Methods of determining binding between Nogo receptor and p75 neurotrophin receptor
Inhibitors of Nogo Receptor (NgR)-p75 binding are used to reduce NgR-p75 binding mediated axon growth inhibition. Mixtures of NgR and p75 are used in pharmaceutical screens to characterize agents as inhibiting binding of NgR to p75 and promoting axon regeneration.
1. A method for assaying Nogo receptor (NgR)-neurotrophin receptor p75NTR (p75) binding, the method comprising the steps of:
providing a mixture comprising a p75-binding NgR domain and an NgR-binding p75 domain; and
directly measuring binding of the NgR domain to the p75 domain as an indication of the assayed NgR-p75 binding.
2. A method according to claim 1 , wherein binding of the NgR domain to the p75 domain is measured in the presence of an inhibitor of NgR-p75 binding and compared to a control amount of binding in the absence of the inhibitor, and wherein binding in the presence of the inhibitor is lower than the control binding.
3. A method according to claim 2 , wherein the inhibitor is selected from the group consisting of: an NgR peptide, a p75 peptide, an NgR peptide-specific antibody fragment and a p75 peptide-specific antibody fragment.
4. A method according to claim 1 , wherein one of the NgR domain and the p75 domain is soluble.
5. A method according to claim 1 , wherein one of the NgR domain and the p75 domain is expressed on the surface of a cell.
6. A method according to claim 1 , wherein the mixture is cell-free.
7. A method according to claim 1 , wherein the mixture comprises an NgR comprising the NgR domain, and a p75 extracellular domain comprising the p75-domain.
8. A method according to claim 1 , wherein one of the NgR domain and p75 domain is soluble and the other is expressed on the surface of a cell.
9. A method according to claim 1 wherein the measuring step is a co-immunoprecipitation assay, a solid phase ELISA-type assay, or a fluorescent polarization assay.
10. A method for characterizing an agent as modulating Nogo receptor (NgR)-neurotrophin receptor p75NTR (p75) binding, the method comprising the steps of:
A) incubating a mixture comprising a p75-binding NgR domain and an NgR-binding p75 domain under conditions that allow for binding between the two domains and directly measuring the degree of binding between the two domains, and
B) incubating a mixture comprising a p75-binding NgR domain, an NgR-binding p75 domain, and an agent under the same conditions as in A), and directly measuring the degree of binding between the two domains;
wherein a difference between the binding measured in A) and the binding measured in B) indicates that the agent modulates NgR-p75 binding.
11. A method according to claim 10 , wherein one of the NgR domain and the p75 domain is soluble.
12. A method according to claim 10 , wherein one of the NgR domain and the p75 domain is recombinantly expressed on the surface of a cell.
13. A method according to claim 10 , wherein the mixture is cell-free.
14. A method according to claim 10 , wherein the mixture comprises an NgR comprising the NgR domain, and a p75 extracellular domain comprising the p75-domain.
15. A method according to claim 10 , wherein one of the NgR domain and p75 domain is soluble and the other is expressed on the surface of a cell.
16. A method according to claim 10 wherein the detecting step is a co-immunoprecipitation assay, a solid phase ELISA-type assay, or a fluorescent polarization assay.
17. A method for assaying Nogo receptor (NgR)-neurotrophin receptor p75NTR (p75) binding, the method comprising the steps of:
recombinantly expressing a p75-binding NgR domain and an NgR-binding p75 domain; and
measuring binding of the NgR domain to the p75 domain.
18. A method according to claim 17 , wherein binding of the NgR domain to the p75 domain is measured in the presence of an inhibitor of NgR-p75 binding and compared to a control amount of binding in the absence of the inhibitor, and wherein binding in the presence of the inhibitor is lower than the control binding.
19. A method according to claim 17 , wherein one of the NgR domain and the p75 domain is soluble.
20. A method according to claim 17 , wherein one of the NgR domain and the p75 domain is expressed on the surface of a cell.
21. A method according to claim 17 , wherein the mixture is cell-free.