Assessment of internalization and endosomal signaling: studies with insulin and EGF.

Endosomes are isolated from rat liver using high-speed centrifugation through sucrose density gradients. They are distinguishable from Golgi elements, with which they coisolate, by their capacity to concentrate internalized protein ligands (viz., insulin and epidermal growth factor (EGF)) in receptor-bound intact form. Endosomal signaling to relevant substrates can be readily shown for insulin and EGF receptor tyrosine kinases (RTKs), respectively. Both RTKs undergo dephosphorylation in endosomes. This can be inhibited by the powerful phosphotyrosine phosphatase inhibitors-the peroxovanadium compounds. In vivo administration of these compounds has been shown to activate selectively the endosomal insulin receptor kinase and promote signaling. Taken together, these observations constitute the basis for the signaling endosome hypothesis for which there is now ample evidence. Furthermore, a substantial body of work has documented the importance of endosomal signaling for growth, development, and disease.