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Molecular Endocrinology 22(12):2583–2595
Copyright © 2008 by The Endocrine Society
doi: 10.1210/me.2008-0209
MINIREVIEW
How Peptide Hormone Vesicles Are Transported to
the Secretion Site for Exocytosis
Joshua J. Park and Y. Peng Loh
Section on Cellular Neurobiology, National Institute of Child Health and Human Development
(NICHD), National Institutes of Health (NIH), Bethesda, Maryland 20892
Post-Golgi transport of peptide hormone-containing vesicles from the site of genesis at the transGolgi network to the release site at the plasma
membrane is essential for activity-dependent hormone secretion to mediate various endocrinological functions. It is known that these vesicles are
transported on microtubules to the proximity of
the release site, and they are then loaded onto an
actin/myosin system for distal transport through
the actin cortex to just below the plasma membrane. The vesicles are then tethered to the
plasma membrane, and a subpopulation of them
are docked and primed to become the readily
releasable pool. Cytoplasmic tails of vesicular
transmembrane proteins, as well as many cytosolic proteins including adaptor proteins, motor
proteins, and guanosine triphosphatases, are involved in vesicle budding, the anchoring of the
vesicles, and the facilitation of movement along
the transport systems. In addition, a set of cytosolic proteins is also necessary for tethering/
docking of the vesicles to the plasma membrane.
Many of these proteins have been identified from
different types of (neuro)endocrine cells. Here,
we summarize the proteins known to be involved
in the mechanisms of sorting various cargo proteins into regulated secretory pathway hormonecontaining vesicles, movement of these vesicles
along microtubules and actin filaments, and their
eventual tethering/docking to the plasma membrane for hormone secretion. (Molecular Endocrinology 22: 2583–2595, 2008)
C
in neurons. Within the cell body, peptidergic vesicles
are formed at the trans-Golgi network (TGN) and transported along processes to the proximity of distal release sites, by a microtubule-based transport mechanism (see Fig. 1). Subsequently, peptidergic vesicles
are transferred to cortical actin filaments. A pool of the
vesicles is retained in the actin cortex as the reserve
pool while another pool is tethered to the plasma
membrane, and a subpopulation of them become
docked (immobilized) and primed to form the readily
releasable pool. During stimulation, docked and
primed peptidergic vesicles in the readily releasable
pool immediately fuse to the plasma membrane to
release their contents into the extracellular space. The
reserve vesicle pool then replenishes the vesicles in
the readily releasable pool that have been depleted by
exocytosis.
In addition to the RSP, (neuro)endocrine cells, like all
other cells, also have a constitutive secretory pathway
(CSP) that supports continuous protein secretion, independent of stimulation (5). In the CSP, small secretory vesicles formed at the TGN are constantly transported to and fused to the plasma membrane without
forming any storage pool (see Fig. 1). CSP vesicles are
either directly transported to the plasma membrane
from the TGN, or they pass through intermediate endosomal compartments (e.g. early/late endosome and
ELLS IN THE endocrine and nervous systems
package peptide hormones, neuropeptide and
specific neurotrophins 关e.g. brain-derived neurotrophic factor (BDNF)兴, into secretory vesicles for release
in a regulated manner upon stimulation. This is known
as the regulated secretory pathway (RSP). Activitydependent secretion of these molecules is critical for
mediating various endocrine functions, neurotransmission, and neuronal plasticity (1–4). Secretion of
these peptidergic vesicles requires them to be transported from the cell body where they are synthesized,
to the secretion sites at the plasma membrane, which
can be some distance away, e.g. at the nerve terminals
First Published Online July 31, 2008
Abbreviations: AP-1A, Adaptor protein 1A; APP, amyloid
precursor protein; BDNF, brain-derived neurotrophic factor;
CAPS, Ca2⫹-dependent activator protein for secretion; CSP,
constitutive secretory pathway; CPE, carboxypeptidase E;
GGA, Golgi localized, ␥-ear-containing ADP-ribosylation factor binding protein; GTPase, guanosine triphosphatase; KIF,
kinesin-like family; Munc18, mammalian homolog of unc-18;
Noc2, no C2 domain; PAM, ␣-amidating monooxygenase;
PC, prohormone convertase; POMC, proopiomelanocortin;
Rim, Rab3-interacting molecule; RSP, regulated secretory
pathway; SgIII, secretogranin III; SNAP25, synaptosomal associated protein of 25 kDa; SNARE, soluble N-ethyl maleimide sensitive-factor attachment protein receptor; SorLA, sorting protein-related receptor containing LDLR class A repeats;
TGN, trans-Golgi network.
2583
2584 Mol Endocrinol, December 2008, 22(12):2583–2595
Park and Loh • Minireview
Peptidergic
vesicle
Golgi
F-actins
a
d
b
c
e
microtubules
Regulated secretory pathway
f
Constitutive secretory pathway
stimulated
secretion
Fig. 1. Steps for post-Golgi RSP Vesicle Transport to the Release Site
Multiple steps are involved in transporting hormone-containing vesicles from the site of biogenesis at the TGN to the release
site in the regulated secretory pathway: a, vesicle budding; b, microtubule-based transport; c, actin-based transport; d, vesicle
tethering; e, docking; and f, fusion with the plasma membrane. These steps share some commonality with the trafficking of
constitutive secretory vesicles, but there are differences as well (see Table 1).
recycling compartments) before reaching the plasma
membrane (6, 7). Because CSP proteins are continuously secreted, the CSP is mainly driven by the biosynthetic rate of secretory proteins at the endoplasmic
reticulum (8). The CSP in (neuro)endocrine cells provide membrane proteins, such as receptors (9), to the
plasma membrane, as well as various secretory proteins (10, 11), for maintenance of cell survival, differentiation, and growth.
In this review we will focus our discussion primarily
on the various cytoplasmic machineries that are involved in mediating post-Golgi RSP vesicle sorting
and transport to the release site, as well as vesicle
tethering to the plasma membrane (see Fig. 1). A comparison with CSP vesicle transport will be made. We
will also discuss the mechanisms of sorting peptide
hormone precursors into the RSP vesicles, but only
briefly, because comprehensive reviews on this subject have been published by Blazquez and Shennan
(12) and, more recently, by Dikeakos and Reudelhuber
(13). The protein machinery involved in the priming,
fusion, and exocytosis of vesicles at the plasma membrane will not be included in this review.
PROTEIN SORTING INTO RSP VESICLES
In this section, we will briefly discuss the various
mechanisms proposed for the sorting of peptide hormone precursors and processing enzymes and
granins into the RSP of (neuro)endocrine cells. Two
modes of RSP protein sorting have been described:
sorting-at-entry and sorting-by-retention 关see reviews
by Blazquez and Shennan (12), and Dikeakos and
Reudelhuber (13)兴.
Mechanisms for sorting-at-entry are divided into different subcatergories (Fig. 2A). One mechanism in-
volves initial aggregation of the cargo proteins such as
the peptide hormone precursors and granins in a pH
(6.0–6.5)- and cation-dependent manner (see “pH/
Ca2⫹-driven aggregation” in Fig. 2). The aggregation
process excludes the constitutive proteins (14–17).
Thereafter, the aggregate binds to the TGN membrane, in some cases via a receptor protein (see “Aggregates bind to sorting receptors in Fig. 2). An example of such a sorting receptor is the membrane form of
carboxypeptidase E (CPE), which has been demonstrated to sort proopiomelanocortin (POMC)/ACTH
(18) and pro-BDNF (19), at the TGN to the RSP in
pituitary cells and hippocampal neurons, respectively.
This function of CPE has been debated and discussed
in a recent review by Dikeakos and Reudelhuber (13).
Secretogranin III (SgIII), a protein associated with cholesterol-sphingolipid-rich membrane microdomains
(lipid rafts), at the TGN has been shown to be an
RSP-sorting receptor for chromogranin A (20). SgIII
was also proposed to play a distal role in sorting
POMC to the RSP by transferring POMC from CPE to
SgIII (21). However, this phenomenon seems redundant given that CPE can carry POMC into RSP
vesicles.
Another sorting-at-entry mechanism that has been
proposed is one used by several prohormone processing enzymes, which involves direct insertion of the
C-terminal domain of the enzyme into lipid rafts at the
TGN. The transmembrane domains of several prohormone-processing enzymes have been shown to mediate their own sorting to the RSP (see “Association
with TGN membrane lipids” in Fig. 2). For example, the
transmembrane domain of ␣-amidating monooxygenase (PAM) contains information for correct routing of
PAM to the RSP (22). Additionally, the C-terminal
transmembrane domains of CPE, prohormone convertase 1/3 (PC1/3), and prohormone convertase 2 (PC2),
Park and Loh • Minireview
Sorting at entry
1. pH/Ca2+-driven aggregation
RSP
protein
CSP
protein
2. Association with
TGN membrane lipids
3. Aggregates bind to
sorting receptors
CGA
PA
M
Sg
III
A
Mol Endocrinol, December 2008, 22(12):2583–2595 2585
CP
E
PC1
PC
POMC
proENK
Proinsulin
BDNF
2
SgIII
TGN membrane
CPE
TGN
membrane
Lipid-raft
B
Sorting by retention
furin
TGN
AP-1/GGA
Constitutivelike secretion
E
CP
Retention receptor
Fig. 2. RSP Protein-Sorting Mechanisms
A, Sorting at entry (at the TGN): 1) Low pH and high Ca2⫹ concentration-drive RSP protein aggregation which excludes
CSP proteins; 3) Protein aggregates associate with the TGN membrane, through direct interaction with lipids, some
specifically at lipid-rafts, or 2) by protein-protein interaction with protein sorting receptors: e.g. CPE is a proposed
RSP-sorting receptor for POMC, proenkephalin (proENK), proinsulin, and BDNF, whereas SgIII is a proposed sorting
receptor for chromogranin A (CgA). The TGN membrane then buds to form the vesicle bringing along the aggregated protein
cargo. B, Sorting by retention: in this model, RSP proteins along with some CSP proteins enter the immature secretory
vesicle formed at the TGN. During maturation of the secretory vesicle, the RSP proteins are retained in the maturing vesicle
by binding to a retention receptor, e.g. in pancreatic ␤-cells immature vesicles, membrane CPE binds and retains insulin (34)
whereas CSP proteins (e.g. furin) are removed from the vesicle by an AP-1/GGA/clathrin-mediated budding mechanism to
yield constitutive-like vesicles for secretion.
are associated with lipid rafts and crucial for their
own targeting to the RSP (23–26). Whereas the evidence for transmembrane orientation of CPE from
two different laboratories is quite clear from biochemical (23) and functional studies (27, 28), the
transmembrane orientation of PC1/3 and PC2 is
considered tentative among some investigators in
the field. Nevertheless, it is generally accepted that
the association of processing enzymes with lipids
at the TGN is necessary for their sorting to the RSP
(29–32).
The mechanism for sorting-by-retention was first
proposed by Arvan and Castle (33) for sorting of proinsulin in pancreatic ␤-cells (Fig. 2B). They observed
that along with proinsulin, other proteins such as lysosomal enzymes were present in the immature granule compartment, which were subsequently removed
by budding off of constitutive-like vesicles, whereas
insulin was retained in the maturing vesicle (33). Our
studies suggest that CPE may act as a retention receptor in this immature vesicle compartment, serving
to retain insulin in pancreatic ␤-cell vesicles (34). In
general, constitutively secreted proteins enter CSP
vesicles by default 关see review by Halban and
Irminger (35)兴. However, certain CSP proteins such
as furin, a transmembrane enzyme, are packaged
with the RSP proteins into immature secretory granules (36, 37). Subsequent removal of furin from the
immature vesicle requires that its cytoplasmic tail be
phosphorylated by casein kinase-2 (38), and bind
the adaptor protein 1 (AP-1) adaptor, phosphofurin
acidic cluster sorting protein-1, in an ADP-ribosylation factor 1-dependent manner to initiate clathrin
coating, followed by budding of furin-containing
constitutive-like vesicles (39).
Hence, RSP proteins are segregated from CSP
proteins and sorted into RSP-secretory vesicles at
the TGN by aggregation, followed by membrane
association of the aggregate either directly with lipids or via protein-sorting receptors. A secondary
sorting step involves removal of CSP proteins that
have inadvertently entered into RSP vesicles, by budding off of constitutive-like vesicles from the maturing
RSP vesicle.
2586 Mol Endocrinol, December 2008, 22(12):2583–2595
CYTOPLASMIC MACHINERY FOR POST-GOLGI
VESICLE TRAFFICKING
Vesicle Coating and Budding at the TGN
Clathrin coating is one of the mechanisms that drive
budding and formation of vesicles at the TGN (see Fig.
3) (40). Both newly formed RSP (41) and CSP (42)
vesicles that have just budded from the TGN contain a
clathrin coat, which is then shed upon maturation of
the vesicles (43, 44). AP-1 and GGA (Golgi localized,
␥-ear-containing ADP-ribosylation factor binding protein) are the major adaptor proteins that mediate clathrin coating on budding CSP vesicles, either independently, or cooperatively (45, 46). In addition to the TGN
vesicle budding, AP-1 also mediates sorting of furin
i
Sorting of
RSP proteins
at the TGN
Park and Loh • Minireview
from intermediate/recycling compartments back to the
TGN (47). Both AP-1 and GGA are also found on
immature RSP-secretory vesicles (48–50), suggesting
that both these molecules might mediate clathrin coating of budding RSP vesicles as well.
Several accessory cytosolic proteins assist with AP1-mediated clathrin coating of CSP vesicles by binding to the cytoplasmic tail of integral membrane CSP
vesicle proteins such as ␤-secretase, sorLA (sorting
protein-related receptor containing LDLR class A repeats), mannose-6-phosphate receptor (40), and furin
(39), which contain AP-1 and GGA binding motifs. For
example, phosphofurin acidic cluster sorting protein-1
facilitates binding of AP-1 to the acidic residues of
furin cytoplasmic tail (39), and epsin-R is needed for
AP-1-based packaging of mannose-6-phosphate re-
ii
Clathrin
coating
Adaptors:
AP-1, GGA
iv
1. Vesicle
anchor/adaptors
interact with MT motor
: CPE cytoplasmic tail /
dynactin, HAP1, Htt,
JIP3
1. Tethering molecules
(+): Rab3A/Rabphilin3A, Rab3A/Rim,
Rab27A, Myosin Va, CAPS
RalA/exocyst
(-): Rab27A/granuphilin, Rab3A/Noc2
2. Vesicle fusion molecules
: SNARE complex
2. Microtubule motors :
kinesin-1 (KIF5), kinesin-2 (KIF3),
kinesin-3 (KIF1A)
PM
Actin
cortex
iii
TGN
Microtubule
Secretory
vesicle
Myosins Va
Actin
Secretory
Vesicles:
fu do tet
sio ck he
n ed re
to to d
PM P to
M PM
1. Shifters to
myosin:
Rab27A/MyRIP
Release sites
Cell body
Fig. 3. Hormone Vesicle Transport in the Regulated Secretory Pathway of (Neuro)Endocrine Cells
Molecules mediating different steps of vesicle transport to the RSP are shown in this model. Peptide hormones and
neuropeptides are sorted and packaged into immature clathrin-coated vesicles at the TGN. The adaptors that might be involved
in clathrin coating of budding RSP vesicles at the TGN are outlined in box i. The immature vesicles are then anchored to the
microtubule (MT)-based transport system via linkers such as the cytoplasmic tail of vesicular transmembrane CPE and adaptors
such as dynactin, which recruits various kinesin motor proteins (box ii) to effect movement to the proximity of the release site. The
vesicles are then shifted from the microtubule-based system to the myosin transport system that moves these vesicles through
the actin cortex to the proximity of the plasma membrane, forming a reserve vesicle pool. The recruitment of the vesicles to the
myosin-based transport system is facilitated by rabGTPases and their effector molecules, outlined in box iii. A population of
vesicles from the reserve pool are then moved and tethered to the plasma membrane via tethering molecules (box iv). In addition
to positive tethering molecules, there are negative ones as well as indicated. A subpopulation of the tethered vesicles are then
immobilized on the plasma membrane by SNARE complex (docking) and primed to become the readily releasable pool. Upon
stimulation, the docked and primed vesicles are exocytosed, releasing the vesicle contents into the extracellular space. PM,
Plasma membrane; MT, microtubule; HAP1, Huntington-associated protein 1; Htt, Huntington; KIF, kinesin-like family; MyRIP,
myosin VIIa- and Rab-interacting protein.
Park and Loh • Minireview
ceptor into clathrin-coated vesicles (51). ␥-Synergin
(52), aftiphilin (53), and adaptin-ear-binding coat-associated protein (54) also interact with AP-1, but their
actual function in clathrin coating of vesicles at the
TGN is unclear. Although likely, a role of these proteins
in recruiting AP-1/GGA or the accessory proteins to
RSP vesicles remains to be substantiated.
Microtubule-Based Vesicle Transport
Subsequent to budding from the TGN, both RSP and
CSP vesicles are transported to the secretion sites at
the plasma membrane via microtubule-based transport systems (see Fig. 3). Microtubules have been
shown to mediate transport of RSP vesicles such as
TRH vesicles in neurons of the paraventricular nucleus
(55) and chromogranin B vesicles from the cell body to
the proximity of the plasma membrane in AtT20 cells,
a corticotroph cell line, and PC12 cells, a neuroendocrine cell line (56). Similarly, CSP vesicles containing
growth factors, such as nerve growth factor, neurotrophin-3 and neurotrophin-4, and membrane proteins
such as amyloid precursor protein (APP), are also
transported by a microtubule-based system (57).
Vesicle anchors and adaptors (see Fig. 3) are required to anchor RSP and CSP vesicles to microtubule
motors. Anterograde transport of BDNF-containing
vesicles is facilitated by the adaptors dynactin and
huntingtin-associated protein-1 (58). Although it has
been shown that CPEs exist as soluble and peripheral
membrane forms (59–63), our work supports the idea
that there is a subpopulation of transmembrane CPE
(23). We demonstrated that CPE has an atypical transmembrane ␣-helical structure (at an acidic pH 5.5–6.5
found in TGN and secretory granules), within the C
terminus, and a cytoplasmic tail (23). Although the
physicochemical properties of this domain would predict an energetically unfavorable presence in a membrane, the existence of other proteins within the cell
that reduce the free energy and which then allow this
transmembrane orientation cannot be ruled out, especially in light of the live cell imaging of the dominantnegative nature of a cytosolically expressed C-terminal tail of CPE (64). However, in the absence of any
further studies to refute this data, the existence of a
CPE C-terminal transmembrane domain and cytoplasmic tail remains a matter of debate with some investigators. The cytoplasmic tail of vesicular transmembrane CPE has been demonstrated not only to
mediate recycling of endocytosed vesicles to the Golgi
(27, 28) but also to anchor ACTH vesicles to microtubule motors via interaction with the adaptor, dynactin
(64). Although the size of the transmembrane pool has
not been determined, only a small number of CPE
cytoplasmic tails need to be present on peptide hormone- and BDNF-containing vesicles because a single
connection between the vesicle and the motor complex is sufficient for transport. In contrast, CPE is not
a vesicle anchor for CSP vesicles (19). However, in
specific CSP vesicles, for example, the cytoplasmic
Mol Endocrinol, December 2008, 22(12):2583–2595 2587
tail of the type I transmembrane protein, APP, was
shown to interact with microtubule motors, via the
adaptor, JNK-interacting protein 1b, to mediate anterograde transport (65, 66).
In general, both RSP and CSP vesicles use the same
type of microtubule motors, such as kinesin, for anterograde transport to the secretion sites, and cytoplasmic
dynein for the retrograde transport back to the cell body
(57). Kinesin-1, a major kinesin in (neuro)endocrine
cells, is shown to transport RSP vesicles (67–69) as
well as CSP vesicles to the microtubule plus ends
located at the proximity of the plasma membrane (65,
66). However, recent studies show that there is a subset of microtubule motors specialized for transport of
RSP vesicles. Kinesin-2 and kinesin-3 关kinesin-loke
family (KIF)1A兴 are reported to mediate anterograde
transport of ACTH vesicles in anterior pituitary cells
(64). In Caenorhabditis elegans, Unc-104, the kinesin-3 ortholog, mediates anterograde trafficking of
synaptic vesicles and peptidergic RSP vesicles to the
nerve terminals (70, 71) via the adaptor, JNK-interacting protein 3 (72).
Interestingly, the activity and/or distribution of kinesin along the microtubules are modulated to favor RSP
vesicle secretion during stimulation. For example, forskolin, the adenyl cyclase activator, increased the velocity of peptidergic vesicle trafficking in neuroblastoma NS20Y cells (73), putatively by affecting the
activity of a yet unidentified kinesin. Another secretagogue, carbachol, induces changes in the activity and
intracellular distribution of kinesin-1 in the acinar cells
of rabbit lacrimal gland (67). Indeed, kinesin-1 purified from carbachol-treated acinar cells shows enhanced in vitro microtubule-gliding activity compared with unstimulated cells. In a partitioning
analysis using a dextran-polyethyleneglycol twophase system, a distributional shift of kinesin-1 from a
Golgi compartment to the ␤-hexosaminidase-containing post-Golgi secretory vesicles was observed in
these cells. This study suggests that carbachol-triggered signaling drives kinesin-1 to facilitate stimulated
secretion of ␤-hexosaminidase. Similar stimulationbased redistribution of kinesin-1 to the RSP zymogen
granules was also observed in pancreatic acinar cells
treated with cholecystokinin or secretin (74).
For CSP vesicles, regulation of APP vesicle transport from the Golgi apparatus to the neurite terminal,
by presenilin-1 found in these vesicles, has been reported (75, 76). Presenilin-1 regulates the transport via
the interaction of its cytoplasmic tail with glycogen
synthase kinase 3b, which phosphorylates kinesin
light chain 1, leading to inhibition of kinesin-1-mediated axonal transport of APP (77, 78).
Thus, during microtubule-based transport, both
RSP and CSP vesicles use a similar microtubule motor
system. Considerable advancement has been made in
identifying RSP and CSP vesicular transmembrane
proteins and which cytoplasmic tails bind to different
adaptors and microtubule motors to mediate transport. Factors that modulate microtubule activity and
2588 Mol Endocrinol, December 2008, 22(12):2583–2595
distribution that enhance RSP vesicle transport during
stimulation of the cell have also been elucidated, thus
increasing our understanding of regulation of vesicular
transport in the RSP.
Transfer of Vesicles from Microtubules to the
Actin Cortex
At the end of microtubule-based transport, both RSP
and CSP vesicles are transferred to the actin cortex
close to the plasma membrane (see Fig. 3). Myosin V,
the F-actin motor protein, has been proposed to mediate the shifting of secretory vesicles from microtubules to the actin cortex via its direct interaction with
microtubule-based motors (79–81), although this proposal needs further investigation because there is
some controversy in the field. Myosin V, then, traffics
both CSP (82, 83) and RSP (84–86) vesicles through
the F-actin-rich cortical region to the secretion sites
proximal to the plasma membrane. In addition to its
role as the transport platform for myosin motors, Factins also appear to function as a physical barrier for
RSP vesicle exocytosis (87), but only transiently for
CSP vesicles (87). Additionally, myosin II has been
reported to be involved in both RSP vesicle transport
and actin caging (88), although its role in this respect
remains controversial.
Much more intensive studies have been conducted
regarding trafficking of RSP vesicles vs. CSP vesicles
through the actin cortex. Myosin Va has been shown
as the major myosin that carries the RSP dense core
vesicles from the reserve pool to the docked/readily
releasable pool compartment for exocytosis. Expression of a headless mutant of myosin Va in PC12 cells
not only prevented secretory vesicles from reaching
the secretion sites at the plasma membrane, resulting
in clustering of the vesicles away from the plasma
membrane, but also decreased motility of the vesicles
in the actin cortex (86). A similar negative effect was
observed in chromaffin cells injected with antimyosin
head antibodies (85). Regulated insulin secretion in
pancreatic ␤-cells also depends on myosin Va (89, 90).
Both silencing of myosin Va and expression of headless myosin mutant significantly suppressed glucoseor depolarization-induced insulin secretion from INS-1
cells by decreasing the number of insulin granules
tethered/docked to the plasma membrane. Thus myosin Va is responsible for coordinating late transport
events of RSP vesicles preceding their exocytosis.
Additionally, recruitment of myosin Va onto RSP vesicles appears to be mediated by the small GTPase
Rab27A and its effectors (see Fig. 3). Myosin Va is
linked to RSP vesicles via melanophilin/Rab27A in
melanocytes (91). In adrenal chromaffin cells and
PC12 cells, myosin-VIIa- and Rab-interacting protein/
Rab27A connects RSP vesicles to myosin Va for transport of these vesicles through the actin cortex (92).
In addition, the integral membrane form of PAM may
play a role in facilitating peptidergic RSP vesicle trafficking in the actin cortex. The cytoplasmic tail of PAM
Park and Loh • Minireview
in peptide hormone vesicles interacts with PAM
COOH-terminal interactor protein 2, a kinase that
phosphorylates PAM, and kalirin, a Rho family GDP/
GTP exchange factor and an F-actin reorganizer (93–
95). The ability of kalirin to reorganize F-actins may
influence the movement of such RSP vesicles through
the actin cortex.
Although both RSP and CSP vesicles use the common actin-based transporter myosin V to reach the
plasma membrane, different molecules appear to influence RSP or CSP vesicle transport through actin
cortex. For instance, Rab27A and its effectors appear
to function only in the RSP actin cortex and enhance
connection of RSP vesicle to myosin V.
Vesicle Tethering to the Plasma Membrane
Movement of the RSP and CSP vesicles through the
actin cortex is followed by tethering of the vesicles to
the plasma membrane for exocytosis. For RSP vesicles there is a reserve pool that is not tethered to the
plasma membrane. The majority of the RSP pool of
vesicles that are tethered remain mobile, but a subpopulation of these are immobilized and docked and
primed at the plasma membrane (the readily releasable pool), ready for exocytosis (see Fig. 3). RSP vesicle tethering and docking are facilitated by a variety of
cytoplasmic molecules, including the small guanosine
triphosphatase (GTPase) Rab proteins and their effectors. A recent study shows that myosin Va mediates
docking (immobilization) of RSP vesicles at the plasma
membrane (96), independent of its motor activity. In
contrast, CSP vesicle tethering is mediated by an
eight-subunit protein complex known as the “exocyst”
(97) that is found to mediate tethering of CSP vesicles
to the plasma membrane in yeast (98) and mammalian
epithelial cells (99), adipocytes (96, 100, 101), and
neurons (102, 103). The adaptor protein AP-1B (104)
and the small GTPase TC10 (100, 101, 105) facilitate
the exocyst-driven CSP vesicle tethering. Additionally,
the exocyst is involved in tethering of insulin-containing RSP vesicles to the plasma membrane of pancreatic ␤-cells (90, 106) via its interaction with the plasma
membrane-associated small GTPase protein, RalA.
However, the exocyst appears to mainly function in
CSP vesicle tethering.
Rab3A and Rab27A are the major Rab proteins in
the regulated secretory pathway which regulate not
only tethering of RSP vesicles to the plasma membrane, but also assembly/disassembly of a fusion
complex between the RSP vesicles and the plasma
membrane during initial membrane contact (107–109).
Both Rab3A and Rab27A are recruited to the newly
synthesized RSP vesicles and associate with the vesicles constantly even after stimulation (110). There is
also a group of Rab3A and Rab27A effectors that
operate in the regulated secretory pathway: Rab3D,
Rabphilin3A, Rab3-interacting molecule (Rim), and
Noc2 for Rab3A, and granuphilin-a for Rab27A.
Rab3D, which was originally found on the zymogen
Park and Loh • Minireview
granules and enhances regulated secretion of amylase
from pancreatic acini (111), is required for association
of Rab3A with RSP vesicles. A mutant form of Rab3D
(N135I) caused a failure in the association of Rab3A
with ACTH vesicles, resulting in defective docking of
ACTH vesicles to the plasma membrane of AtT20 cells
(112, 113). Rabphilin3A (114, 115) and Rim1 (116) appear to bind to Rab3A that is already associated with
RSP vesicles and enhance its activity via an unknown
mechanism. Rabphilin3A is reported to potentiate
Ca2⫹-induced exocytosis of GH from bovine adrenal
chromaffin cells (115, 117), whereas Rim1 is shown to
facilitate Rab3A-mediated formation of a fusion complex between synaptic vesicles and the presynaptic
membrane in the synapses of hippocampal neurons
(116). Especially, Rim1 appears to scaffold the formation of the fusion complex by recruiting a number of
components such as mammalian homolog of unc(Munc)13-1 (118), synaptosomal associated protein
SNAP25 (synaptosomal associated protein of 25 kDa),
and synaptotagmin (119). Rim1 also interacts with Rimbinding protein, a Ca2⫹ channel coupler (120), as well as
␣-liprins (121) and ELKS (protein containing the high
levels of the aminoacids E, L, K, S) Rab3-interacting
molecules/(cytomatrix at the active zone) [CAST CAZassociated structural protein] (122), although their functions are unclear.
Conversely, Noc2 and granuphilin-a negatively regulate the function of Rab3A (123) and Rab27A (124,
125), respectively. Additionally, granuphilin-a has
been shown to control Rab27A-dependent regulated
secretion of neuropeptide Y from PC-12 cells (124),
and insulin from the pancreatic ␤ cell line, MIN6
(125) by controlling interaction of Rab27A with syntaxin 1a and Munc18-1, proteins involved in the
SNARE (soluble N-ethyl maleimide sensitive-factor
attachment protein receptor) complex and vesicle
fusion (126, 127).
In addition to Rabs and their effectors, Ca2⫹-dependent activator protein for secretion 1 (CAPS1) and
CAPS 2 are involved in the recruitment, perhaps by
enhancing tethering of insulin-containing vesicles to
the plasma membrane (128, 129). Chromaffin cells
from embryonic CAPS1- or CAPS2-knockout mice
show decreases in the number of docked insulin vesicles and the level of insulin secretion. The mechanism
of vesicle recruitment mediated by CAPSs, however, is
unknown.
Subsequent to tethering, RSP vesicles are docked,
primed, and fused to the plasma membrane via a
series of steps as follows (130–134). The GTP-bound
form of Rab3A on RSP vesicles tethers vesicles to the
plasma membrane via its binding to Rim1 on plasma
membrane. Rim1 is then released from Rab3A upon
GTP hydrolysis, which activates Munc13, which, in
turn, initiates the interaction between syntaxin-1,
SNAP25, and vesicle-associated membrane protein
(also known as synaptobrevin) to form the SNARE
complex. Upon Ca2⫹ influx by stimulation, the Ca2⫹
sensor synaptotagmin binds the SNARE complex and
Mol Endocrinol, December 2008, 22(12):2583–2595 2589
triggers membrane fusion between vesicles and the
plasma membrane. After fusion, the SNARE complex
is disassembled by NSF and ␣-SNAP for recycling.
Thus, many molecules have been identified and
shown to be necessary for mediating RSP vesicle
tethering, docking, priming, and fusion.
SUMMARY
In this review, we have highlighted the cytoplasmic
proteins that play a role in the transport of regulated
secretory pathway vesicles, primarily those containing
peptide hormones, from the site of genesis at the TGN
to the secretion site at the plasma membrane. A model
showing the cytoplasmic proteins that participate in
the transport and tethering of RSP hormone-containing vesicles drawn from studies using different cell
systems is shown in Fig. 3. At the TGN (Fig. 3, box i),
sorting of RSP proteins into immature vesicle takes
place. The various mechanisms of protein sorting into
vesicles via aggregation followed by association of the
aggregates to the TGN membrane via interaction with
lipid rafts, or protein-sorting receptors are discussed
in section entitled “Protein Sorting into RSP Vesicles”
and Fig. 2. Simultaneously, clathrin-driven vesicle
budding occurs at the TGN, facilitated by the recruitment of adaptors such as AP-1 and GGA found on
RSP vesicles. Post-Golgi transport of RSP vesicles is
mediated by a microtubule-based machinery (Fig. 3,
box ii). How are RSP vesicles anchored to microtubules? Our recent studies have indicated that the
cytoplasmic tail of transmembrane CPE on ACTH
vesicles in pituitary cells interacts with the adaptor
protein dynactin, which in turn recruits the motor kinesin-2 (KIF3) and kinesin-3 (KIF1A) (64). Others have
shown that kinesin-1, -2, and -3 are involved in RSP
vesicle transport in various types of endocrine cells.
The microtubule-based transport system carries the
RSP vesicles to the proximity of the release site at the
plasma membrane. There the RSP vesicles are loaded
on to the myosin V transport system (Fig. 3, box iii).
The GTPase, rab27A, found associated with RSP vesicles, in conjunction with myosin-VIIa- and Rab-interacting protein, has been identified to play an important
role in recruiting RSP vesicles on to the myosin transport system, which then moves them to the actin cortex where they are held as a reserve pool. Subsequently, a subpopulation of RSP vesicles are moved
by the myosin system through the actin cortex, arriving
just below the plasma membrane where they are tethered and some are docked to become the readily
releasable pool. Thus, microtubules and F-actins function in a coordinated manner during vesicle transport
to achieve precise delivery of hormone-containing
RSP vesicles to the plasma membrane (56). The RSP
vesicles are tethered to the plasma membrane via
various molecules highlighted in Fig. 3, box iv. These
include Rabs, primarily Rab3 and various effector mol-
2590 Mol Endocrinol, December 2008, 22(12):2583–2595
ecules such as rabphillin and Rim1, that facilitate
exocytosis.
Excessive membrane on the plasma membrane produced by exocytosis is removed by endocytosis 关see
review by Gundelfinger et al. (135)兴. Thus, coupling
between endocytosis and exocytosis is important to
maintain constant membrane mass on the plasma
membrane.
Comparison of cytoplasmic proteins involved in the
transport of RSP and CSP vesicles revealed many
commonalities, both in the clathrin-mediated budding
process and the microtubule and actin-based trafficking mechanisms (Table 1). However, there are also
differences, most significantly in the transport and
tethering mechanisms within the actin cortex and at
the plasma membrane, respectively (see Table 1). During post-Golgi transport, both RSP and CSP vesicles
use microtubule-based motor systems, but each type
of vesicle heads for distinctive destinations, e.g. to a
reserve pool distant from the plasma membrane or
directly to the plasma membrane for secretion, respectively. It has been speculated that variations in
microtubule-associated proteins and posttranslational
modification of microtubules (136) may distinguish the
different routes. Hence, the different destinations of
the RSP and CSP vesicles may be determined by
selection of different subsets of microtubule motors
that show high affinity to a certain route 关e.g. kinesin-1
binds glutamylated microtubules more strongly (136)兴.
Indeed, the cytoplasmic tail of transmembrane CPE on
RSP vesicles anchors on to dynactin, which interacts
with kinesins, KIF1A and KIF3A, and a subset of modulators different from those present on CSP vesicles
for movement (see Table 1). At the proximity of the
plasma membrane, only RSP vesicles are captured in
a reserve pool in the actin cortex. A subpopulation of
Park and Loh • Minireview
RSP vesicles is tethered and docked to the plasma
membrane using GTPase Rab proteins and a number of effectors (Fig. 3, box iv) and released upon
stimulation. In contrast, CSP vesicles are tethered to
the plasma membrane using the exocyst machinery
and exocytose mostly in an unregulated manner (98,
99, 137).
It is also intriguing how multiple cytoplasmic molecules can fit and work together within a limited space
beneath a single RSP vesicle without any steric hindrance to mediate trafficking and tethering to the
plasma membrane. Studies now show that this phenomenon can be accomplished by usage of multisubunit protein complexes such as dynactin (138), or a
scaffolding protein such as Rim1, which interacts with
multiple proteins (118, 122, 139, 140). A multisubunit
protein complex or scaffold protein capable of binding
several proteins simultaneously can function as a multiarm adaptor to allow even a single cytoplasmic tail to
interact with multiple proteins at the same time. This is
exemplified by our recent study showing that the CPE
cytoplasmic tail found in all peptide hormone-containing vesicles interacts with dynactin (64), which consists of 11 different subunits. This enables various
adaptor proteins (138) to be recruited to the CPE tail to
mediate transport, tethering, and docking of RSP vesicles to the plasma membrane for exocytosis in (neuro)endocrine cells.
Another question is how do different molecules corroborate to drive vesicles in one direction vs. another.
So far, there are various proposed hypotheses regarding this issue. For example, in the tug-of-war model
(141, 142), microtubule motors of opposite polarity
compete with each other whereas motors of the
same polarity increase the speed of vesicle movement. One recent study shows a cooperative effort
Table 1. Comparison of Molecules Involved in Vesicle Traffic in the RSP and CSP
Differences
Commonalities RSP/CSP
RSP
CSP
AP-1 effectors: PACS-1,
␥-synerigin, aftiphilin,
NECAP
MT motor/modulators:
kinesin-1/PS1/APH-1/PEN2/nicastrin kinesin-1/JIP1b
Vesicle budding from
TGN
Adaptors: AP-1, GGA
Clathrin coat
AP-1/GGA effectors: unknown
Vesicle transport along
MTs
MT motors: kinesin-1
cytoplasmic dynein
Vesicle transport in actin
cortex
F-actin motor; myosin Va
Vesicle tethering to PM
F-actins-based stabilization
of tethered/docked
vesicles on PM
MT motors/modulators:
kinesin-3/JIP3
kinesin/dynactin-HAP1-Htt
kinesin-2 and -3/dynactin
Myosin Va recruiter: Rab27A/
MyRIP Rab27A/
melanophilin
Interaction of PAM with actins
Rab3A with Rab3D, Rim-1,
Rabphilin3A, Noc2
Rab27A with granuphilin-1
CAPS
RalA/exocyst
Myosin Va recruiter: unknown
but maybe Rabs
Exocyst
PM, Plasma membrane; TGN, trans-Golgi network; JIP, JNK-interacting protein; PACS-1, phosphofurin acidic cluster sorting
protein-1; NECAP, adaptin-ear-binding coat-associated protein; PS1, presenilin 1; APH-1, anterior pharynx-defective-1; PEN-2,
presenilin enhancer-2.
Park and Loh • Minireview
between kinesin-1 and myosin V (143) for processive
vesicle movement. Myosin V confers processivity to
kinesin-driven movement on microtubules whereas kinesin-1 confers it to myosin-mediated movement on
F-actins. However, because this study is based on in
vitro experiments, the cooperation might not occur in
vivo. Thus, further study is necessary to verify this
phenomenon in vivo.
In conclusion, the understanding of the cytoplasmic
mechanism(s) that coordinate the trafficking and tethering of hormone-containing vesicles to the plasma
membrane in the regulated secretory pathway of (neuro)endocrine cells is still at its infancy. The field has
only just begun to identify the different vesicular transmembrane and cytosolic proteins that mediate these
transport and tethering processes, and much of the
data are derived and assembled in our model (Fig. 3)
from studies of different (neuro)endocrine cell types.
To this end, these players serve as excellent candidates for studying vesicle trafficking in the specific
neuroendocrine cell system of interest. However, as a
cautionary note, it cannot be assumed that all RSP
peptidergic vesicles use the same vesicular anchors,
motors, and cytosolic effector proteins in different
neuroendocrine cell types for trafficking and tethering
to the plasma membrane, although there is much in
common as the literature indicates. What is certain is
that the differences between RSP and CSP vesicle
transport (Table 1) will stand.
It is clear that there are still many unanswered questions. How are the different cytosolic proteins selectively recruited to RSP vesicles but not CSP vesicles?
What initiates and mediates the recruitment of different proteins to assemble the transport machinery
complex? How are the recruited proteins switched
during post-Golgi vesicle traffic through different junctions: microtubules, actin cortex, and finally the
plasma membrane? Are the protein complexes and
transport machinery modulated or reorganized in an
activity-dependent manner? If so, how does stimulation induce the change? Ultimately, answers to these
questions will fully uncover the transport and tethering
mechanisms and the proteins that govern regulated
secretion of peptide hormones and neuropeptides.
This will eventually facilitate identification of endocrinological and neurological diseases associated with
defects 关e.g. mutations in the kinesin and dynactin
genes (144–148)兴 in the proteins of the RSP vesicle
transport system.
Acknowledgments
We thank Dr. Niamh Cawley 关National Institute of Child
Health and Human Development (NICHD), National Institutes
of Health (NIH)兴, and Dr. Andre Phillips (NICHD, NIH) for their
suggestions and critical reading of the manuscript.
Received June 30, 2008. Accepted July 22, 2008.
Address all correspondence and requests for reprints to:
Peng Loh, National Institutes of Health, Building 49, Room
Mol Endocrinol, December 2008, 22(12):2583–2595 2591
5A22, 49 Convent Drive, Bethesda, Maryland 20892. E-mail:
[email protected].
This work was supported by the Intramural Research Program of the Eunice Kennedy Shriver, NICHD, NIH.
Disclosure Statement: The authors have nothing to disclose.
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