Integrin trafficking plays an important role in cellular motility and 
cytokinesis. Integrins undergo constant endo/exocytic shuttling to facilitate 
the dynamic regulation of cell adhesion. Integrin activity toward the components 
of the extracellular matrix is regulated by the ability of these receptors to 
switch between active and inactive conformations. Several cellular signalling 
pathways have been described in the regulation of integrin traffic under 
different conditions. However, the interrelationship between integrin activity 
conformations and their endocytic fate have remained incompletely understood. 
Here, we have investigated the endocytic trafficking of active and inactive β1 
integrins in cancer cells. Both conformers are endocytosed in a clathrin- and 
dynamin-dependent manner. The net endocytosis rate of the active β1 integrins is 
higher, whereas endocytosis of the inactive β1 integrin is counteracted by rapid 
recycling back to the plasma membrane via an ARF6- and early endosome antigen 
1-positive compartment in an Rab4a- and actin-dependent manner. Owing to these 
distinct trafficking routes, the two receptor pools display divergent 
subcellular localization. At steady state, the inactive β1 integrin is mainly on 
the plasma membrane, whereas the active receptor is predominantly intracellular. 
These data provide new insights into the endocytic traffic of integrins and 
imply the possibility of a previously unappreciated crosstalk between pathways 
regulating integrin activity and traffic.