Abstract
The low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor (LRP/alpha 2MR) binds and internalizes several plasma proteins including tissue-type plasminogen activator (t-PA) and alpha 2-macroglobulin-protease complexes (alpha 2M*). A 39-kDa protein that copurifies with LRP/alpha 2MR inhibits the binding and uptake of ligands by LRP/alpha 2MR, including t-PA and alpha 2M*. To define domains on the 39-kDa protein which are essential for inhibition of t-PA and alpha 2M* binding to LRP/alpha 2MR, we have generated bacterial expression constructs encoding discrete regions of the 39-kDa protein as fusion proteins with glutathione S-transferase. Inhibition of t-PA and alpha 2M* binding to LRP/alpha 2MR on rat hepatoma MH1C1 cells are shown to require amino acid residues 18-24 and 100-107 on the 39-kDa protein. Inhibition of t-PA but not alpha 2M* binding to LRP/alpha 2MR is also mediated by residues 200-225 and 311-319. The same 39-kDa protein constructs that inhibit alpha 2M* and t-PA binding to MH1C1 cells are able to bind directly to purified LRP/alpha 2MR immobilized on nitrocellulose. Thus, our studies demonstrate several specific regions on the 39-kDa protein which are required for the inhibition of t-PA and alpha 2M* binding to LRP/alpha 2MR.
Highlights
Theseresults suggest that in addition to residues 200-225, amino acid(s) within residues 311-319 of the 39-kDa protein are important for inhibiting lZ5I-t-PbAinding to LRP/a2MRon MHICl cells
Each symbol represents the average of duplicate determinations from two to four separate experiments
Each symbol represents the average of duplicate determinations from two tfoour separate experiments
Summary
47.1 and purified various regions of the 39-kDa protein and used the recombinant protein constructs to define residues on the 39-kDa protein which are required in the inhibition of t-PA and azM* binding to LRP/a2MR on rat hepatoma MHlCl. Identical to theresults seen for the inhibition of 1251-t-PAbinding to MHIClcells, the constructs GST/12-114, GST/18-114, and GST/12-107 inhibit "'IazM*binding to MHIC1 cells, whereas the constructs GST/ 24-114, GST/1-81, and GST/1-100 are unable toinhibit binding These results suggest that amino acid(s) withinresidues 18-24 and 100-107 on the 39-kDa protein are required for inhibition of both 12'I-t-PA and lz'I-azM*binding to LRP/ a2MRon MHICl cells. Because mixtures of GST/ 115-224 and GST/225-319 were unable to reconstitute the inhibition seen withGST/115-319, this may suggest that the conformation of 39-kDa protein is important for the inhibition of ligand binding to LRP/a2MR. dTeofine those essential
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have