Comparative Assessment of the Ligand and Metal Ion Binding Properties of Integrins α9β1 and α4β1

Integrins α9β1 and α4β1 form a distinct structural class, but while α4β1 has been subjected to extensive study, α9β1 remains poorly characterized. We have used the small molecule N-(benzenesulfonyl)-(l)-prolyl-(l)-O-(1-pyrrolidinylcarbonyl)tyrosine (3) to investigate the biochemical properties of α9β1 and directly compare these properties with those of α4β1. Compound 3 has a high affinity for both integrins with K D values of ≤3 and 180 pM for α9β1 in 1 mM Mn2+(activating) and 1 mM Ca2+ and 1 mM Mg2+ (nonactivating) conditions and ≤5 and 730 pM for α4β1 under the corresponding conditions. Ca2+ treatment promoted the binding of 3 to both integrins (EC50 = 30 μM Ca2+ in both cases). Compound 3 binding to both integrins was also stimulated by the addition of the activating monoclonal antibody TS2/16. These findings indicate that the mechanisms by which metal ions and TS2/16 regulate ligand binding to α9β1 and α4β1 are similar. The binding of 3 to both integrins induced the mAb 9EG7 LIBS epitope, a property consistent with occupancy of the receptor's ligand binding site by 3. But whereas EGTA treatment inhibited the binding of 9EG7 to α4β1, it stimulated the binding of 9EG7 to α9β1. The 9EG7 and TS2/16 effects point to contributions of the β1-chains on binding. Cross-linking data revealed that the integrin α-chains are also involved in binding the small molecule, as stable linkages were observed on both the α9 chain of α9β1 and the α4 chain of α4β1. Extensive structure−activity analyses with natural and synthetic ligands indicate distinct features of the ligand binding pockets. Most notable was the estimated >1000-fold difference in the affinity of the integrins for VCAM-1, which binds α4β1with an apparent K D of 10 nM and α9β1 with an apparent K D of >10 μM. Differences were also seen in the binding of α9β1 and α4β1 to osteopontin. Compound 3 competed effectively for the binding of VCAM-1 and osteopontin to both integrins. While these studies show many similarities in the biochemical properties of α9β1 and α4β1, they identify important differences in their structure and function that can be exploited in the design of selective α9β1 and α4β1 inhibitors.