Glucagon receptor recycling: role of carboxyl terminus, {beta}-arrestins, and cytoskeleton

1 Gastroenterology Research Laboratory, Digestive Diseases Center, Department of Biochemistry and Molecular Biology, The George Washington University, Washington, District of Columbia; 2 Laboratory of Molecular Biology and Biochemistry, The Rockefeller University, New York, New York; and 3 Department of Medicine, The George Washington University, Washington, District of Columbia Submitted 2 May 2008 ; accepted in final form 20 August 2008 Glucagon receptor (GR) activity and expression are altered in several diseases, including Type 2 diabetes. Previously, we investigated the mechanism of GR desensitization and internalization. The present study focused on the fate of internalized GR. Using both hamster hepatocytes and human embryonic kidney (HEK)-293 cells, we showed that internalized GR recycled to the plasma membrane within 30–60 min following stimulation of the cells with 100 nM glucagon. In HEK-293 cells and during recycling, GR colocalized with Rab4, Rab11, β-arrestin1, β-arrestin2, and actin filaments, in the cytosolic and/or perinuclear domains. Glucagon treatment triggered redistribution of actin filaments from the plasma membrane to the cytosol. GR coimmunoprecipitated with β-actin in both hepatocytes and HEK-293 cells. Downregulation of β-arrestin1 and β-arrestin2 or disruption of the cytoskeleton inhibited recycling, but not internalization of GR. Deletion of the GR carboxyl-terminal 70 amino acids abolished internalization of GR in response to glucagon while deletion of the last 40 amino acids only did not affect GR internalization and recycling. After exposure of the cells to either high concentrations or prolonged duration of glucagon, GR colocalized with lysosomes. GR degradation was inhibited by lysosomal, but not proteosomal, inhibitors. In conclusion, GR recycles through Rab4- and Rab11- positive vesicles. The actin cytoskeleton, β-arrestin1, β-arrestin2, and the receptor's carboxyl terminus are involved in recycling. Prolonged stimulation with glucagon targets GR for degradation in lysosomes. Therefore, the present study provides a better understanding of the GR recycling mechanism, which could become useful in the treatment of certain diseases, including diabetes. actin; G protein-coupled receptor; lysosome; Rab4; Rab11 Address for reprint requests and other correspondence: B. Bouscarel, Dept. of Biochemistry and Molecular Biology, George Washington Univ., 2300 Eye St. NW, Washington, DC 20037 (e-mail: bbouscarel{at}mfa.gwu.edu )