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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.
Highlights
In mammals, two highly homologous non-allelic isoforms of the enzyme glutamate decarboxylase (GAD), GAD65 and GAD67, synthesize the major inhibitory neurotransmitter GABAPLOS ONE | DOI:10.1371/journal.pone.0117130 February 3, 2015Targeting of GAD67 and GAD65 in Pancreatic Beta Cells by a JFRF Advanced Postdoctoral Fellowship (3APF-2014-208-A-N)
We assessed whether GAD67-GFP and GAD65-GFP acquire membrane anchoring in the insulinoma
We have previously shown in neurons, that the hydrophilic GAD67 isoform is anchored to membranes and robustly targeted to the Golgi compartment and presynaptic clusters either by association with the hydrophobic GAD65 isoform [22, 54] or by a distinct GAD65-independent mechanism that involves association with a different membrane-anchoring moiety [23]
Summary
Two highly homologous non-allelic isoforms of the enzyme glutamate decarboxylase (GAD), GAD65 and GAD67, synthesize the major inhibitory neurotransmitter GABA. In addition to serving as an intracellular source of fuel in β-cells [28], GABA is released from rat β-cells by regulated Ca+2-dependent exocytosis during membrane depolarization in response to physiological voltages [4] Consistent with this finding, the islets of Langerhans express components required for GABA signaling. In order to elucidate the membrane trafficking and fate of GAD65 and GAD67 in β-cells, we have developed a method to grow and transfect primary cultures of islet cells in order to study the GAD isoforms in β-cells by high-resolution confocal microscopy This approach has revealed that GAD65 membrane trafficking is similar to neurons, but that β-cells lack the neuronal mechanism for GAD65-independent mechanism of GAD67 membrane anchoring [23]. In β-cells, GAD67 can only be anchored to membranes by dimerization with GAD65 and the β-cell is distinct from neurons in only expressing soluble GAD67 in the absence of GAD65
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