Glucagon receptor recycling: role of carboxyl terminus, beta-arrestins, and cytoskeleton.
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, beta-arrestin1, beta-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 beta-actin in both hepatocytes and HEK-293 cells. Downregulation of beta-arrestin1 and beta-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, beta-arrestin1, beta-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.
Krilov
Lada
L
Gastroenterology Research Laboratory. Digestive Diseases Center, Dept. of Biochemistry and Molecular Biology, George Washington Univ., 2300 Eye St. NW, Washington, DC 20037, USA.
Nguyen
Amy
A
Miyazaki
Teruo
T
Unson
Cecilia G
CG
Bouscarel
Bernard
B
D000818:Animals; D019390:Arrestins / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D006224:Cricetinae; D003599:Cytoskeleton / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D005934:Glucagon / Q000378:metabolism*; D022781:Hepatocytes / Q000378:metabolism; D006801:Humans; D008247:Lysosomes / Q000378:metabolism; D008647:Mesocricetus; D009154:Mutation; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D018027:Receptors, Glucagon / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013997:Time Factors; D014162:Transfection; D022161:Transport Vesicles / Q000378:metabolism; D000071557:beta-Arrestins; D020691:rab GTP-Binding Proteins / Q000378:metabolism; D020695:rab4 GTP-Binding Proteins / Q000378:metabolism; D000099193:rab11 GTP-Binding Proteins
D019390:Arrestins; D005934:Glucagon; D018027:Receptors, Glucagon; D011993:Recombinant Fusion Proteins; D000071557:beta-Arrestins; D020691:rab GTP-Binding Proteins; D020695:rab4 GTP-Binding Proteins; D000099193:rab11 GTP-Binding Proteins
Trends Pharmacol Sci. 2001 Feb;22(2):91-6
11166853
Eur J Cell Biol. 1995 Apr;66(4):309-23
7544728
J Biol Chem. 1999 Nov 5;274(45):32248-57
10542263
J Immunol. 2001 Jan 15;166(2):1272-84
11145710
Nat Rev Mol Cell Biol. 2002 Apr;3(4):296-304
11994749
Mol Endocrinol. 2002 Dec;16(12):2720-32
12456793
J Biol Chem. 1988 Mar 15;263(8):3784-9
3346222
Endocrinology. 1992 Jul;131(1):447-57
1319325
J Biol Chem. 2003 Oct 24;278(43):41581-4
12947104
Nature. 2003 Mar 6;422(6927):37-44
12621426
J Clin Endocrinol Metab. 2000 Nov;85(11):4053-9
11095432
J Biol Chem. 2001 Jun 1;276(22):19452-60
11279203
J Cell Biol. 2000 May 15;149(4):901-14
10811830
Traffic. 2001 Mar;2(3):149-59
11260520
J Clin Endocrinol Metab. 1987 Jan;64(1):106-10
3536980
Metabolism. 1978 Nov;27(11):1691-709
360007
Am J Physiol Cell Physiol. 2001 Oct;281(4):C1396-402
11546678
FEBS Lett. 2006 Oct 16;580(24):5697-704
17010343
Am J Physiol. 1995 Feb;268(2 Pt 1):G300-10
7864127
J Biol Chem. 1995 Nov 17;270(46):27720-7
7499239
Nature. 1999 Sep 16;401(6750):286-90
10499588
Life Sci. 2003 Dec 5;74(2-3):225-35
14607250
Diabetes. 1997 Sep;46(9):1400-5
9287038
FEBS Lett. 2003 Jul 3;546(1):65-72
12829238
J Biol Chem. 2006 Sep 8;281(36):26391-9
16818493
Traffic. 2000 May;1(5):411-24
11208127
Nat Rev Mol Cell Biol. 2001 Feb;2(2):107-17
11252952
Biopolymers. 2008;90(3):287-96
18260137
J Cell Sci. 1999 Feb;112 ( Pt 3):329-38
9885286
J Biol Chem. 2000 Sep 1;275(35):27221-8
10854436
J Biol Chem. 2000 Jun 2;275(22):17201-10
10748214