Inhibition of Rab5 Activation During Insulin Receptor-Mediated Endocytosis.
Activation of receptor tyrosine kinases is a key feature in receptor signaling and membrane trafficking processes. In this study, we found that the insulin receptor tyrosine kinase activity is required for fusion between early endosomes. AG1024, a receptor tyrosine kinase inhibitor, blocked thein vitroendosome fusion in a concentration-dependent manner. We observed that Rab5: wild type partially rescued the fusion reaction, whereas Rab5: Q79L mutant fully rescued it. We also observed that treatment of cells with insulin receptor kinase inhibitor HNMPA-(AM)3blocked the formation of Rab5-positive endosomes as well as the activation of Rab5 upon addition of insulin in intact cells. HNMPA-(AM)3inhibitor also affected the endosomal co-localization of Rab5 and insulin receptor. However, the formation of Rab5: Q79L mutant-positive endosomes were not affected by the HNMPA-(AM)3inhibitor. In addition, HNMPA-(AM)3inhibitor affected the association of Rin1 to membrane upon insulin stimulation. Furthermore, Rin1 did not fully support endosome fusion in the presence of the AG1024 inhibitor. These results constitute the first evidence that, at least in part, the enzymatic activity of insulin receptor is required for the fusion events via the activation of Rab5.
Department of Biological Sciences, Florida International University, Miami, FL 33199.
Ivan
I
Jozic
Department Molecular & Integrative Physiology, University of Kansas Medical Center, Kansas City, KS 66160.
Gustavo
G
Blanco
M Alejandro
MA
Barbieri
Endosome fusion; Kinase Inhibitors; Receptor tyrosine kinase; small GTPases