SNARE Proteins Underpin Insulin‐Regulated GLUT4 Traffic

Molecular and Cellular Biology

Gould

Bryant

2014

Self Cite

Insulin stimulates glucose transport into fat and muscle cells by increasing the exocytic trafficking rate of the GLUT4 facilitative glucose transporter from intracellular stores to the plasma membrane. Delivery of GLUT4 to the plasma membrane is mediated by formation of functional SNARE complexes containing syntaxin4, SNAP23, and VAMP2. Here we have used an in situ proximity ligation assay to integrate these two observations by demonstrating for the first time that insulin stimulation causes an increase in syntaxin4-containing SNARE complex formation in adipocytes. Furthermore, we demonstrate that insulin brings about this increase in SNARE complex formation by mobilizing a pool of syntaxin4 held in an inactive state under basal conditions. Finally, we have identified phosphorylation of the regulatory protein Munc18c, a direct target of the insulin receptor, as a molecular switch to coordinate this process. Hence, this report provides molecular detail of how the cell alters membrane traffic in response to an external stimulus, in this case, insulin.A major consequence of insulin binding its receptor on fat and muscle cells is a change in localization of the GLUT4 facilitative glucose transporter. In the absence of insulin, ϳ95% of the transporter is retained intracellularly. Activation of the insulin receptor tyrosine kinase culminates in a 10-to 20-fold increase in the amount of GLUT4 present at the cell surface, accounting for the increased rate of glucose transport into these cells (1). GLUT4 is sequestered away from the cell surface in the absence of insulin by continually cycling through two interrelated endosomal cycles (1). The first operates between the plasma membrane and early and recycling endosomes. This is a fast-trafficking loop that keeps steady-state levels of transporter at the cell surface low under basal conditions. GLUT4 is further sorted from this cycle into a more slowly operating loop between recycling endosomes, the transGolgi network, and a population of vesicles termed GSVs (GLUT4 storage vesicles). It is from GSVs that GLUT4 is mobilized to the cell surface in response to insulin (1).Like all eukaryotic membrane trafficking events, insulin-regulated trafficking of GLUT4 is mediated by formation of specific SNARE complexes. Cell surface delivery of GLUT4 is mediated by a SNARE complex containing the plasma membrane-localized syntaxin4 (Sx4) and SNAP23 t-SNAREs and the VAMP2 v-SNARE (2). SNARE complex formation contributes to specificity of membrane traffic and also provides energy for bilayer fusion (3). Thus, regulating SNARE complex assembly allows the cell to regulate membrane traffic, but whether insulin stimulates SNARE complex formation remains unknown. Sec1/Munc18 (SM) proteins are key regulators of membrane traffic exerting their effect through their cognate SNARE proteins, but their precise mode of action is not understood (3). The Munc18c SM protein binds to Sx4 and is required for insulin-regulated delivery of GLUT4 to the surface of fat and muscle cells (2). Intriguingly, M...

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confidence: 99%

“…Cell surface delivery of GLUT4 is mediated by a SNARE complex containing the plasma membrane-localized syntaxin4 (Sx4) and SNAP23 t-SNAREs and the VAMP2 v-SNARE (2). SNARE complex formation contributes to specificity of membrane traffic and also provides energy for bilayer fusion (3).…”

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confidence: 99%

Insulin Stimulates Syntaxin4 SNARE Complex Assembly via a Novel Regulatory Mechanism

Molecular and Cellular Biology

Gould

Bryant

2014

Self Cite

“…The identification of v-SNARE proteins within purified GSVs [8] provided the first evidence that GLUT4 translocation requires SNARE proteins (reviewed in [9]). Subsequent studies have indicated the VAMP2 (vesicleassociated membrane protein 2) isoform as being the key v-SNARE in GSVs (reviewed in [9], but, for an alternative view, see [10]).…”

Section: Glut4 Trafficking Uses Snare Proteinsmentioning

confidence: 99%

“…Disrupting the function of these molecules (e.g. using antibody microinjection, transgenic knockout or siRNA approaches) supports a role for these proteins in the insulin-stimulated delivery of GLUT4 to the cell surface (see, for example, [9,[11][12][13]). Consistent with this, Sx4/SNAP23-containing liposomes could fuse with liposomes containing VAMP2, providing functional evidence that this SNARE combination is sufficient to drive membrane fusion in vitro [14].…”

mentioning

confidence: 98%

“…Subsequent studies have indicated the VAMP2 (vesicleassociated membrane protein 2) isoform as being the key v-SNARE in GSVs (reviewed in [9], but, for an alternative view, see [10]). Studies from numerous laboratories identified Sx4 (syntaxin 4) and SNAP23 (23 kDa synaptosome-associated protein) as the key t-SNAREs on the plasma membrane of adipocytes [9,11]. Disrupting the function of these molecules (e.g.…”

mentioning

confidence: 99%

See 1 more Smart Citation

Studies of the regulated assembly of SNARE complexes in adipocytes

Biochemical Society Transactions

Sadler

Black

et al. 2014

Insulin plays a fundamental role in whole-body glucose homeostasis. Central to this is the hormone's ability to rapidly stimulate the rate of glucose transport into adipocytes and muscle cells [1]. Upon binding its receptor, insulin stimulates an intracellular signalling cascade that culminates in redistribution of glucose transporter proteins, specifically the GLUT4 isoform, from intracellular stores to the plasma membrane, a process termed 'translocation' [1,2]. This is an example of regulated membrane trafficking [3], a process that also underpins other aspects of physiology in a number of specialized cell types, for example neurotransmission in brain/neurons and release of hormone-containing vesicles from specialized secretory cells such as those found in pancreatic islets. These processes invoke a number of intriguing biological questions as follows. How is the machinery involved in these membrane trafficking events mobilized in response to a stimulus? How do the signalling pathways that detect the external stimulus interface with the trafficking machinery? Recent studies of insulin-stimulated GLUT4 translocation offer insight into such questions. In the present paper, we have reviewed these studies and draw parallels with other regulated trafficking systems.

Proximity Ligation Assay to Study the GLUT4 Membrane Trafficking Machinery

Methods in Molecular Biology

Gould

Bryant

2017

In this chapter a detailed protocol of proximity ligation assay (PLA) is described thoroughly. PLA is a technique that allows detection of protein associations in situ, providing a sensitive and selective approach for protein-protein interaction studies. We demonstrate the technique by applying it for trafficking studies of the facilitative glucose transporter GLUT4. Trafficking of GLUT4 from perinuclear depots to the plasma membrane is regulated by insulin in adipocytes and muscle cells, and mediated by formation of functional SNARE complexes containing Syntaxin4, SNAP23, and VAMP2. The Sec1/Munc18 (SM) protein Munc18c also plays a key role in insulin-stimulated GLUT4 translocation via a series of different interactions with the SNARE complex and/or with the SNARE proteins individually. Studying the interactions that occur between SNARE proteins themselves and also with Munc18c in insulin-responsive cells is critical to further understand SNARE protein function and GLUT4 trafficking mechanism in general.