Figure 5.

Test of membrane microdomains involved in leptin-induced internalization of the ligand-receptor complex in HEK293 cells. A) Effects of Dynasore and chlorpromazine on ¹²⁵I-leptin endocytosis. Dynasore inhibited leptin endocytosis in both ObRa- and ObR860-overexpressing cells in a dose-dependent manner. *P < 0.05; ***P < 0.005. B) Cellular distribution of Alexa555-leptin was similar in HEK293 cells overexpressing either ObRa or ObR860. In DMSO vehicle-treated cells, 20 min after initiation of endocytosis, leptin fluorescence was seen at both cell surface and vesicular compartments in cytoplasm. High dose of Dynasore (160 μM) blocked internalization in both ObRa- and ObR860-overexpressing cells. Chlorpromazine also blocked internalization of leptin mediated by ObR860 but not by ObRa. Lower dose of Dynasore (80 μM), nystatin, or filipin did not alter trafficking pattern of leptin.