β-granule transport and exocytosis

seminars in CELL & DEVELOPMENTAL BIOLOGY, Vol. 11, 2000: pp. 253–266 doi: 10.1006/scdb.2000.0174, available online at http://www.idealibrary.com on

β-granule transport and exocytosis Richard A. Easom

Regulated β-granule exocytosis is critical for the ability of the β-cell to finely control body glucose homeostasis. This is now understood to be a multistage process whereby β-granules are transported from biosynthetic/storage sites in the cell cytoplasm and targeted to specific regions of the plasma membrane. Exocytosis is achieved when these granules are triggered to fuse with the membrane by an elevated cytosolic Ca2+ . Dramatic advances have been made recently in our understanding of the protein–protein interactions and regulatory signals that govern intracellular transport and fusion. Although best understood for exocytosis from neurons and neuroendocrine cells, similar processes are thought to be conserved in the β-cell.

Accordingly, more than 99% of endogenous insulin is released via a tightly regulated pathway.3 Insulin release is ultimately achieved when βgranules fuse with the plasma membrane. This process, called exocytosis, is mediated by a set of highly conserved membrane proteins known as SNAp REceptors (SNAREs), [for soluble NSF attachment protein (SNAP) receptors]4 which form the core of the fusion machinery.5 As in regulated exocytosis from neurons and neuroendocrine cells, this process is highly Ca2+ -dependent.2 Granule fusion is, however, only the final event of a complex multistage process that necessarily commences with the directed transport of β-granules from the biosynthetic and/or storage pools in the cell cytosol to exocytotically defined regions of the β-cell. At the plasma membrane, β-granules must also proceed through several additional steps including docking and ATP-dependent priming before they are competent to fuse with the plasma membrane (Figure 1). Each of these necessary steps is likely regulated for the optimal control of insulin exocytosis. D-glucose is the primary physiological stimulator of insulin secretion and controls β-cell function via the generation of multiple signals derived from its metabolism.6 Among these is an elevation in cytosolic ATP:ADP ratio which, via sequential closing and activation of KATP channels and voltage-dependent (L-type) Ca2+ channels, promotes an elevation in intracellular Ca2+ ([Ca2+ ]i ). A tight control of β-granule fusion by glucose is achieved via the localization of these L-type Ca2+ channels to exocytotically active regions of the plasmalemma.7 Ca2+ (and ATP) is also necessary for the processes of granule recruitment which may, under physiological conditions, be reliant on the mobilization of Ca2+ from intracellular stores induced by incretin hormones such as acetylcholine (ACh) or cholecystakinin (CCK). Ca2+ is by no means the only signal required for insulin exocytosis as GTP (via monomeric and heterotrimeric G-proteins)8 and fatty acyl CoAs,9 derived from glucose metabolism, are capable of modulating this process. However, for

Key words: β-cell / insulin / exocytosis / calcium / protein phosphorylation c 2000 Academic Press

Beta (β)-cells of the endocrine pancreas are critical in the regulation of the body’s energy balance in that they provide the only source of the necessary anabolic hormone, insulin. As described in prior articles of this series, the β-cell is dedicated to the biosynthesis and accurate processing of hormone pre-cursors to produce mature insulin which it then stores in large dense core secretory vesicles (LDCV), herein called β-granules. The healthy β-cell maintains in excess of 13 000 granules1 but only a fraction of these are released even under maximal stimulatory conditions.2 The fine control of glucose homeostasis is thus dependent not so much on the acute regulation of insulin biosynthesis, but rather on the appropriate and timely release of mature hormone from the β-cell.

From the Department of Molecular Biology & Immunology, University of North Texas Health Science Center at Fort Worth, Fort Worth, TX 76107-2699, USA. E-mail: [email protected] . c

2000 Academic Press 1084–9521 / 00 / 040253+ 14 / $35.00/0 / 0

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R. A. Easom

Granule Recruitment

actin microfilament

(1)

Granule Fusion

ATP

(3)

(2) docked

primed

Figure 1. Multistage process of insulin exocytosis. An overview of insulin exocytosis describing distinct pools of granules at various stages of progression towards fusion. The elevation of Ca2+ at the plasma membrane induces the fusion of granules in a primed, readily releasable pool.3 This pool is replenished rapidly from granules docked at the plasma membrane by ATP-dependent priming mechanisms2 but then more slowly by granule recruitment from cytosolic storage and biosynthetic pools.1

The former, revealed by the use of caged Ca2+ , is thought to represent the rapid exocytosis of a fusion competent pool of granules, a so-called readily releasable pool (RRP). Exocytosis of these granules is independent of ATP indicating that they have progressed past ATP-dependent priming12 (Figure 1). In contrast, the slower recruitment phase is both Ca2+ and ATP-dependent and likely represents the delayed exocytosis of granules that have to be recruited into the RRP from reserve pools and require maturation, via priming, to achieve fusion-competency. At higher Ca2+ concentrations (17–87 µM), the early phase capacitance increase can be further resolved into three distinct kinetic components.13 Only the latter two of these, however, correspond to β-granule fusion based on simultaneous capacitance and amperometry measurements, with the most rapid ‘burst’ of exocytosis occurring independently of 5-HT release.13, 14 This burst may correspond to the fusion of SLMVs although this suggestion has yet to be confirmed. That early phase β-granule exocytosis retains two kinetic components, referred to as mode 1 and mode 2, implies that the readily releasable pool may itself be internally comprised of distinct granule populations.13, 15 Whether these are released by a sequential, linear pathway or via parallel pathways with inherently different rates is not clear.13, 14 It may be that both populations of granules are equally competent to fuse but that the distinct kinetics are explained by the position of granules relative to Ca2+ channels localized to the exocytotic complexes (see below) as recently documented in chromaffin cells.16 Either way,

the sake of clarity, most of the discussion here centers around events leading to Ca2+ -triggered insulin exocytosis.

Insulin exocytosis; an electrophysiological perspective The study of regulated exocytosis mechanisms has been greatly enhanced by the advent of electrophysiological techniques which provide high-time resolution and quantitation of granule fusion events.10 Insulin exocytosis has been studied by both patch-clamp capacitance, which measures exocytosis based on transient increases in membrane surface area, and amperometry which measures the actual release of a charged monoamine (5-hydroxytryptamine, 5-HT) pre-loaded specifically into β-granules. The latter technique offers several advantages over capacitance, among which is the ability to distinguish fusion of β-granules from smaller, clear vesicles such as the synaptic-like microvesicles (SLMV) which are prominent in the β-cell and secrete GABA.11 Collectively, these studies reveal insulin exocytosis to exhibit multiple kinetic components which are commonly interpreted as the sequential release of distinct pools of β-granules in different functional states relative to exocytosis. By cell capacitance, insulin exocytosis provoked by the modest elevation of [Ca2+ ]i (to 2–3 µM) can be resolved into a rapid (<200 ms) release phase followed by a slower, sustained release phase.12 254

β-granule transport and exocytosis

Storage Pool

MAP-2 Synapsin I

Microtubules

Myosin ER

CaMKII

MAP-2

P IP3

Ca2+

Actin Microfilaments

P

MLCK P

ACh

Ca2+ VDCC

Morphologicallydocked primed

fused

Figure 2. Regulation of granule recruitment by Ca2+ -dependent protein kinases. A model for the Ca2+ /ATP-dependent process of β-granule transport is illustrated. The elevation of intracellular Ca2+ via mobilization from intracellular stores, or via diffusion from Ca2+ influx, activates CaM Kinase II (CaMKII) on the β-granule or myosin light chain kinase (MLCK). The phosphorylation of cytoskeletal proteins (P) is hypothesized to facilitate the navigation of β-granules through the microfilament cell web to permit docking at the plasma membrane.

Molecular mechanisms of insulin exocytosis

a challenge for the future is to determine the molecular basis for each of these putative granule pools. These studies have suggested that the exocytosis of distinct granule pools contributes to the well characterized biphasic insulin release induced by glucose physiologically. Only a small fraction of β-granules (estimated to be as low as 0.05% or ∼40 granules12 ) resides in the primed, RRP in the β-cell consistent with infrequent visualization of granules in close proximity to the plasma membrane in morphological studies.17 While seemingly smaller than equivalent pools in neuroendocrine cells,18 the β-cell RRP is calculated to have a capacity sufficient to support first-phase secretion.12 Biphasic insulin secretion may therefore be the result of the sequential exocytosis of a readily releasable pool, triggered by Ca2+ influx, followed by the energy-dependent recruitment and release of granules from reserve pools.12, 13 The validity of this hypothesis requires further substantiation, but it is consistent with a number of observations. For instance, first-phase secretion is mimicked by secretagogues (e.g. high K+ ) whose primary action is to elevate [Ca2+ ]i . In addition, cooling (to 24 ◦ C), which affects granule mobilization primarily, profoundly suppresses the slower phase of insulin exocytosis, more so than most other neuroendocrine/endocrine cells.19 Collectively, these observations argue that granule recruitment is critical to sustained insulin exocytosis expected of the physiologically effective β-cell.

β-granule transport The transport of β-granules from biosynthetic or storage pools in the cytoplasm to the plasma membrane is thought to be facilitated by their interaction first with microtubules and then with microfilament networks of the β-cell cytoskeleton.20 β-granule interactions with microtubules have been observed both in vitro 21 and in situ 20 and use microtubules as a guide to provide directional control to this movement. The necessary motive force is most likely provided by ATP-dependent motor proteins such as kinesin also associated with the β-granule.22 The best evidence supporting a role for microtubules in insulin secretion remains the observations that mitotic spindle inhibitors, that either disrupt or stabilize microtubule polymers, uniformly suppress hormone release. It is significant, however, that where tested, these drugs preferentially affect second-phase insulin release.20, 23 Granule mobilization may therefore be dependent on localized areas of microtubule catastrophe and rescue, a process that may be regulated by phosphorylation of microtubule-associated proteins (see below) and motor proteins. The actin microfilament network is visualized as a cortical band of fine filaments just beneath and often parallel to the plasma membrane.17 Referred to as the ‘cell web’, this network may function in two ways. First, since its disintegration using cytochalasin 255

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the microtubule-associated protein, MAP-2,36 and the actin-binding protein, synapsin I,37, 38 are in situ substrates for CaM Kinase II in β-cells. Both proteins are also known substrates for cyclic AMP-dependent protein kinase (PKA)39, 40 suggesting that they may also mediate the ability of PKA to facilitate granule recruitment.30, 41 The phosphorylation of MAP-2 by CaM Kinase II has a profound impact on microtubule stability42, 43 and thus may mediate dynamic changes in the microtubule network shown to be necessary for insulin secretion.44 The significance of CaM Kinase II phosphorylation of synapsin I is less obvious37, 45 but it seems reasonable to hypothesize that it may be important in the control of microfilament network integrity. CaM Kinase II is activated by both acetylcholine46 and glucose44, 47 in a manner that correlates with insulin release. Moreover, the inhibition of CaM Kinase II activation prevents the enhancement of granule replenishment by acetylcholine.31 Based on the partial localization of CaM Kinase II to the β-granule45, 48 and its unique regulatory properties to retain activity beyond periods of [Ca2+ ]i elevation,44 this kinase would seem perfectly poised for the regulation of granule mobilization and the maintenance of granule pool capacity.44, 47

or Clostridium botulinum toxin C2 enhances insulin secretion,17, 24 this actin microfilament appears to act as a barrier denying granule access to the plasma membrane. In this role, the recruitment of granules would require the physical dissolution, or at least rearrangement, of this barrier during active secretion (Figure 2). Indeed, localized patches of microfilament disruption, promoted by scinderin or gelsolin, have been observed in stimulated chromaffin cells25 but similar responses have not been detected in cultured β-cells stimulated to secrete insulin.24 Under alternative conditions, this microfilament network appears proactive,24 suggesting that microfilaments also harbor the capacity to promote granule movement towards the plasma membrane, presumably via actin–myosin interactions. It is conceivable that both mechanisms are important to the process of β-granule recruitment but information regarding the dynamic control of microfilament organization is lacking. In this light, it is interesting that a novel zinc-finger-containing protein, NoC2, is expressed in the β-cell26 and it has the capacity to reorganize actin microfilaments via interaction with zyxin.27 A functional relationship between this protein and insulin exocytosis, however, has yet to be demonstrated. How cytoskeleton mechanics are harnessed to direct β-granule transport remains unclear although protein phosphorylation has long been implicated.28 This concept is certainly consistent with the known dependence of granule recruitment on ATP12, 29 and Ca2+30, 31 but is further reinforced in observations that granule movement provoked by various insulin secretagogues is dependent on Ca2+ and cyclic AMP-dependent protein kinases.32, 33 More specifically, acetylcholine enhances the rate and extent of replenishment of the readily-releasable granule pool by a mechanism dependent on the activation of Ca2+ /calmodulin-dependent protein kinase II, CaM Kinase II.31 These effects are supported by Ca2+ mobilized from intracellular stores and not by Ca2+ influx at the cell periphery implying that the intracellular location of Ca2+ increase is critical. The functional resolution of these mechanisms hinges on the identification of substrates targeted by these kinases. The enhanced β-granule mobility promoted by acetylcholine correlates with myosin light chain (MLC) phosphorylation which could potentially fuel granule movement via the modulation of actin–myosin interactions.32, 33 However, other studies suggest that myosin heavy chain, not MLC, phosphorylation is more important for insulin secretion.34, 35 Clearer, perhaps, are determinations that

β-granule docking and priming Capacitance studies indicate that β-granules arriving at the plasma membrane are required to proceed through two primary pre-fusion steps, namely docking and priming, in preparation for eventual exocytosis as described for neurons and neuroendocrine cells12 (Figure 1). These steps are likely important for the organization of SNARE proteins into fusion complexes. SNAREs and the SNARE hypothesis SNAREs were originally identified as synaptic proteins but it is now understood that they are universally involved as the core machinery in all intracellular fusion events.4 In the β-cell, SNAREs are required for Ca2+ -triggered insulin exocytosis since their destruction via proteolysis abolishes this process. The role of SNAREs in exocytosis was initially implied from observations that a complex of these proteins formed the binding site for two factors, N-ethylmaleimide-sensitive factor (NSF) and a soluble NSF-attachment factor (α-SNAP)49 known to be required for constitutive fusion.49 Interestingly, this 7S complex was found to be constituted by 256

β-granule transport and exocytosis

Key: - tethering molecule - VAMP (v-SNARE) - syntaxin (t-SNARE) - SNAP-25 (t-SNARE) - synaptotagmin

N

N C

C

N

N C

C

NSF/αSNAP PKA

PI Kinases ATP

Docked "Tethered"

Ca2+

ATP

ATP

Primed

Fused

Figure 3. Mechanism of insulin exocytosis. The three primary stages of insulin exocytosis at the plasma membrane are illustrated. Docking mechanisms remain undefined and may involve tethering proteins independent of SNAREs. ATPdependent granule priming mechanisms include NSF-ATP hydrolysis (via α-SNAP) and the generation of PtdIns(4,5)P2 by PI kinases. The fusion of primed granules is triggered by Ca2+ binding to sensor proteins such as synaptotagmin which is thought to permit the ‘constructive’ formation of trans-SNARE complexes between v-SNAREs of the β-granule and tSNAREs of the plasma membrane. This organized interaction of SNAREs is sufficient to overcome the energy required to fuse the membranes (inset). New information suggests that PKA intervenes at a post-priming step, perhaps to increase the number of granules that can be rapidly released in response to Ca2+ .

a vesicle membrane protein, synaptobrevin [or a vesicle-attached membrane protein (VAMP), a vesicle or v-SNARE] and two plasma membrane proteins, SNAP-25 and syntaxin (called target or t-SNAREs), assembled with 1 : 1 : 1 stoichiometry. Furthermore, this complex was unusually stable to heat or SDS50 but its disassembly was mediated via the ATPase activity of NSF.50 The SNARE hypothesis formulated on these observations proposed that the specificity of vesicle to target membrane interaction was regulated via cognitive interactions of v-SNAREs with t-SNAREs and that the disassembly of the formed complex via ATP hydrolysis by NSF drives the fusion process.51 As delineated below, both principles are now known to be inaccurate although SNARE complex formation is necessary for fusion (reviewed in Reference 4). The β-cell expresses a full repertoire of SNARE proteins that are either identical to the neuronal proteins or very similar. Synaptobrevin (VAMP-2) and cellubrevin are expressed on the β-granules as v-SNAREs whereas syntaxin I and SNAP-25 are expressed on the plasma membrane as primary t-SNAREs.52–55 It is noted that multiple isoforms of syntaxin and SNAP-25 are expressed in the β-cell but the significance of this remains unclear (refer to Table 2).

Docking Docking describes the initial reversible interaction of β-granules with the plasma membrane and constitutes a pool of unprimed granules in close proximity to the exocytotic site (Figure 3). Although most clearly observed for synaptic vesicles docked at the active zone, fluorescence imaging studies56, 57 and electron microscopic analyses18, 58 have established similar pools of LDCVs in chromaffin cells. This pool is also defined biochemically in PC12 cells as granules that sediment with plasma membrane fractions and which require MgATP and cytosol for Ca2+ -activated fusion.59 The presence of this pool in β-cells is implied from the speed with which the readily releasable pool can be replenished (estimated at <400 ms) following the addition of ATP to nucleotide-depleted β-cells12 but there is little morphological evidence for β-granule docking.17 A ‘docked pool’ of granules has putatively been identified in β-cells based on the co-precipitation of complex including both v- and t-SNAREs that is reversibly disrupted on the stimulation of insulin exocytosis,60 but these complexes do not necessarily define granules at this specific stage of exocytosis. Granule docking is presumably important for the efficiency of insulin exocytosis and the molecular 257

R. A. Easom

sion of recombinant α-SNAP to cytosol-depleted cells partially restores Ca2+ -sensitive exocytosis.72 This is presumably via the activation of NSF since no other function has been ascribed to α-SNAP. NSF functions as a molecular chaperone to activate SNARE proteins destined for the fusion (core) complex.74 This role is not disputed but the precise site of NSF action is currently a matter of debate. In the exocytotic scheme of insulin exocytosis illustrated in Figure 3, NSF is implicated in a post-docking priming step. This is modeled after LDCV exocytosis of neuroendocrine cells71 but is also supported from genetic studies in Drosophila that show that synaptic vesicle docking is unaltered in NSF-1 function-deficient mutants (comatose).75 Other studies of slower yeast vacuole homofusion suggest, however, that NSF-mediated priming occurs earlier in the exocytotic process at some pre-docking step.76 This is notable because NSF is found on the surface of secretory vesicles and LDCVs along with a full complement of SNAREs, i.e. both vand t-SNAREs.77 Thus, NSF may be necessary to dissociate SNARE complexes formed within a single membrane (cis-SNARE complexes) in favor of productive trans-SNARE complexes between membranes necessary perhaps for docking, but at least fusion. An association of NSF with β-granules has not been confirmed but is a possible explanation as to why the provision of exogenous NSF fails to reconstitute exocytosis in permeabilized β-cells.72 Similarly, NSF action is predicted to be necessary for the dissociation of cis-SNARE complexes that remain on the plasma membrane post-fusion although this is likely to be more important for synaptic vesicles that are rapidly recycled.74, 78 These apparently distinct functions may not be mutually exclusive and it is conceivable that NSF functions in multiple steps of exocytosis, and endocytosis, to ensure that SNARE proteins conform to the orientation appropriate to each stage.67 As an added note, a cysteine string protein (CSP) harboring a J-domain is also expressed on β-granules79, 80 which may have chaperone activity toward exocytotic proteins.

mechanisms involved need to be understood. It would seem logical that the stable recognition of vto t-SNAREs might be important for granule docking and indeed SNAP-25 appears to be necessary for this process in chromaffin cells.61 However, recent observations, among which is an inability of clostridial toxins or the genetic ablation of synaptobrevin or syntaxin to prevent vesicle docking in neurons,62, 63 questions the involvement of SNAREs in docking mechanisms. Even if SNARE recognition is involved, it is now clear that the assembly of the core complex is insufficient to direct specific targeting of trafficking granules.64, 65 Additional non-SNARE proteins/complexes, perhaps equivalent to Rab and p115 proteins of the Golgi,66, 67 are therefore deemed necessary for precision of granule docking at the plasma membrane68 but candidate proteins have not been identified in the β-cell. Priming Biochemical studies in neuroendocrine cells have demonstrated that post-docking ATP-dependent priming steps are essential for exocytosis.59 Electrophysiological studies also imply that granule priming is a pre-requisite for Ca2+ -activated insulin exocytosis12 but again the molecular mechanisms involved in the β-cell have not been characterized. Priming may encompass a series of ATP-dependent events (see Figure 3) but two major mechanisms are predicted to be important for the β-cell. 1. NSF ATPase NSF, along with its partner α-SNAP, is an ATPase whose hydrolytic activity is essential for membrane fusion both in intracellular trafficking through the Golgi complex69 and for exocytosis.70 It is now established that NSF action does not energize membrane fusion, as initially proposed in the SNARE hypothesis, but is rather required at a prior priming step.71 In the β-cell, such a role is indicated from capacitance studies in which the inactivation of NSF by NEM inhibited the delayed phase of Ca2+ -induced insulin exocytosis in a manner similar to the withdrawal of ATP.12 However, attempts to demonstrate a direct functional role of NSF in insulin exocytosis have proved not to be informative.72 On the other hand, α-SNAP, which mediates NSF’s interaction with the SNARE complex and stimulates NSF ATPase activity, is clearly important since interference with its function, via dominant negative mutant expression,73 inhibits insulin exocytosis.72 Furthermore, the provi-

2. Phospholipid phosphorylation NEM-sensitive steps only account for approximately 50% of the restorative effect of cell cytosol on insulin exocytosis from permeabilized β-cells.72 A second potential role for ATP in β-granule priming, therefore, is as a substrate in the synthesis of phosphatidylinositol (4,5)-bisphosphate (PtdIns(4,5)P2 ), a phospholipid mediator required for Ca2+ -triggered 258

β-granule transport and exocytosis

Table 1.

Targets and consequences of Clostridual botulinum (BoNT) or Tetanus toxins (TeTx) on insulin exocytosis

Toxin

SNARE targeted

Effect on Ca2+ -dependent insulin exocytosis

Reference

TeTx BoNT/A BoNT/B BoNT/C1a BoNT/C2 BoNT/E BoNT/F

VAMP-2/cellubrevin SNAP-25 VAMP-2/cellubrevin Syntaxin Actin microfilaments SNAP-25 VAMP-2/cellubrevin

Inhibitione Inhibitionb Inhibition Inhibitione Inhibition/Stimulationc Inhibition No Effect (?d )

(53, 55, 138) (139) (53) (89) (24) (139–141) (142)

a Not absolutely specific and can also proteolyse SNAP-25; b secretion can be reconstituted by BoNT-resistant SNAP-23;141 c differential effects are noted depending on the phase of secretion studied; d Botulinum toxin B also failed to inhibit insulin secretion in this RINm5F cell model;142 e these toxins are reported to have no effect on exocytosis induced by GTPγ S;8

exocytosis in neuroendocrine cells.81 This is a specific requirement since exocytosis is prevented by enzymatic degradation of, or antibody interference with, PtdIns(4,5)P2 , but not by inhibitors of PtdIns 3-kinase which further phosphorylates this phospholipid.82 PtdIns(4,5)P2 is generated by the sequential action of two kinases, PtdIns 4-kinase (PI4K) and PtdIns 4-phosphate 5-kinase (PI4P5K), the former of which is localized to LDCVs, at least in chromaffin cells.83 PtdIns(4,5)P2 is emphasized because its known effectors include the Ca2+ -binding proteins, synaptotagmin84 and/or CAPS,85 which are heavily implicated in β-granule fusion (see below). It is speculated that the production of PtdIns(4,5)P2 may recruit and then modulate the interaction of these proteins with the fusion apparatus.85 Importantly, three isoforms of PI4K5P are expressed in β-cells (MIN-6)86, 87 although the production of PtdIns(4,5)P2 has not been demonstrated in this cell.

by the ability of monoclonal anti-syntaxin antibodies90 or peptides derived from a C-terminal domain of syntaxin91 to similarly inhibit insulin exocytosis. Consideration of the orientation of SNAREs within the core complex suggest that it is the formation, rather than the disassembly of this complex that fuels membrane fusion. The crystal structure of the cytosolic portion of the core complex has recently been solved revealing that helical portions (SNARE motifs), one each from VAMP-2 and syntaxin and two from SNAP-25, bundle together to form an extended coiled-coil rod-like structure92 (illustrated in the inset in Figure 3). The SNARE motifs in these bundles associate in a parallel orientation, rather than the more intuitive anti-parallel orientation, such that their C-terminal membrane anchors align.78 This parallel orientation can only be achieved with the physical bending of the SNAREs leading to the proposition that as SNAREs associate (‘zipperup’) the granule is brought into close proximity with the plasma membrane, thus overcoming the free energy barrier for fusion93, 94 (Figure 3). Such a model is supported by findings that v- and tSNAREs reconstituted into separate liposomes are sufficient to produce full membrane fusion5, 95 in a timely manner, particularly if the N-terminal section of syntaxin is removed prior to lipid mixing.96 In situ, exocytosis of LDCV correlates with the formation of core complex.97, 98 However, it is acknowledged that the absolute dependence of granule fusion on core complex formation is still a matter of debate because of observations, in models of yeast vacuolar fusion, that v- to t-(trans)SNARE complexes are formed long before fusion and can be dissociated without detrimental effects on subsequent fusion (see Reference 99).

Mechanisms of β-granule fusion SNAREs as the fusion apparatus The transition of the primed granule to the fully fused state is most important to the exocytotic process since this step results in the release of insulin. As indicated previously, SNAREs are critical to this step since Ca2+ -activated insulin exocytosis is abolished by Clostridial neurotoxins, endoproteases that cleave specific SNAREs.88 As detailed in Table 1, the independent cleavage of core SNAREs by toxins of botulinum and tetanus serotypes all abolish insulin exocytosis. This is less definitive for syntaxin cleavage by BoNT C1 since this toxin tends to also proteolyse SNAP-25,89 but a role for this t-SNARE is reinforced 259

R. A. Easom

Table 2.

β-cell proteins predicted to play important roles in insulin exocytosis

β-cell protein Core SNARE complex Synaptobrevin (VAMP-2) Cellubrevina Syntaxinb SNAP-25c Regulatory Proteins Synaptotagmin III Rab 3Ad munc18 (nSec1) CAPS (p145) Syncollin Granuphilin

Functional property of protein

Cellular location

Reference

v-SNAREe v-SNAREe t-SNARE f t-SNARE f

β-granule β-granule Plasma membrane Plasma membrane

(52, 53, 55) (52, 53, 55) (55, 143) (52, 55, 139, 140)

Ca2+ sensor GTPase syntaxin-binding protein Ca2+ /phospholipid binding protein Ca2+ /syntaxin binding protein Phospholipid binding protein

β-granule β-granule Plasma membrane N.D.

(55, 107) (114, 138, 144, 145) (52, 55) (108)

N.D.

(125)

β-granule

(124)

a Cellubrevin is more ubiquitously expressed than VAMP and may mediate constitutive exocytosis;53, 146 b multiple syntaxins have been shown to be expressed in β-cells including syntaxins 2,4,5.52, 55, 147 The significance of this is unclear although they may direct fusion events at different regions of the cell; c β-cells express both α- and β-isoforms.140 β-cells also express SNAP-23, a ubiquitous member of this family of proteins;141 d not expressed in human islets;115 e also classified as R-SNARE based on the conserved presence of an arginine residue in the heart of the SNARE motif;148 f also classified as Q-SNARE based on the conserved presence of a glutamine residue in the heart of the SNARE motif;148

N.D. Expressed but location in the β-cell unknown.

Ca2+ -activated fusion

binding to the core SNARE complex, via syntaxin,102 and membrane phospholipids,103 the latter being implicated for Ca2+ -dependent insertion into the lipid bilayer.104 The second domain, C2B, promotes synaptotagmin polymerization,105 its binding to polyphosphoinositide lipids84 and its interaction with other vesicle-associated proteins such as SV2.66 The interchange of synaptotagmin with itself and other proteins regulated by Ca2+ may supply both negative and positive control to exocytosis.66 The interference with synaptotagmin function in permeabilized β-cells, using antibodies targeted against the C2A domain or by the mutation of this domain, inhibits Ca2+ -triggered insulin secretion.106, 107 However, despite the expression of synaptotagmin I in certain insulinoma cell lines,106 it is the general consensus that this isoform is not expressed to any significant degree in primary β-cells;52, 55, 106, 107 synaptotagmin I is confined to non-β-cells of the islet mantle.52 Rather, the principal isoform of the β-cell appears to be synaptotagmin III,55, 107 an isoform that is expressed on the β-granule, and supports syntaxin binding at lower Ca2+ concentrations (<1 µM) relative to synaptotagmin I (>200 µM).101 This sensitivity seems more appropriate for insulin

Induced β-granule fusion is highly dependent on Ca2+ although at ion concentrations (1–10 µM) significantly lower than those required for synaptic vesicle exocytosis (100–200 µM).2 How Ca2+ triggers these events remains a major challenge in both systems but it is envisioned to work through regulatory proteins functioning as Ca2+ sensors. Candidate proteins in the β-cell are summarized in Table 2. Synaptotagmin Synaptotagmin represents the best and most thoroughly characterized exocytotic Ca2+ sensor. Knockout in mice have revealed that synaptotagmin I is essential for fast Ca2+ -triggered neurotransmitter release.100 It is equally clear, however, that other proteins are required in the neuron, since the slow Ca2+ -dependent release mechanisms remain unaltered.100 Synaptotagmin is now a large family of proteins, and its role in exocytosis is mediated via two conserved Ca2+ -binding regions, the C2 domains C2A and C2B, each sharing homology with the C2 regulatory domain of protein kinase C.101 C2A mediates Ca2+ -dependent synaptotagmin 260

β-granule transport and exocytosis

exocytosis that is triggered by Ca2+ concentrations in a low micromolar range (∼1–10 µM). Just as synaptotagmin I is insufficient to direct neurotransmitter release, the simultaneous activation of multiple Ca2+ -binding proteins will likely be required to achieve optimal exocytosis in β-cells. Accordingly, circumstantial evidence supports a potential role for a number of other proteins.

More important perhaps are Rab3A interactions with calmodulin2, 120 since the region of the protein to which calmodulin binds is necessary for the ability of Rab3A to inhibit insulin exocytosis.120 Furthermore, the binding of Ca2+ /calmodulin causes Rab3A to dissociate from cell membranes121 providing a mechanism whereby the inhibitory action of Rab3A on exocytosis may be relieved. In addition, Rab3A may function to provide calmodulin for other regulatory purposes involving the fusion apparatus and the cytoskeleton.122, 123 It is noteworthy that a novel rabphilin-like protein, granuphilin, that binds phospholipids is expressed on β-granules but this protein is reported not to interact with Rab3A.124

CAPS is a low-affinity Ca2+ -binding protein required for Ca2+ -triggered fusion from neuroendocrine cells.108, 109 Importantly, CAPS is also expressed in clonal β-cells RINm5F108 and primary rat islets (Martin, T. F. J., personal communication) but it is the properties exhibited by this protein that attract most interest. First, CAPS harbors a pleckstrin homology domain that exhibits binding specificity towards PtdIns(4,5)P2 implying that it is a selective target for phospholipid mediators specifically formed during granule priming.85 Interestingly, at elevated Ca2+ concentrations sufficient to trigger exocytosis (10–100 µM), this lipid specificity of CAPS is switched in favor of membrane phosphatidylcholine/phosphatidylethanolamine-containing phospholipids85 providing a means by which the plasma membrane bilayer may be disrupted to permit fusion. Second, CAPS is selective for LDCV exocytosis over neurotransmitter release,110 an observation that is corroborated by its localization to LDCV (and plasma) membranes but not to small synaptic vesicles.111 CAPS.

Syncollin was identified as a zymogen granule-associated protein that binds to syntaxin in a Ca2+ -sensitive manner.125 Its association with syntaxin inhibits granule fusion in vitro, but is reversed as Ca2+ concentrations increase consistent with its potential role as a Ca2+ -sensitive fusion ‘clamp’. Interest in this protein is sparked by recent determinations that syncollin is also expressed in the endocrine β-cell (Wickstead, B. & Rhodes, C. J., personal communication). Syncollin.

Although not a Ca2+ -sensitive protein, munc18 (nSec1) is essential for all intracellular membrane fusion events: its expression in the βcell55 is therefore sufficient to implicate it in insulin exocytosis. Munc18 binds to syntaxin in a closed conformation and may therefore provide an important restraint to fusion mechanisms by preventing the productive incorporation of this t-SNARE into the core complex.126 Interestingly, munc18 interaction with the SNARE complex is reversed by protein kinase C-catalyzed phosphorylation127 offering a possible mechanism whereby this kinase can influence insulin release. munc18.

Rab proteins represent a large class of ras-like GTPases that are implicated in mechanisms of vesicle trafficking in eukaryotic cells.112 Members of the Rab3 subfamily, in particular, are implicated in LDCV fusion mechanisms in neuronal and endocrine cells113 and all four known isoforms, Rab3A through D, are expressed on β-granules in the β-cell.114 The most studied isoform, Rab3A, acts as a negative modulator of Ca2+ -triggered exocytosis in β-cells,114, 115 as in neuroendocrine cells116 and neurons.117 Functionally, Rab3A does not interact directly with the SNARE complex but is reasoned to mediate its effects on exocytosis via interaction with downstream effector proteins, such as rabphilin and RIM in the neuron.113 However, based on the marginal expression of rabphilin in the β-cell,115, 118 and accumulating reports that Ca2+ -triggered exocytosis is independent of rabphilin or RIM,119, 120 these proteins are unlikely to represent important targets of Rab3A involvement in insulin exocytosis. Rab3A.

Emerging regulatory mechanisms SNARE interaction with L-type Ca2+ channels As depicted in Figure 1, cell depolarization and the activation of Ca2+ influx through L-type Ca2+ channels triggers the release of granules from the readily releasable pool12 and is likely the mechanism by which glucose stimulates early insulin exocytosis. This functional relationship is strengthened by recent observations that components of the SNARE 261

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complex interact directly with α-subunits of L-type Ca2+ channels in β-cells,128, 129 just as with N- and P/Q-type Ca2+ channels in the neuron.130 Presumably this arrangement ensures optimal efficiency of Ca2+ -triggered granule fusion and, indeed, the disruption of this interaction via the introduction of a competing peptide dramatically attenuates depolarization-induced insulin exocytosis.128 This complex, called the excitosome,128 may, however, also regulate Ca2+ entry and its ability to support exocytosis.129 This new information indicates that the slower time resolution of LDCV exocytosis (tens of milliseconds) relative to neurotransmitter release (fractions of milliseconds) is not a function of a remote location of the fusion apparatus from the point of Ca2+ entry as previously suggested.131 This does not, however, eliminate the possibility that the location of β-granules relative to the Ca2+ channel may contribute to multi-kinetic release from the readily releasable pool discussed earlier13, 14 and as shown recently to be the case in chromaffin cells.16

Concluding remarks There has been an exponential increase in our knowledge of the mechanisms of regulated exocytosis over the last few years. However relative to the well studied mechanisms of synaptic vesicle or LDCV exocytosis from the synapse and neuroendocrine cells, respectively, β-granule exocytosis from the β-cell is poorly understood. Current information points to a high level of conservation between these mechanisms but the unique dependence of β-cell secretion on nutrient metabolism may require specialized mechanisms to coordinate an optimal physiological response. The importance of this information is emphasized in a recent study that reported the dysfunction of the exocytotic apparatus as a contributing factor to the secretory incapacity of the diabetic β-cell.137 In this case the evidence was that the inadequate expression of core SNARE proteins led to the deficiency but considering the complexity of this process and the number of regulatory proteins required, it is likely that other abnormalities in this process will be found to contribute to the etiology of Type II diabetes.

Regulation of insulin exocytosis by cAMP/protein kinase A

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Insulin secretion is profoundly sensitive to cAMP and accounts for the tonic regulation of β-cell exocytosis by incretins such as glucagon-like peptide (GLP-1). As identified earlier, this may be due in part to the ability of PKA to enhance pre-priming granule recruitment mechanisms.30 However, capacitance30 and amperometry studies15 have also identified a potential role for cAMP to facilitate the release of primed granules either directly through some undefined receptor30 or via the activation of PKA and protein phosphorylation.15 This latter study provocatively suggests that the activation of PKA may be sensitive to changes in ATP concentration that are anticipated to occur within a cell activated by glucose.15, 132 This mechanism is attractive in that it identifies a pathway by which glucose, via its metabolism and increased generation of ATP, may directly influence insulin exocytosis by increasing the efficiency of granule fusion induced by Ca2+ .

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