title: Compartmentalization of GABA synthesis by GAD67 differs between pancreatic beta cells and neurons.
abstract: The inhibitory neurotransmitter GABA is synthesized by the enzyme glutamic acid decarboxylase (GAD) in neurons and in pancreatic β-cells in islets of Langerhans where it functions as a paracrine and autocrine signaling molecule regulating the function of islet endocrine cells. The localization of the two non-allelic isoforms GAD65 and GAD67 to vesicular membranes is important for rapid delivery and accumulation of GABA for regulated secretion. While the membrane anchoring and trafficking of GAD65 are mediated by intrinsic hydrophobic modifications, GAD67 remains hydrophilic, and yet is targeted to vesicular membrane pathways and synaptic clusters in neurons by both a GAD65-dependent and a distinct GAD65-independent mechanism. Herein we have investigated the membrane association and targeting of GAD67 and GAD65 in monolayer cultures of primary rat, human, and mouse islets and in insulinoma cells. GAD65 is primarily detected in Golgi membranes and in peripheral vesicles distinct from insulin vesicles in β-cells. In the absence of GAD65, GAD67 is in contrast primarily cytosolic in β-cells; its co-expression with GAD65 is necessary for targeting to Golgi membranes and vesicular compartments. Thus, the GAD65-independent mechanism for targeting of GAD67 to synaptic vesicles in neurons is not functional in islet β-cells. Therefore, only GAD65:GAD65 homodimers and GAD67:GAD65 heterodimers, but not the GAD67:GAD67 homodimer gain access to vesicular compartments in β-cells to facilitate rapid accumulation of newly synthesized GABA for regulated secretion and fine tuning of GABA-signaling in islets of Langerhans.
authors: Departments of Medicine and Microbiology/Immunology, Diabetes Center, University of California San Francisco, San Francisco, California, United States of America.
authors: Jamil
authors: J
authors: Kanaani
authors: Institute of Bioengineering, School of Life Sciences, École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland.
authors: Chiara
authors: C
authors: Cianciaruso
authors: Institute of Bioengineering, School of Life Sciences, École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland.
authors: Edward A
authors: EA
authors: Phelps
authors: Institute of Bioengineering, School of Life Sciences, École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland.
authors: Miriella
authors: M
authors: Pasquier
authors: Cell Isolation and Transplantation Center, Department of Surgery, Geneva University Hospitals and University of Geneva, Geneva, Switzerland.
authors: Estelle
authors: E
authors: Brioudes
authors: Institute of Biomedical Sciences, University of Copenhagen, Copenhagen, Denmark.
authors: Nils
authors: N
authors: Billestrup
authors: Departments of Medicine and Microbiology/Immunology, Diabetes Center, University of California San Francisco, San Francisco, California, United States of America; Institute of Bioengineering, School of Life Sciences, École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland.
authors: Steinunn
authors: S
authors: Baekkeskov
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D005968:Glutamate Decarboxylase / Q000032:analysis / Q000378:metabolism*; D006056:Golgi Apparatus / Q000378:metabolism; D006801:Humans; D050417:Insulin-Secreting Cells / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D009474:Neurons / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013572:Synaptic Vesicles / Q000378:metabolism; D005680:gamma-Aminobutyric Acid / Q000378:metabolism*
chemical_list: D005680:gamma-Aminobutyric Acid; D005968:Glutamate Decarboxylase; C402565:glutamate decarboxylase 1
references: Nature. 1989 Sep 21;341(6239):233-6
references: 2550826
references: Diabetes. 2007 Feb;56(2):320-7
references: 17259375
references: Neuron. 1991 Jul;7(1):91-100
references: 2069816
references: Diabetes. 1981 Feb;30(2):168-71
references: 6110597
references: Diabetes. 1991 Nov;40(11):1365-74
references: 1936599
references: J Cell Biol. 1992 Jul;118(2):309-20
references: 1321158
references: Science. 1976 Nov 5;194(4265):620-2
references: 185693
references: Cell Metab. 2006 Jan;3(1):47-58
references: 16399504
references: Proc Natl Acad Sci U S A. 1992 Mar 15;89(6):2115-9
references: 1549570
references: J Biol Chem. 2003 Jan 17;278(3):1966-74
references: 12414805
references: J Neurosci Res. 1998 Jun 15;52(6):736-41
references: 9669322
references: Diabetes. 1993 Dec;42(12):1799-808
references: 8243826
references: Diabetes. 2010 Jul;59(7):1694-701
references: 20413510
references: J Neurosci. 1988 Jun;8(6):2123-30
references: 3385490
references: J Cell Sci. 2004 Apr 15;117(Pt 10):2001-13
references: 15039456
references: Proc Natl Acad Sci U S A. 2003 Apr 1;100(7):4293-8
references: 12634427
references: Proc Natl Acad Sci U S A. 1997 Dec 9;94(25):14060-5
references: 9391152
references: J Gen Physiol. 2004 Mar;123(3):191-204
references: 14769845
references: J Cell Biol. 2002 Sep 30;158(7):1229-38
references: 12356867
references: Neurosci Lett. 2013 Mar 14;537:50-4
references: 23376695
references: J Cell Sci. 2004 Aug 1;117(Pt 17):3749-58
references: 15252115
references: J Biol Chem. 1999 Dec 24;274(52):37200-9
references: 10601283
references: Diabetes. 2004 Apr;53(4):1038-45
references: 15047619
references: Proc Natl Acad Sci U S A. 2011 Jul 12;108(28):11692-7
references: 21709230
references: Diabetologia. 2012 Jul;55(7):1985-94
references: 22538358
references: J Neurosci. 2006 Mar 29;26(13):3404-11
references: 16571747
references: Proc Natl Acad Sci U S A. 1999 Feb 16;96(4):1698-703
references: 9990087
references: J Cell Biol. 2010 Sep 6;190(5):911-25
references: 20805323
references: Diabetes Obes Metab. 2012 Oct;14 Suppl 3:143-51
references: 22928575
references: Neuron. 2004 Dec 16;44(6):977-86
references: 15603740
references: J Vis Exp. 2007;(7):255
references: 18989427
references: Endocrinology. 1992 Jan;130(1):167-78
references: 1370150
references: Diabetes. 1993 Mar;42(3):484-95
references: 8432419
references: J Cell Biol. 1994 Mar;124(6):927-34
references: 8132714
references: Neuroscience. 1999;93(4):1475-82
references: 10501472
references: J Neurochem. 2006 Apr;97(2):385-96
references: 16539672
references: Proc Natl Acad Sci U S A. 1993 Apr 1;90(7):2832-6
references: 7681990
references: Neuron. 2002 Dec 19;36(6):989-91
references: 12495613
references: Eur J Pharmacol. 2012 Feb 29;677(1-3):188-96
references: 22210053
references: Mol Membr Biol. 2009 Jan;26(1):42-54
references: 19169934
references: Endocrinology. 1991 Nov;129(5):2521-9
references: 1682137
references: Am J Physiol Endocrinol Metab. 2000 Sep;279(3):E684-94
references: 10950838
references: Nat Struct Mol Biol. 2007 Apr;14(4):280-6
references: 17384644
references: PLoS One. 2013 Jun 24;8(6):e67228
references: 23826240
references: J Biol Chem. 1995 Feb 3;270(5):2241-6
references: 7836456
references: Proc Natl Acad Sci U S A. 2014 Jun 24;111(25):E2524-9
references: 24927554
references: Diabetes. 1998 Dec;47(12):1848-56
references: 9836515
references: Diabetologia. 2002 Feb;45(2):242-52
references: 11935156
references: Nature. 1990 Sep 13;347(6289):151-6
references: 1697648
references: J Biol Chem. 1991 Nov 5;266(31):21257-64
references: 1939164
references: Diabetes. 2002 Jun;51(6):1763-71
references: 12031963
references: Clin Exp Immunol. 1994 Apr;96(1):129-37
references: 8149657
references: J Neurosci Res. 1993 Aug 1;35(5):567-76
references: 8377226
references: N Engl J Med. 1990 May 31;322(22):1555-60
references: 2135382
references: J Cell Sci. 2008 Feb 15;121(Pt 4):437-49
references: 18230651
references: Am J Physiol Endocrinol Metab. 2000 Oct;279(4):E773-81
references: 11001758
references: Proc Natl Acad Sci U S A. 1999 Oct 26;96(22):12911-6
references: 10536022