Abstract
A20FMDV2 is a peptide derived from the foot-and-mouth disease virus with a high affinity and selectivity for the alpha-v beta-6 (alpha v beta 6) arginyl-glycinyl-aspartic acid (RGD)-binding integrin. It has been shown to be an informative tool ligand in pre-clinical imaging studies for selective labelling of the alpha v beta 6 integrin in a number of disease models. In a radioligand binding assay using a radiolabelled form of the peptide ([H-3] A20FMDV2), its high affinity (K-D : 0.22 nmol/l) and selectivity (at least 85-fold) for alpha v beta 6 over the other members of the RGD integrin family was confirmed. [H-3] A20FMDV2 alpha v beta 6 binding could be fully reversed only in the presence of EDTA, whereas a partial reversal was observed in the presence of excess concentrations of an RGD-mimetic small molecule (SC-68448) or unlabelled A20FMDV2. Using flow cytometry on bronchial epithelial cells, the ligand-induced internalization of alpha v beta 6 by A20FMDV2 and latency-associated peptide-1 was shown to be fast (t(1/2): 1.5 and 3.1 min, respectively), concentration-dependent (EC50 : values 1.1 and 3.6 nmol/l, respectively) and was followed by a moderately slow return of integrin to the surface. The results of the radioligand binding studies suggest that the binding of A20FMDV2 to the RGD-binding site on alpha v beta 6 is required to maintain its engagement with the hypothesised A20FMDV2 synergy site on the integrin. In addition, there is evidence from flow cytometric studies that the RGD-ligand engagement of alpha v beta 6 post-internalization plays a role in delaying recycling of the integrin to the cell surface. This mechanism may act as a homeostatic control of membrane alpha v beta 6 following RGD ligand engagement. (C) 2016 S. Karger AG, Basel