thiol ester mapping in human alpha 2-macroglobulin. FEBS Lett 367:137-40
Gettins, P G (1994) 1H- and 19F-NMR approaches to the study of the structure of proteins larger than 25 kDa. Int J Biol Macromol 16:227-35
Gettins, P G; Crews, B C (1994) Binding of epidermal growth factor to human alpha 2-macroglobulin. Significance for cytokine alpha 2-macroglobulin interactions. Ann N Y Acad Sci 737:383-98
Gettins, P G; Boel, E; Crews, B C (1994) Thiol ester role in correct folding and conformation of human alpha 2-macroglobulin. Properties of recombinant C949S variant. FEBS Lett 339:276-80
Gettins, P G; Crews, B C (1993) Human alpha 2-macroglobulin structure. Location of Cys-949 residues within a half-molecule measured by fluorescence energy transfer. FEBS Lett 332:211-4
Gettins, P G; Crews, B C (1993) Epidermal growth factor binding to human alpha 2-macroglobulin. Implications for alpha 2-macroglobulin-growth factor interactions. Biochemistry 32:7916-21
Gettins, P G; Beechem, J M; Crews, B C (1993) alpha 2-Macroglobulin bait region integrity. Role in determining fast-form structure. FEBS Lett 325:267-70