PUBLICATION
Mapping the ligand-binding pocket of integrin alpha5beta1 using a gain-of-function approach
- Authors
- Mould, A.P., Koper, E.J., Byron, A., Zahn, G., and Humphries, M.J.
- ID
- ZDB-PUB-090921-2
- Date
- 2009
- Source
- The Biochemical journal 424(2): 179-189 (Journal)
- Registered Authors
- Mould, Paul
- Keywords
- none
- MeSH Terms
-
- Animals
- Binding Sites
- CHO Cells
- Cricetinae
- Cricetulus
- Fibronectins/genetics
- Fibronectins/metabolism
- Humans
- Integrin alpha5beta1/antagonists & inhibitors
- Integrin alpha5beta1/chemistry
- Integrin alpha5beta1/metabolism*
- Models, Molecular
- Mutation
- Protein Multimerization
- Protein Structure, Quaternary
- Structural Homology, Protein
- Zebrafish/metabolism*
- PubMed
- 19747169 Full text @ Biochem. J.
Citation
Mould, A.P., Koper, E.J., Byron, A., Zahn, G., and Humphries, M.J. (2009) Mapping the ligand-binding pocket of integrin alpha5beta1 using a gain-of-function approach. The Biochemical journal. 424(2):179-189.
Abstract
Integrin alpha5beta1 is a key receptor for the extracellular matrix protein fibronectin. Antagonists of human alpha5beta1 have therapeutic potential as anti-angiogenic agents in cancer and diseases of the eye. However, the structure of the integrin is unsolved and the atomic basis of fibronectin and antagonist binding by alpha5beta1 is poorly understood. Here we demonstrate that zebrafish alpha5beta1 integrins do not interact with human fibronectin or the human alpha5beta1 antagonists JSM6427 and cyclic peptide CRRETAWAC. Zebrafish alpha5beta1 integrins do bind zebrafish fibronectin-1, and mutagenesis of residues on the upper surface and side of the zebrafish alpha5 subunit beta-propeller domain shows that these residues are important for the recognition of RGD and synergy sites in fibronectin. Using a gain-of-function analysis involving swapping regions of the zebrafish alpha5 subunit with the corresponding regions of human alpha5 we show that blades 1-4 of the beta-propeller are required for human fibronectin recognition, suggesting that fibronectin binding involves a broad interface on the side and upper face of the beta-propeller domain. We find that the loop connecting blades 2 and 3 of the beta-propeller (D3-A3 loop) contains residues critical for antagonist recognition, with a minor role played by residues in neighbouring loops. A new homology model of human alpha5beta1 supports an important function for D3-A3 loop residues Trp-157 and Ala-158 in the binding of antagonists. These results will aid the development of reagents that block alpha5beta1 functions in vivo.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping