Downregulation of the v6 Integrin via RGD Engagement Is Affinity and Time Dependent.

The arginyl-glycinyl-aspartic acid (RGD) integrin alpha-v beta-6 (v6) has been identified as playing a key role in the activation of transforming growth factor- (TGF) that is hypothesized to be pivotal in the development of fibrosis and other diseases. In this study, v6 small molecule inhibitors were characterized in a range of in vitro systems to determine affinity, kinetics, and duration of TGF inhibition. High v6 binding affinity was shown to be correlated with slow dissociation kinetics. Compound (high v6 affinity, slow dissociation) and SC-68448 (low v6 affinity, fast dissociation) induced concentration- and time-dependent internalization of v6 in normal human bronchial epithelial (NHBE) cells. After washout, the v6 cell surface repopulation was faster for SC-68448 compared with compound In addition, v6-dependent release of active TGF from NHBE cells was inhibited by compound and SC-68448. After washout of SC-68448, release of active TGF was restored, whereas after washout of compound the inhibition of TGF activation was maintained and only reversible in the presence of a lysosomal inhibitor (chloroquine). However, SC-68448 was able to reduce total levels of v6 in NHBE cells if present continuously. These observations suggest v6 can be degraded after high affinity RGD binding that sorts the integrin for lysosomal degradation after internalization, likely due to sustained engagement as a result of slow dissociation kinetics. In addition, the v6 integrin can also be downregulated after sustained engagement of the RGD binding site with low affinity ligands that do not sort the integrin for immediate lysosomal degradation. SIGNIFICANCE STATEMENT: The fate of RGD integrin after ligand binding has not been widely investigated. Using the αvβ6 integrin as a case study, we have demonstrated that RGD-induced downregulation of αvβ6 is both affinity and time dependent. High affinity ligands induced downregulation via lysosomal degradation, likely due to slow dissociation, whereas sustained low affinity ligand engagement was only able to decrease αvβ6 expression over longer periods of time. Our study provides a potential unique mechanism for obtaining duration of action for drugs targeting integrins.