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1.
Yuusuke Maruyama Toshihiko Ogura Kazuhiro Mio Kenta Kato Takeshi Kaneko Shigeki Kiyonaka Yasuo Mori Chikara Sato 《The Journal of biological chemistry》2009,284(20):13676-13685
The Ca2+ release-activated Ca2+ channel is a
principal regulator of intracellular Ca2+ rise, which conducts
various biological functions, including immune responses. This channel,
involved in store-operated Ca2+ influx, is believed to be composed
of at least two major components. Orai1 has a putative channel pore and
locates in the plasma membrane, and STIM1 is a sensor for luminal
Ca2+ store depletion in the endoplasmic reticulum membrane. Here we
have purified the FLAG-fused Orai1 protein, determined its tetrameric
stoichiometry, and reconstructed its three-dimensional structure at 21-Å
resolution from 3681 automatically selected particle images, taken with an
electron microscope. This first structural depiction of a member of the Orai
family shows an elongated teardrop-shape 150Å in height and 95Å in
width. Antibody decoration and volume estimation from the amino acid sequence
indicate that the widest transmembrane domain is located between the round
extracellular domain and the tapered cytoplasmic domain. The cytoplasmic
length of 100Å is sufficient for direct association with STIM1. Orifices
close to the extracellular and intracellular membrane surfaces of Orai1 seem
to connect outside the molecule to large internal cavities.Ca2+ is an intracellular second messenger that plays important
roles in various physiological functions such as immune response, muscle
contraction, neurotransmitter release, and cell proliferation. Intracellular
Ca2+ is mainly stored in the endoplasmic reticulum
(ER).2 This ER system
is distributed through the cytoplasm from around the nucleus to the cell
periphery close to the plasma membrane. In non-excitable cells, the ER
releases Ca2+ through the inositol 1,4,5-trisphosphate
(IP3) receptor channel in response to various signals, and the
Ca2+ store is depleted. Depletion of Ca2+ then induces
Ca2+ influx from outside the cell to help in refilling the
Ca2+ stores and to continue Ca2+ rise for several
minutes in the cytoplasm (1,
2). This Ca2+ influx
was first proposed by Putney
(3) and was named
store-operated Ca2+ influx. In the immune system, store-operated
Ca2+ influx is mainly mediated by the Ca2+
release-activated Ca2+ (CRAC) current, which is a highly
Ca2+-selective inwardly rectified current with low conductance
(4,
5). Pathologically, the loss of
CRAC current in T cells causes severe combined immunodeficiency
(6) where many Ca2+
signal-dependent gene expressions, including cytokines, are interrupted
(7). Therefore, CRAC current is
necessary for T cell functions.Recently, Orai1 (also called CRACM1) and STIM1 have been physiologically
characterized as essential components of the CRAC channel
(8–12).
They are separately located in the plasma membrane and in the ER membrane;
co-expression of these proteins presents heterologous CRAC-like currents in
various types of cells (10,
13–15).
Both of them are shown to be expressed ubiquitously in various tissues
(16–18).
STIM1 senses Ca2+ depletion in the ER through its EF hand motif
(19) and transmits a signal to
Orai1 in the plasma membrane. Although Orai1 is proposed as a regulatory
component for some transient receptor potential canonical channels
(20,
21), it is believed from the
mutation analyses to be the pore-forming subunit of the CRAC channel
(8,
22–24).
In the steady state, both Orai1 and STIM1 molecules are dispersed in each
membrane. When store depletion occurs, STIM1 proteins gather into clusters to
form puncta in the ER membrane near the plasma membrane
(11,
19). These clusters then
trigger the clustering of Orai1 in the plasma membrane sites opposite the
puncta (25,
26), and CRAC channels are
activated (27).Orai1 has two homologous genes, Orai2 and Orai3
(8). They form the Orai family
and have in common the four transmembrane (TM) segments with relatively large
N and C termini. These termini are demonstrated to be in the cytoplasm,
because both N- and C-terminally introduced tags are immunologically detected
only in the membrane-permeabilized cells
(8,
9). The subunit stoichiometry
of Orai1 is as yet controversial: it is believed to be an oligomer, presumably
a dimer or tetramer even in the steady state
(16,
28–30).Despite the accumulation of biochemical and electrophysiological data,
structural information about Orai1 is limited due to difficulties in
purification and crystallization. In this study, we have purified Orai1 in its
tetrameric form and have reconstructed the three-dimensional structure from
negatively stained electron microscopic (EM) images. 相似文献
2.
3.
Congmin Li Jenny Chan Franciose Haeseleer Katsuhiko Mikoshiba Krzysztof Palczewski Mitsuhiko Ikura James B. Ames 《The Journal of biological chemistry》2009,284(4):2472-2481
Calcium-binding protein 1 (CaBP1), a neuron-specific member of the
calmodulin (CaM) superfamily, modulates Ca2+-dependent activity of
inositol 1,4,5-trisphosphate receptors (InsP3Rs). Here we present
NMR structures of CaBP1 in both Mg2+-bound and
Ca2+-bound states and their structural interaction with
InsP3Rs. CaBP1 contains four EF-hands in two separate domains. The
N-domain consists of EF1 and EF2 in a closed conformation with Mg2+
bound at EF1. The C-domain binds Ca2+ at EF3 and EF4, and exhibits
a Ca2+-induced closed to open transition like that of CaM. The
Ca2+-bound C-domain contains exposed hydrophobic residues
(Leu132, His134, Ile141, Ile144,
and Val148) that may account for selective binding to
InsP3Rs. Isothermal titration calorimetry analysis reveals a
Ca2+-induced binding of the CaBP1 C-domain to the N-terminal region
of InsP3R (residues 1-587), whereas CaM and the CaBP1 N-domain did
not show appreciable binding. CaBP1 binding to InsP3Rs requires
both the suppressor and ligand-binding core domains, but has no effect on
InsP3 binding to the receptor. We propose that CaBP1 may regulate
Ca2+-dependent activity of InsP3Rs by promoting
structural contacts between the suppressor and core domains.Calcium ion (Ca2+) in the cell functions as an important
messenger that controls neurotransmitter release, gene expression, muscle
contraction, apoptosis, and disease processes
(1). Receptor stimulation in
neurons promotes large increases in intracellular Ca2+ levels
controlled by Ca2+ release from intracellular stores through
InsP3Rs (2). The
neuronal type-1 receptor
(InsP3R1)2
is positively and negatively regulated by cytosolic Ca2+
(3-6),
important for the generation of repetitive Ca2+ transients known as
Ca2+ spikes and waves
(1). Ca2+-dependent
activation of InsP3R1 contributes to the fast rising phase of
Ca2+ signaling known as Ca2+-induced Ca2+
release (7).
Ca2+-induced inhibition of InsP3R1, triggered at higher
cytosolic Ca2+ levels, coordinates the temporal decay of
Ca2+ transients (6).
The mechanism of Ca2+-dependent regulation of InsP3Rs is
complex (8,
9), and involves direct
Ca2+ binding sites
(5,
10) as well as remote sensing
by extrinsic Ca2+-binding proteins such as CaM
(11,
12), CaBP1
(13,
14), CIB1
(15), and NCS-1
(16).Neuronal Ca2+-binding proteins (CaBP1-5
(17)) represent a new
sub-branch of the CaM superfamily
(18) that regulate various
Ca2+ channel targets. Multiple splice variants and isoforms of
CaBPs are localized in different neuronal cell types
(19-21)
and perform specialized roles in signal transduction. CaBP1, also termed
caldendrin (22), has been
shown to modulate the Ca2+-sensitive activity of InsP3Rs
(13,
14). CaBP1 also regulates
P/Q-type voltage-gated Ca2+ channels
(23), L-type channels
(24), and the transient
receptor potential channel, TRPC5
(25). CaBP4 regulates
Ca2+-dependent inhibition of L-type channels in the retina and may
be genetically linked to retinal degeneration
(26). Thus, the CaBP proteins
are receiving increased attention as a family of Ca2+ sensors that
control a variety of Ca2+ channel targets implicated in neuronal
degenerative diseases.CaBP proteins contain four EF-hands, similar in sequence to those found in
CaM and troponin C (18)
(Fig. 1). By analogy to CaM
(27), the four EF-hands are
grouped into two domains connected by a central linker that is four residues
longer in CaBPs than in CaM. In contrast to CaM, the CaBPs contain
non-conserved amino acids within the N-terminal region that may confer target
specificity. Another distinguishing property of CaBPs is that the second
EF-hand lacks critical residues required for high affinity Ca2+
binding (17). CaBP1 binds
Ca2+ only at EF3 and EF4, whereas it binds Mg2+ at EF1
that may serve a functional role
(28). Indeed, changes in
cytosolic Mg2+ levels have been detected in cortical neurons after
treatment with neurotransmitter
(29). Other neuronal
Ca2+-binding proteins such as DREAM
(30), CIB1
(31), and NCS-1
(32) also bind Mg2+
and exhibit Mg2+-induced physiological effects. Mg2+
binding in each of these proteins helps stabilize their Ca2+-free
state to interact with signaling targets.Open in a separate windowFIGURE 1.Amino acid sequence alignment of human CaBP1 with CaM. Secondary
structural elements (α-helices and β-strands) were derived from NMR
analysis. The four EF-hands (EF1, EF2, EF3, and EF4) are highlighted
green, red, cyan, and yellow. Residues in the 12-residue
Ca2+-binding loops are underlined and chelating residues
are highlighted bold. Non-conserved residues in the hydrophobic patch
are colored red.Despite extensive studies on CaBP1, little is known about its structure and
target binding properties, and regulation of InsP3Rs by CaBP1 is
somewhat controversial and not well understood. Here, we present the NMR
solution structures of both Mg2+-bound and Ca2+-bound
conformational states of CaBP1 and their structural interactions with
InsP3R1. These CaBP1 structures reveal important
Ca2+-induced structural changes that control its binding to
InsP3R1. Our target binding analysis demonstrates that the C-domain
of CaBP1 exhibits Ca2+-induced binding to the N-terminal cytosolic
region of InsP3R1. We propose that CaBP1 may regulate
Ca2+-dependent channel activity in InsP3Rs by promoting
a structural interaction between the N-terminal suppressor and ligand-binding
core domains that modulates Ca2+-dependent channel gating
(8,
33,
34). 相似文献
4.
Nanako Masada Antonio Ciruela David A. MacDougall Dermot M. F. Cooper 《The Journal of biological chemistry》2009,284(7):4451-4463
Nine membrane-bound mammalian adenylyl cyclases (ACs) have been identified.
Type 1 and 8 ACs (AC1 and AC8), which are both expressed in the brain and are
stimulated by Ca2+/calmodulin (CaM), have discrete neuronal
functions. Although the Ca2+ sensitivity of AC1 is higher than that
of AC8, precisely how these two ACs are regulated by Ca2+/CaM
remains elusive, and the basis for their diverse physiological roles is quite
unknown. Distinct localization of the CaM binding domains within the two
enzymes may be essential to differential regulation of the ACs by
Ca2+/CaM. In this study we compare in detail the regulation of AC1
and AC8 by Ca2+/CaM both in vivo and in vitro and
explore the different role of each Ca2+-binding lobe of CaM in
regulating the two enzymes. We also assess the relative dependence of AC1 and
AC8 on capacitative Ca2+ entry. Finally, in real-time fluorescence
resonance energy transfer-based imaging experiments, we examine the effects of
dynamic Ca2+ events on the production of cAMP in cells expressing
AC1 and AC8. Our data demonstrate distinct patterns of regulation and
Ca2+ dependence of AC1 and AC8, which seems to emanate from their
mode of regulation by CaM. Such distinctive properties may contribute
significantly to the divergent physiological roles in which these ACs have
been implicated.Nine membrane-bound mammalian adenylyl cyclases
(ACs),2 AC1–AC9,
have been identified (1). They
possess a common predicted structure
(2)3
and are stimulated by forskolin (FSK; except AC9) and Gsα,
although they are distributed and regulated differently
(1,
3,
4). Four ACs are regulated by
physiological concentrations of Ca2+ and thereby provide a critical
link between the Ca2+- and cAMP-signaling pathways
(3,
5); AC5 and AC6 are directly
inhibited by Ca2+, whereas AC1 and AC8 are stimulated by
Ca2+ in a calmodulin (CaM)-dependent manner
(5). AC3 is also regulated by
CaM in vitro, although this requires supramicromolar concentration of
Ca2+ (6), and in
vivo AC3 is inhibited by Ca2+ via CaM kinase II
(7), unlike AC1 and AC8.AC1 is closely related in sequence to the Ca2+/CaM-stimulable
rutabaga AC from Drosophila, which is important in
Drosophila learning tasks
(8–10).
AC1 and the other Ca2+/CaM-stimulable mammalian AC, AC8, have also
been implicated in learning and memory
(11). As a means of
establishing their proposed roles, single and/or double AC1 and AC8 knockout
mice have been generated. Mouse models have demonstrated that
Ca2+/CaM-stimulable ACs are involved in long-term potentiation and
long-term memory (12).
However, despite the general view that AC1 and AC8 can behave similarly,
discrete physiological actions of each isoform are becoming apparent. Recent
studies by Zhuo''s group demonstrated that AC1 specifically participates in
N-methyl-d-aspartic acid receptor-induced neuronal
excitotoxicity (13) and an
increase in GluR1 synthesis induced by blocking AMPA receptors
(14). Furthermore, Nicol and
colleagues (15,
16) showed a contribution of
AC1, but not AC8, in axon terminal refinement in the retina. On the other
hand, AC8 specifically was seen to be responsible for retrieval from adaptive
presynaptic silencing (17) and
the acquiring of new spatial information
(18). These differences in
physiological roles must reflect not only differences in their distributions
but also presumably in their regulatory properties. Both enzymes are expressed
in brain; AC1 is neuro-specific, whereas the expression of AC8 is more
widespread (1,
12). Within the central
nervous system, AC1 is abundant in the hippocampus, the cerebral cortex, and
the granule cells of the cerebellum, whereas AC8 has a high expression level
in the thalamus and the cerebral cortex
(19). Studies of mouse brain
revealed that AC1 is distributed pre-synaptically and AC8 post-synaptically
(18,
20).Although physiological differences in the roles of these two enzymes are
suggested from the studies outlined above, the regulatory mechanisms that
might underlie these differences are not. AC1 is more sensitive to
Ca2+ than is AC8 in vitro
(21,
22), yet details on how these
two enzymes are regulated by Ca2+/CaM are sparse. In non-excitable
cells, a Ca2+ elevation caused by capacitative Ca2+
entry (CCE), the mode of Ca2+ entry triggered by emptying
Ca2+ from internal stores
(23), preferentially
stimulates AC1 and AC8 (21).
Although stimulation of AC8 by CCE has been shown to be at least partially
dependent on its localization at lipid rafts
(24), whether AC1 is also
targeted to this region of plasma membranes has never been addressed. In
addition, CaM regulation of AC1 and AC8 has not been compared in detail,
although CaM appears to bind to different domains of the two enzymes. AC8
utilizes two CaM binding domains: a classic amphipathic “1-5-8-14”
motif at the N terminus and an IQ-like motif in the C2b domain
(25). A recent study indicates
that CaM pre-associates with the N terminus of AC8, where it becomes fully
saturated upon a Ca2+ rise, and activates the enzyme via a
C-terminally mediated relief of auto-inhibitory mechanisms
(26). By contrast, only
residues 495–522 of the C1b region of AC1 have been shown to bind CaM in
a Ca2+-dependent manner
(27,
28). With the presence of only
one CaM binding domain in AC1, a simpler mechanism of CaM regulation might be
expected.CaM mediates the regulation of numerous Ca2+-dependent processes
in eukaryotic cells (29). The
protein possesses N- and C-terminal lobes, both of which contain two
Ca2+ binding EF hands (EF1 and EF2 in the N lobe, and EF3 and EF4
in the C lobe (30)). Mutations
in the EF hands have been valuable for investigating the interaction of CaM
with its targets. Alanine substitutions in the EF12 pair or EF34 pair have
generated CaM12 and CaM34 to investigate the independent
function of the C and N lobes of CaM, respectively
(31,
32).Against the background of the distinct physiological roles carried out by
AC1 and AC8, we performed a detailed comparison of the two enzymes expressed
in HEK 293 cells. Their sensitivity to Ca2+/CaM was compared both
in vitro and in vivo; the possibility that they might be
expressed in different domains of the plasma membrane was addressed; and
putative lobe-specific CaM regulation was assessed using
Ca2+-binding mutants of CaM. Single cell measurements using a
FRET-based cAMP sensor were performed to compare the kinetic responses of the
enzymes to physiological elevations of [Ca2+]i.
The results demonstrate superficial similarities in the regulation of AC1 and
AC8 but critical disparities in their mechanism of activation by the lobes of
CaM and in the speed and pattern of their responsiveness to
[Ca2+]i. These discrete behaviors provide a
physiological opportunity for different outcomes to elevation of
[Ca2+]i, depending on the AC that is expressed
in particular contexts. 相似文献
5.
Quang-Kim Tran Jared Leonard D. J. Black Owen W. Nadeau Igor G. Boulatnikov Anthony Persechini 《The Journal of biological chemistry》2009,284(18):11892-11899
We have investigated the possible biochemical basis for enhancements in NO
production in endothelial cells that have been correlated with agonist- or
shear stress-evoked phosphorylation at Ser-1179. We have found that a
phosphomimetic substitution at Ser-1179 doubles maximal synthase activity,
partially disinhibits cytochrome c reductase activity, and lowers the
EC50(Ca2+) values for calmodulin binding and enzyme
activation from the control values of 182 ± 2 and 422 ± 22
nm to 116 ± 2 and 300 ± 10 nm. These are
similar to the effects of a phosphomimetic substitution at Ser-617 (Tran, Q.
K., Leonard, J., Black, D. J., and Persechini, A. (2008) Biochemistry
47, 7557–7566). Although combining substitutions at Ser-617 and Ser-1179
has no additional effect on maximal synthase activity, cooperativity between
the two substitutions completely disinhibits reductase activity and further
reduces the EC50(Ca2+) values for calmodulin binding and
enzyme activation to 77 ± 2 and 130 ± 5 nm. We have
confirmed that specific Akt-catalyzed phosphorylation of Ser-617 and Ser-1179
and phosphomimetic substitutions at these positions have similar functional
effects. Changes in the biochemical properties of eNOS produced by combined
phosphorylation at Ser-617 and Ser-1179 are predicted to substantially
increase synthase activity in cells at a typical basal free Ca2+
concentration of 50–100 nm.The nitric-oxide synthases catalyze formation of NO and
l-citrulline from l-arginine and O2, with
NADPH as the electron donor
(1). The role of NO generated
by endothelial nitricoxide synthase
(eNOS)2 in the
regulation of smooth muscle tone is well established and was the first of
several physiological roles for this small molecule that have so far been
identified (2). The
nitric-oxide synthases are homodimers of 130–160-kDa subunits. Each
subunit contains a reductase and oxygenase domain
(1). A significant difference
between the reductase domains in eNOS and nNOS and the homologous P450
reductases is the presence of inserts in these synthase isoforms that appear
to maintain them in their inactive states
(3,
4). A calmodulin (CaM)-binding
domain is located in the linker that connects the reductase and oxygenase
domains, and the endothelial and neuronal synthases both require
Ca2+ and exogenous CaM for activity
(5,
6). When CaM is bound, it
somehow counteracts the effects of the autoinhibitory insert(s) in the
reductase. The high resolution structure for the complex between
(Ca2+)4-CaM and the isolated CaM-binding domain from
eNOS indicates that the C-ter and N-ter lobes of CaM, which each contain a
pair of Ca2+-binding sites, enfold the domain, as has been observed
in several other such CaM-peptide complexes
(7). Consistent with this
structure, investigations of CaM-dependent activation of the neuronal synthase
suggest that both CaM lobes must participate
(8,
9).Bovine eNOS can be phosphorylated in endothelial cells at Ser-116, Thr-497,
Ser-617, Ser-635, and Ser-1179
(10–12).
There are equivalent phosphorylation sites in the human enzyme
(10–12).
Phosphorylation of the bovine enzyme at Thr-497, which is located in the
CaM-binding domain, blocks CaM binding and enzyme activation
(7,
11,
13,
14). Ser-116 can be basally
phosphorylated in cells (10,
11,
13,
15), and dephosphorylation of
this site has been correlated with increased NO production
(13,
15). However, it has also been
reported that a phosphomimetic substitution at this position has no effect on
enzyme activity measured in vitro
(13). Ser-1179 is
phosphorylated in response to a variety of stimuli, and this has been reliably
correlated with enhanced NO production in cells
(10,
11). Indeed, NO production is
elevated in transgenic endothelium expressing an eNOS mutant containing an
S1179D substitution, but not in tissue expressing an S1179A mutant
(16). Shear stress or insulin
treatment is correlated with Akt-catalyzed phosphorylation of Ser-1179 in
endothelial cells, and this is correlated with increased NO production in the
absence of extracellular Ca2+
(17–19).
Akt-catalyzed phosphorylation or an S1179D substitution has also been
correlated with increased synthase activity in cell extracts at low
intracellular free [Ca2+]
(17). Increased NO production
has also been observed in cells expressing an eNOS mutant containing an S617D
substitution, and physiological stimuli such as shear-stress, bradykinin,
VEGF, and ATP appear to stimulate Akt-catalyzed phosphorylation of Ser-617 and
Ser-1179 (12,
13,
20). Although S617D eNOS has
been reported to have the same maximum activity in vitro as the wild
type enzyme (20), in our hands
an S617D substitution increases the maximal CaM-dependent synthase activity of
purified mutant enzyme ∼2-fold, partially disinhibits reductase activity,
and reduces the EC50(Ca2+) values for CaM binding and
enzyme activation (21).In this report, we describe the effects of a phosphomimetic Asp
substitution at Ser-1179 in eNOS on the Ca2+ dependence of CaM
binding and CaM-dependent activation of reductase and synthase activities. We
also describe the effects on these properties of combining this substitution
with one at Ser-617. Finally, we demonstrate that Akt-catalyzed
phosphorylation and Asp substitutions at Ser-617 and Ser-1179 have similar
functional effects. Our results suggest that phosphorylation of eNOS at
Ser-617 and Ser-1179 can substantially increase synthase activity in cells at
a typical basal free Ca2+ concentration of 50–100
nm, while single phosphorylations at these sites produce smaller
activity increases, and can do so only at higher free Ca2+
concentrations. 相似文献
6.
7.
8.
Sean R. Stowell Moonjae Cho Christa L. Feasley Connie M. Arthur Xuezheng Song Jennifer K. Colucci Sougata Karmakar Padmaja Mehta Marcelo Dias-Baruffi Rodger P. McEver Richard D. Cummings 《The Journal of biological chemistry》2009,284(8):4989-4999
Galectin-1 (Gal-1) regulates leukocyte turnover by inducing the cell
surface exposure of phosphatidylserine (PS), a ligand that targets cells for
phagocytic removal, in the absence of apoptosis. Gal-1 monomer-dimer
equilibrium appears to modulate Gal-1-induced PS exposure, although the
mechanism underlying this regulation remains unclear. Here we show that
monomer-dimer equilibrium regulates Gal-1 sensitivity to oxidation. A mutant
form of Gal-1, containing C2S and V5D mutations (mGal-1), exhibits impaired
dimerization and fails to induce cell surface PS exposure while retaining the
ability to recognize carbohydrates and signal Ca2+ flux in
leukocytes. mGal-1 also displayed enhanced sensitivity to oxidation, whereas
ligand, which partially protected Gal-1 from oxidation, enhanced Gal-1
dimerization. Continual incubation of leukocytes with Gal-1 resulted in
gradual oxidative inactivation with concomitant loss of cell surface PS,
whereas rapid oxidation prevented mGal-1 from inducing PS exposure.
Stabilization of Gal-1 or mGal-1 with iodoacetamide fully protected Gal-1 and
mGal-1 from oxidation. Alkylation-induced stabilization allowed Gal-1 to
signal sustained PS exposure in leukocytes and mGal-1 to signal both
Ca2+ flux and PS exposure. Taken together, these results
demonstrate that monomer-dimer equilibrium regulates Gal-1 sensitivity to
oxidative inactivation and provides a mechanism whereby ligand partially
protects Gal-1 from oxidation.Immunological homeostasis relies on efficient contraction of activated
leukocytes following an inflammatory episode. Several factors, including
members of the galectin and tumor necrosis factor families
(1,
2), regulate leukocyte turnover
by inducing apoptotic cell death. In contrast, several galectin family
members, in particular galectin-1
(Gal-1),2 uniquely
regulate neutrophil turnover by inducing phosphatidylserine (PS) exposure,
which normally sensitizes apoptotic cells to phagocytic removal
(3,
4), independent of apoptosis, a
process recently termed preaparesis
(5).Previous studies suggested that dimerization may be required for
Gal-1-induced PS exposure, as a mutant form of Gal-1 (mGal-1) containing two
point mutations within the dimer interface, C2S and V5D (C2S,V5D), displays
impaired Gal-1 dimerization and fails to induce PS exposure
(6). However, the manner in
which monomer-dimer equilibrium regulates Gal-1 signaling remains unclear.
Previous studies suggest that dimerization may be required for efficient
cross-linking of functional receptors or the formation of signaling lattices
(7–9).
Consistent with this, monomeric mutants of several other galectins fail to
induce PS exposure or signal leukocytes
(4,
8). Gal-1 signaling of PS
exposure requires initial signaling events, such as mobilization of
intracellular Ca2+ followed by sustained receptor engagement
(10). Although mGal-1 fails to
induce PS exposure (6), whether
mGal-1 can induce these initial signaling events remains unknown
(10).In addition to directly regulating signaling, monomer-dimer equilibrium may
also regulate other aspects of Gal-1 function. Unlike many other proteins
involved in the regulation of immunity, Gal-1 displays unique sensitivity to
oxidative inactivation
(11–15).
Although engagement of ligand partially protects Gal-1 from oxidation
(15), the impact of Gal-1
oxidation on signaling remains enigmatic. During oxidation, Gal-1 forms three
distinct intramolecular disulfide bridges that facilitate profound
conformational changes that preclude ligand binding and Gal-1 dimerization
(12–14),
suggesting that monomerdimer equilibrium may also regulate Gal-1 sensitivity
to oxidative inactivation.Previous studies utilized dithiothreitol (DTT) in treatment conditions to
protect Gal-1 from oxidative inactivation
(16,
17). Indeed, failure to
include DTT precluded Gal-1-induced death in T cells
(3,
18), suggesting that Gal-1
undergoes rapid oxidation in vivo in the absence of reducing
conditions. However, DTT itself can induce apoptosis in leukocytes
(19), leaving questions
regarding the impact of Gal-1 oxidation on these signaling events. In
contrast, recent studies utilizing iodoacetamide-alkylated Gal-1 (iGal-1),
previously shown to protect Gal-1 from oxidative inactivation
(20–29),
demonstrated that DTT actually primes cells to become sensitive to
Gal-1-induced apoptosis regardless of Gal-1 sensitivity to oxidation
(5).As the engagement of leukocyte ligands requires glycan recognition and
oxidation precludes this binding
(11,
15), understanding the impact
of oxidation on Gal-1 signals will facilitate a greater appreciation of the
factors that govern Gal-1 oxidation and therefore function. Our results
demonstrate that Gal-1 monomer-dimer equilibrium provides a key regulatory
point controlling both Gal-1 sensitivity to oxidation and its ability to
signal PS exposure in leukocytes. These results provide novel insights into
Gal-1 function and explain at a biochemical level the mechanisms regulating
Gal-1 oxidative inactivation and signaling. 相似文献
9.
Danielle M. Paul Edward P. Morris Robert W. Kensler John M. Squire 《The Journal of biological chemistry》2009,284(22):15007-15015
The troponin complex on the thin filament plays a crucial role in the
regulation of muscle contraction. However, the precise location of troponin
relative to actin and tropomyosin remains uncertain. We have developed a
method of reconstructing thin filaments using single particle analysis that
does not impose the helical symmetry of actin and is independent of a starting
model. We present a single particle three-dimensional reconstruction of the
thin filament. Atomic models of the F-actin filament were fitted into the
electron density maps and troponin and tropomyosin located. The structure
provides evidence that the globular head region of troponin labels the two
strands of actin with a 27.5-Å axial stagger. The density attributed to
troponin appears tapered with the widest point toward the barbed end. This
leads us to interpret the polarity of the troponin complex in the thin
filament as reversed with respect to the widely accepted model.Regulation of actin filament function is a fundamental biological process
with implications ranging from cell migration to muscle contraction. Skeletal
and cardiac muscle thin filaments consist of actin and the regulatory proteins
troponin and tropomyosin. Contraction is initiated by release of
Ca2+ into the sarcomere and the consequent binding of
Ca2+ to regulatory sites on troponin. Troponin is believed to
undergo a conformational change leading to an azimuthal movement of
tropomyosin, which allows myosin heads to interact with actin, hydrolyze ATP,
and generate force. The molecular basis by which troponin acts to regulate
muscle contraction is only partly understood. It is essential that the
structure of troponin in the thin filament at high and low Ca2+ is
determined to properly understand the mechanism of regulation.The basic structure of the thin filament was described by Ebashi in 1972
(1). In this structure each
tropomyosin molecule covers seven actin monomers, and there is a 27.5-Å
stagger between troponin molecules. The 7-Å tropomyosin structure
(2), the atomic model of
F-actin (3), and the troponin
“core domain” (4)
have recently been used to generate atomic models of the thin filament in low
and high Ca2+ states
(5). While the position of
troponin in these models was constrained by known distance measurements
between filament components, the exact arrangement of the complex on the
filament has not been determined a priori. Although recently
published crystal structures of partial troponin complexes
(4,
6) have provided valuable
insights into the arrangement of the globular head or core domain, the complex
in its entirety has not been crystallized.Troponin is believed to consist of a globular core domain with an extended
tail (7). The globular core
contains the Ca2+-binding subunit
(TnC),2 the inhibitory
subunit (TnI), and the C-terminal part (residues 156–262) of the
tropomyosin-binding subunit (TnT). The extended tail consists of the
N-terminal part of TnT (residues 1–155). A structural rearrangement
associated with Ca2+ dissociation from the troponin core has been
observed (4) such that the
helix connecting the two domains of TnC collapses, releasing the TnI
inhibitory segment. It is postulated that the TnI inhibitory segment then
becomes able to bind actin, in so doing biasing tropomyosin
(8). To understand properly how
Ca2+ binding to TnC leads to movement of tropomyosin, it is
necessary to determine a high resolution structure of troponin attached to the
thin filament, allowing unambiguous docking of the available crystal
structures and direct observation of any changes at a molecular level caused
by Ca2+ binding.Direct visualization of the thin filament is possible using electron
microscopy. Tropomyosin strands have been resolved in the low and high
Ca2+ states confirming the movement of tropomyosin and the steric
blocking model (9,
10). Until recently the actin
helical repeat has been imposed in the majority of reconstructions of the thin
filament causing artifacts. Helical averaging using the actin repeat spreads
troponin density over every actin monomer, which prevents the detailed
position and shape of the troponin complex from being found
(11). It is possible to avoid
this effect by applying a single particle approach. Individual filament images
are divided into segments and each segment treated as a particle.
Three-dimensional reconstruction may then be carried out by single particle
techniques of alignment, classification
(12,
13), Euler angle assignment
(14–16)
and exact filter back-projection
(17,
18).Two forms of single particle analysis have emerged: helical single particle
analysis (19), where the
determined helical symmetry is applied to the final reconstruction, and
non-helical single particle analysis, which treats the complex as a truly
asymmetric particle. Helical single particle analysis has been used to
successfully reconstruct a myosin containing invertebrate thick filament to a
resolution of 25 Å (20),
and non-helical single particle analysis has been applied to the vertebrate
skeletal muscle thick filament allowing azimuthal perturbations of the myosin
heads to be observed (21).Model-based single particle image processing methods have recently been
applied to the structural analysis of the vertebrate
(5,
22,
23) and the insect thin
filament (24). We have
deliberately avoided starting with a model and any potential model bias by
using a reference-free alignment procedure. The adaptation of conventional
procedures and their application to the structural study of the muscle thin
filament has been documented
(25). 相似文献
10.
Rebecca M. Dixon Jack R. Mellor Jonathan G. Hanley 《The Journal of biological chemistry》2009,284(21):14230-14235
Oxygen and glucose deprivation (OGD) induces delayed cell death in
hippocampal CA1 neurons via Ca2+/Zn2+-permeable,
GluR2-lacking AMPA receptors (AMPARs). Following OGD, synaptic AMPAR currents
in hippocampal neurons show marked inward rectification and increased
sensitivity to channel blockers selective for GluR2-lacking AMPARs. This
occurs via two mechanisms: a delayed down-regulation of GluR2 mRNA expression
and a rapid internalization of GluR2-containing AMPARs during the OGD insult,
which are replaced by GluR2-lacking receptors. The mechanisms that underlie
this rapid change in subunit composition are unknown. Here, we demonstrate
that this trafficking event shares features in common with events that mediate
long term depression and long term potentiation and is initiated by the
activation of N-methyl-d-aspartic acid receptors. Using
biochemical and electrophysiological approaches, we show that peptides that
interfere with PICK1 PDZ domain interactions block the OGD-induced switch in
subunit composition, implicating PICK1 in restricting GluR2 from synapses
during OGD. Furthermore, we show that GluR2-lacking AMPARs that arise at
synapses during OGD as a result of PICK1 PDZ interactions are involved in
OGD-induced delayed cell death. This work demonstrates that PICK1 plays a
crucial role in the response to OGD that results in altered synaptic
transmission and neuronal death and has implications for our understanding of
the molecular mechanisms that underlie cell death during stroke.Oxygen and glucose deprivation
(OGD)3 associated with
transient global ischemia induces delayed cell death, particularly in
hippocampal CA1 pyramidal cells
(1–3),
a phenomenon that involves Ca2+/Zn2+-permeable,
GluR2-lacking AMPARs (4).
AMPARs are heteromeric complexes of subunits GluR1–4
(5), and most AMPARs in the
hippocampus contain GluR2, which renders them calcium-impermeable and results
in a marked inward rectification in their current-voltage relationship
(6–8).
Ischemia induces a delayed down-regulation of GluR2 mRNA and protein
expression (4,
9–11),
resulting in enhanced AMPAR-mediated Ca2+ and Zn2+
influx into CA1 neurons (10,
12). In these neurons,
AMPAR-mediated postsynaptic currents (EPSCs) show marked inward rectification
1–2 days following ischemia and increased sensitivity to 1-naphthyl
acetyl spermine (NASPM), a channel blocker selective for GluR2-lacking AMPARs
(13–16).
Blockade of these channels at 9–40 h following ischemia is
neuroprotective, indicating a crucial role for Ca2+-permeable
AMPARs in ischemic cell death
(16).In addition to delayed changes in AMPAR subunit composition as a result of
altered mRNA expression, it was recently reported that
Ca2+-permable, GluR2-lacking AMPARs are targeted to synaptic sites
via membrane trafficking at much earlier times during OGD
(17). This subunit
rearrangement involves endocytosis of AMPARs containing GluR2 complexed with
GluR1/3, followed by exocytosis of GluR2-lacking receptors containing GluR1/3
(17). However, the molecular
mechanisms behind this trafficking event are unknown, and furthermore, it is
not known whether these trafficking-mediated changes in AMPAR subunit
composition contribute to delayed cell death.AMPAR trafficking is a well studied phenomenon because of its crucial
involvement in long term depression (LTD) and long term potentiation (LTP),
activity-dependent forms of synaptic plasticity thought to underlie learning
and memory. AMPAR endocytosis, exocytosis, and more recently subunit-switching
events (brought about by trafficking that involves endo/exocytosis) are
central to the necessary changes in synaptic receptor complement
(7,
18–20).
It is possible that similar mechanisms regulate AMPAR trafficking during
OGD.PICK1 is a PDZ and BAR (Bin-amphiphysin-Rus) domain-containing protein that
binds, via the PDZ domain, to a number of membrane proteins including AMPAR
subunits GluR2/3. This interaction is required for AMPAR internalization from
the synaptic plasma membrane in response to Ca2+ influx via NMDAR
activation in hippocampal neurons
(21–23).
This process is the major mechanism that underlies the reduction in synaptic
strength in LTD. Furthermore, PICK1-mediated trafficking has recently emerged
as a mechanism that regulates the GluR2 content of synaptic receptors, which
in turn determines their Ca2+ permeability
(7,
20). This is likely to be of
profound importance in both plasticity and pathological mechanisms.
Importantly, PICK1 overexpression has been shown to induce a shift in synaptic
AMPAR subunit composition in hippocampal CA1 neurons, resulting in inwardly
rectifying AMPAR EPSCs via reduced surface GluR2 and no change in GluR1
(24). This suggests that PICK1
may mediate the rapid switch in subunit composition occurring during OGD
(17). Here, we demonstrate
that the OGD-induced switch in AMPAR subunit composition is dependent on PICK1
PDZ interactions, and importantly, that this early trafficking event that
occurs during OGD contributes to the signaling that results in delayed
neuronal death. 相似文献
11.
Kuen-Feng Chen Pei-Yen Yeh Chiun Hsu Chih-Hung Hsu Yen-Shen Lu Hsing-Pang Hsieh Pei-Jer Chen Ann-Lii Cheng 《The Journal of biological chemistry》2009,284(17):11121-11133
Hepatocellular carcinoma (HCC) is one of the most common and aggressive
human malignancies. Recombinant tumor necrosis factor-related
apoptosis-inducing ligand (TRAIL) is a promising anti-tumor agent. However,
many HCC cells show resistance to TRAIL-induced apoptosis. In this study, we
showed that bortezomib, a proteasome inhibitor, overcame TRAIL resistance in
HCC cells, including Huh-7, Hep3B, and Sk-Hep1. The combination of bortezomib
and TRAIL restored the sensitivity of HCC cells to TRAIL-induced apoptosis.
Comparing the molecular change in HCC cells treated with these agents, we
found that down-regulation of phospho-Akt (P-Akt) played a key role in
mediating TRAIL sensitization of bortezomib. The first evidence was that
bortezomib down-regulated P-Akt in a dose- and time-dependent manner in
TRAIL-treated HCC cells. Second, , a PI3K inhibitor, also sensitized
resistant HCC cells to TRAIL-induced apoptosis. Third, knocking down Akt1 by
small interference RNA also enhanced TRAIL-induced apoptosis in Huh-7 cells.
Finally, ectopic expression of mutant Akt (constitutive active) in HCC cells
abolished TRAIL sensitization effect of bortezomib. Moreover, okadaic acid, a
protein phosphatase 2A (PP2A) inhibitor, reversed down-regulation of P-Akt in
bortezomib-treated cells, and PP2A knockdown by small interference RNA also
reduced apoptosis induced by the combination of TRAIL and bortezomib,
indicating that PP2A may be important in mediating the effect of bortezomib on
TRAIL sensitization. Together, bortezomib overcame TRAIL resistance at
clinically achievable concentrations in hepatocellular carcinoma cells, and
this effect is mediated at least partly via inhibition of the PI3K/Akt
pathway.Hepatocellular carcinoma
(HCC) LY2940022 is currently
the fifth most common solid tumor worldwide and the fourth leading cause of
cancer-related death. To date, surgery is still the only curative treatment
but is only feasible in a small portion of patients
(1). Drug treatment is the
major therapy for patients with advanced stage disease. Unfortunately, the
response rate to traditional chemotherapy for HCC patients is unsatisfactory
(1). Novel pharmacological
therapy is urgently needed for patients with advanced HCC. In this regard, the
approval of sorafenib might open a new era of molecularly targeted therapy in
the treatment of HCC patients.Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), a
type II transmembrane protein and a member of the TNF family, is a promising
anti-tumor agent under clinical investigation
(2). TRAIL functions by
engaging its receptors expressed on the surface of target cells. Five
receptors specific for TRAIL have been identified, including DR4/TRAIL-R1,
DR5/TRAIL-R2, DcR1, DcR2, and osteoprotegerin. Among TRAIL receptors, only DR4
and DR5 contain an effective death domain that is essential to formation of
death-inducing signaling complex (DISC), a critical step for TRAIL-induced
apoptosis. Notably, the trimerization of the death domains recruits an adaptor
molecule, Fas-associated protein with death domain (FADD), which subsequently
recruits and activates caspase-8. In type I cells, activation of caspase-8 is
sufficient to activate caspase-3 to induce apoptosis; however, in another type
of cells (type II), the intrinsic mitochondrial pathway is essential for
apoptosis characterized by cleavage of Bid and release of cytochrome
c from mitochondria, which subsequently activates caspase-9 and
caspase-3 (3).Although TRAIL induces apoptosis in malignant cells but sparing normal
cells, some tumor cells are resistant to TRAIL-induced apoptosis. Mechanisms
responsible for the resistance include receptors and intracellular resistance.
Although the cell surface expression of DR4 or DR5 is absolutely required for
TRAIL-induced apoptosis, tumor cells expressing these death receptors are not
always sensitive to TRAIL due to intracellular mechanisms. For example, the
cellular FLICE-inhibitory protein (c-FLIP), a homologue to caspase-8 but
without protease activity, has been linked to TRAIL resistance in several
studies (4,
5). In addition, inactivation
of Bax, a proapoptotic Bcl-2 family protein, resulted in resistance to TRAIL
in MMR-deficient tumors (6,
7), and reintroduction of Bax
into Bax-deficient cells restored TRAIL sensitivity
(8), indicating that the Bcl-2
family plays a critical role in intracellular mechanisms for resistance of
TRAIL.Bortezomib, a proteasome inhibitor approved clinically for multiple myeloma
and mantle cell lymphoma, has been investigated intensively for many types of
cancer (9). Accumulating
studies indicate that the combination of bortezomib and TRAIL overcomes the
resistance to TRAIL in various types of cancer, including acute myeloid
leukemia (4), lymphoma
(10–13),
prostate
(14–17),
colon (15,
18,
19), bladder
(14,
16), renal cell carcinoma
(20), thyroid
(21), ovary
(22), non-small cell lung
(23,
24), sarcoma
(25), and HCC
(26,
27). Molecular targets
responsible for the sensitizing effect of bortezomib on TRAIL-induced cell
death include DR4 (14,
27), DR5
(14,
20,
22–23,
28), c-FLIP
(4,
11,
21–23,
29), NF-κB
(12,
24,
30), p21
(16,
21,
25), and p27
(25). In addition, Bcl-2
family also plays a role in the combinational effect of bortezomib and TRAIL,
including Bcl-2 (10,
21), Bax
(13,
22), Bak
(27), Bcl-xL
(21), Bik
(18), and Bim
(15).Recently, we have reported that Akt signaling is a major molecular
determinant in bortezomib-induced apoptosis in HCC cells
(31). In this study, we
demonstrated that bortezomib overcame TRAIL resistance in HCC cells through
inhibition of the PI3K/Akt pathway. 相似文献
12.
13.
Isabel Molina-Ortiz Rub��n A. Bartolom�� Pablo Hern��ndez-Varas Georgina P. Colo Joaquin Teixid�� 《The Journal of biological chemistry》2009,284(22):15147-15157
Melanoma cells express the chemokine receptor CXCR4 that confers high
invasiveness upon binding to its ligand CXCL12. Melanoma cells at initial
stages of the disease show reduction or loss of E-cadherin expression, but
recovery of its expression is frequently found at advanced phases. We
overexpressed E-cadherin in the highly invasive BRO lung metastatic cell
melanoma cell line to investigate whether it could influence CXCL12-promoted
cell invasion. Overexpression of E-cadherin led to defective invasion of
melanoma cells across Matrigel and type I collagen in response to CXCL12. A
decrease in individual cell migration directionality toward the chemokine and
reduced adhesion accounted for the impaired invasion. A p190RhoGAP-dependent
inhibition of RhoA activation was responsible for the impairment in
chemokine-stimulated E-cadherin melanoma transfectant invasion. Furthermore,
we show that p190RhoGAP and p120ctn associated predominantly on the plasma
membrane of cells overexpressing E-cadherin, and that E-cadherin-bound p120ctn
contributed to RhoA inactivation by favoring p190RhoGAP-RhoA association.
These results suggest that melanoma cells at advanced stages of the disease
could have reduced metastatic potency in response to chemotactic stimuli
compared with cells lacking E-cadherin, and the results indicate that
p190RhoGAP is a central molecule controlling melanoma cell invasion.Cadherins are a family of Ca2+-dependent adhesion molecules that
mediate cell-cell contacts and are expressed in most solid tissues providing a
tight control of morphogenesis
(1,
2). Classical cadherins, such
as epithelial (E) cadherin, are found in adherens junctions, forming core
protein complexes with β-catenin, α-catenin, and p120 catenin
(p120ctn). Both β-catenin and p120ctn directly interact with E-cadherin,
whereas α-catenin associates with the complex through its binding to
β-catenin, providing a link with the actin cytoskeleton
(1,
2). E-cadherin is frequently
lost or down-regulated in many human tumors, coincident with morphological
epithelial to mesenchymal transition and acquisition of invasiveness
(3-6).Although melanoma only accounts for 5% of skin cancers, when metastasis
starts, it is responsible for 80% of deaths from skin cancers
(7). Melanocytes express
E-cadherin
(8-10),
but melanoma cells at early radial growth phase show a large reduction in the
expression of this cadherin, and surprisingly, expression has been reported to
be partially recovered by vertical growth phase and metastatic melanoma cells
(9,
11,
12).Trafficking of cancer cells from primary tumor sites to intravasation into
blood circulation and later to extravasation to colonize distant organs
requires tightly regulated directional cues and cell migration and invasion
that are mediated by chemokines, growth factors, and adhesion molecules
(13). Solid tumor cells
express chemokine receptors that provide guidance of these cells to organs
where their chemokine ligands are expressed, constituting a homing model
resembling the one used by immune cells to exert their immune surveillance
functions (14). Most solid
cancer cells express CXCR4, a receptor for the chemokine CXCL12 (also called
SDF-1), which is expressed in lungs, bone marrow, and liver
(15). Expression of CXCR4 in
human melanoma has been detected in the vertical growth phase and on regional
lymph nodes, which correlated with poor prognosis and increased mortality
(16,
17). Previous in vivo
experiments have provided evidence supporting a crucial role for CXCR4 in the
metastasis of melanoma cells
(18).Rho GTPases control the dynamics of the actin cytoskeleton during cell
migration (19,
20). The activity of Rho
GTPases is tightly regulated by guanine-nucleotide exchange factors
(GEFs),4 which
stimulate exchange of bound GDP by GTP, and inhibited by GTPase-activating
proteins (GAPs), which promote GTP hydrolysis
(21,
22), whereas guanine
nucleotide dissociation inhibitors (GDIs) appear to mediate blocking of
spontaneous activation (23).
Therefore, cell migration is finely regulated by the balance between GEF, GAP,
and GDI activities on Rho GTPases. Involvement of Rho GTPases in cancer is
well documented (reviewed in Ref.
24), providing control of both
cell migration and growth. RhoA and RhoC are highly expressed in colon,
breast, and lung carcinoma
(25,
26), whereas overexpression of
RhoC in melanoma leads to enhancement of cell metastasis
(27). CXCL12 activates both
RhoA and Rac1 in melanoma cells, and both GTPases play key roles during
invasion toward this chemokine
(28,
29).Given the importance of the CXCL12-CXCR4 axis in melanoma cell invasion and
metastasis, in this study we have addressed the question of whether changes in
E-cadherin expression on melanoma cells might affect cell invasiveness. We
show here that overexpression of E-cadherin leads to impaired melanoma cell
invasion to CXCL12, and we provide mechanistic characterization accounting for
the decrease in invasion. 相似文献
14.
Richard L. Daniels Yoshio Takashima David D. McKemy 《The Journal of biological chemistry》2009,284(3):1570-1582
Cold temperatures robustly activate a small cohort of somatosensory nerves,
yet during a prolonged cold stimulus their activity will decrease, or adapt,
over time. This process allows for the discrimination of subtle changes in
temperature. At the molecular level, cold is detected by transient receptor
potential melastatin 8 (TRPM8), a nonselective cation channel expressed on a
subset of peripheral afferent fibers. We and others have reported that TRPM8
channels also adapt in a calcium-dependent manner when activated by the
cooling compound menthol. Additionally, TRPM8 activity is sensitive to the
phospholipid phosphoinositol 4,5-bisphosphate (PIP2), a substrate
for the enzyme phospholipase C (PLC). These results suggest an adaptation
model whereby TRPM8-mediated Ca2+ influx activates PLC, thereby
decreasing PIP2 levels and resulting in reduced TRPM8 activity.
Here we tested this model using pharmacological activation of PLC and by
manipulating PIP2 levels independent of both PLC and
Ca2+. PLC activation leads to adaptation-like reductions in cold-
or menthol-evoked TRPM8 currents in both heterologous and native cells.
Moreover, PLC-independent reductions in PIP2 had a similar effect
on cold- and menthol-evoked currents. Mechanistically, either form of
adaptation does not alter temperature sensitivity of TRPM8 but does lead to a
change in channel gating. Our results show that adaptation is a shift in
voltage dependence toward more positive potentials, reversing the trend toward
negative potentials caused by agonist. These data suggest that PLC activity
not only mediates adaptation to thermal stimuli, but likely underlies a more
general mechanism that establishes the temperature sensitivity of
somatosensory neurons.The detection of temperature is a fundamental task of the nervous system.
Temperature-sensing sensory afferent neurons reside in either the trigeminal
(TG)2 or dorsal root
(DRG) sensory ganglia and project peripherally, terminating as free nerve
endings that innervate areas of the head or trunk, respectively
(1,
2). Subpopulations of these
afferents respond to distinct sub-modalities of thermal stimuli, including
noxious heat, innocuous cooling and warmth, and painfully cold temperatures.
Each carries thermal information to the dorsal horn of the spinal cord,
synapsing with neurons that project centrally
(1,
3).The discovery of thermosensitive ion channels of the transient receptor
potential (TRP) family demonstrated an underlying molecular mechanism for
temperature detection (4). Cold
temperature sensation is largely mediated by TRPM8, a nonselective cation
channel expressed on a small subset of neurons
(5,
6). TRPM8 is activated by
cooling compounds, such as menthol, as well as cold temperatures below ∼28
°C, in vitro (7,
8). Recent reports on the
behavioral phenotype of TRPM8-null mice suggest that this lone channel is
required for the majority of cold sensing in vivo
(5,
9–11).
These and other data strongly implicate TRPM8 in not only the detection of
both innocuous cool and some aspects of noxious cold but also injury-induced
hypersensitivity to cold and, paradoxically, cooling-mediated analgesia
(11,
12). Thus, understanding
regulatory mechanisms that alter TRPM8 activity will provide keen insights
into temperature sensation, nociception, and analgesia.One fundamental property of cold-sensitive neurons is an intrinsic ability
to adapt to prolonged cold stimuli, a mechanism that is likely critical for
discrimination of changing environmental conditions
(13,
14). We and others have shown
that cold-sensitive neurons adapt to cold and menthol over time in
vitro (6,
15), a phenomenon also
observed with recombinant TRPM8 channels activated by menthol
(7). During sustained exposure
to menthol, TRPM8 currents adapt in a manner that is dependent upon the
presence of external calcium
(7). Interestingly, cold- and
menthol-evoked currents are highly sensitive to cellular manipulation. In
heterologous cells, TRPM8 currents quickly decrease or run down upon membrane
patch excision (16,
17). Moreover, in membrane
patches excised from cold- and menthol-sensitive DRG neurons, cold thresholds
for current activation exhibit a shift of ∼10 °C to colder
temperatures in comparison with thresholds recorded in intact cells
(18).Phosphatidylinositol 4,5-bisphosphate (PIP2) is a membrane
phospholipid that accounts for ∼1% of all lipids in the inner leaflet of
the plasma membrane and is known to regulate a variety of ion channels,
including TRPM8 (16,
17). When applied to the
cytoplasmic face of excised membrane patches containing TRPM8 channels,
PIP2 can recover menthol-evoked currents to near pre-rundown levels
(16,
17). PIP2 is
proposed to interact with channels either through electrostatic interactions
or by binding to target proteins at specific phosphoinositide-binding sites
(19,
20). Membrane PIP2
levels are a product of enzymatic activity, such as phosphoinositide kinases
that synthesize PIP2 from membrane precursors and phospholipase C
(PLC) that hydrolyzes it, creating membrane-bound diacylglycerol (DAG) and
cytosolic inositol trisphosphate (IP3), both of which function as
second messengers. Of the three different PLC isotypes, PLCδ isoforms
are modulated by increases in intracellular calcium
(21).When taken in context with the sensitivity of TRPM8 currents to
PIP2 levels, a model has been proposed whereby adaption is a result
of channel-mediated Ca2+ influx activating one or more PLCδ
isoforms (16,
17). The subsequent reductions
in PIP2 levels then promote reduced or adapted TRPM8 currents.
However, this hypothesis has not been conclusively shown in intact
heterologous cells or in somatosensory neurons expressing TRPM8. Moreover,
other alternative hypotheses for TRPM8 adaptation have been proposed,
including Ca2+-dependent kinase activity mediated by protein kinase
C (22,
23). Thus, the cellular and
molecular mechanisms for Ca2+-mediated TRPM8 adaptation are
unclear.Here we show, in both heterologous cells and native TRPM8-expressing
neurons, that Ca2+-independent activation of PLC results in adapted
TRPM8 currents. Moreover, PLC- and Ca2+-independent PIP2
depletion in heterologous cells produces similar effects on TRPM8 activity,
again reducing both cold- and menthol-evoked currents. Mechanistically, we
find that all such manipulations do not alter the temperature sensitivity of
the channel but do lead to a shift in the voltage dependence of TRPM8 channel
gating. 相似文献
15.
Qinli Wang Bo Chen Peng Liu Maozhong Zheng Yuqing Wang Sujuan Cui Daye Sun Xiaohong Fang Chun-Ming Liu William J. Lucas Jinxing Lin 《The Journal of biological chemistry》2009,284(18):12000-12007
Calmodulin (CaM) is a highly conserved intracellular calcium sensor. In
plants, CaM also appears to be present in the apoplasm, and application of
exogenous CaM has been shown to influence a number of physiological functions
as a polypeptide signal; however, the existence and localization of its
corresponding apoplasmic binding sites remain controversial. To identify the
site(s) of action, a CaM-conjugated quantum dot (QD) system was employed for
single molecule level detection at the surface of plant cells. Using this
approach, we show that QD-CaM binds selectively to sites on the outer surface
of the plasma membrane, which was further confirmed by high resolution
transmission electron microscopy. Measurements of Ca2+ fluxes
across the plasma membrane, using ion-selective microelectrodes, demonstrated
that exogenous CaM induces a net influx into protoplasts. Consistent with
these flux studies, calcium-green-dextran and FRET experiments confirmed that
applied CaM/QD-CaM elicited an increase in cytoplasmic Ca2+ levels.
These results support the hypothesis that apoplasmic CaM can act as a
signaling agent. These findings are discussed in terms of CaM acting as an
apoplasmic peptide ligand to mediate transmembrane signaling in the plant
kingdom.Calmodulin (CaM)2
is a conserved multifunctional calcium sensor that mediates intracellular
Ca2+ signaling and regulates diverse cellular processes by
interacting with calmodulin-binding proteins
(1–3).
Interestingly, in both animals and plants, CaM may also act as an
extracellular agent to regulate physiological events
(4). Consistent with this
notion, extracellular CaM has been detected within the cell walls of a broad
range of plant species (4,
5).Functional studies have established that exogenously applied CaM can
stimulate the proliferation of suspension-cultured plant cells
(6) as well as affect
intracellular activities of heterotrimeric G proteins and phospholipases in
protoplasts (7,
8). Based on these findings, it
has been proposed that, in plants, extracellular CaM may function as a
signaling agent involved in the regulation of cell growth and development
(4). However, as a 17-kDa
hydrophilic protein, exogenously applied CaM could well be retrieved from the
apoplasmic space and then exert its effects on components within the
cytoplasm. Evidence against this hypothesis was provided by studies with
Arabidopsis thaliana suspension-cultured cells in which it was shown
that 24 h of incubation in exogenous CaM did not result in protein uptake or
degradation (4).To exert an effect from the apoplasm, it would seem logical to assume that
a protein(s) within the plant plasma membrane would have a CaM-binding site
exposed to the apoplasm. Although a number of studies have addressed the
molecular mechanism(s) by which extracellular CaM might act as a signal
(6,
9) and attempts have been made
to identify extracellular CaM-binding proteins
(4,
6), currently there is no
direct evidence in support of the hypothesis that specific CaM-binding sites
exist at the surface of plant cells.To address this question, a CaM-conjugated quantum dot (QD) system was
employed for single molecule level detection
(10–13)
at the surface of plant cells. These nanoparticles have several advantages
over conventional fluorophores for light microscopic imaging, including their
higher brightness and photostability
(14,
15). In addition, because of
their electron dense nature, QDs can be used for single labeling studies at
the transmission electron microscope level
(16,
17). Using this QD-CaM system,
we demonstrate that QD-CaM binds selectively to sites on the outer surface of
the plant plasma membrane. We also show by three independent methods that
applied CaM can modulate Ca2+ fluxes across the plasma membrane,
leading to alterations in cytoplasmic Ca2+ status. These findings
support the hypothesis that, in plants, apoplasmic CaM can act as a signaling
agent. 相似文献
16.
Ruben K. Dagda Salvatore J. Cherra III Scott M. Kulich Anurag Tandon David Park Charleen T. Chu 《The Journal of biological chemistry》2009,284(20):13843-13855
Mitochondrial dysregulation is strongly implicated in Parkinson disease.
Mutations in PTEN-induced kinase 1 (PINK1) are associated with familial
parkinsonism and neuropsychiatric disorders. Although overexpressed PINK1 is
neuroprotective, less is known about neuronal responses to loss of PINK1
function. We found that stable knockdown of PINK1 induced mitochondrial
fragmentation and autophagy in SH-SY5Y cells, which was reversed by the
reintroduction of an RNA interference (RNAi)-resistant plasmid for PINK1.
Moreover, stable or transient overexpression of wild-type PINK1 increased
mitochondrial interconnectivity and suppressed toxin-induced
autophagy/mitophagy. Mitochondrial oxidant production played an essential role
in triggering mitochondrial fragmentation and autophagy in PINK1 shRNA lines.
Autophagy/mitophagy served a protective role in limiting cell death, and
overexpressing Parkin further enhanced this protective mitophagic response.
The dominant negative Drp1 mutant inhibited both fission and mitophagy in
PINK1-deficient cells. Interestingly, RNAi knockdown of autophagy proteins
Atg7 and LC3/Atg8 also decreased mitochondrial fragmentation without affecting
oxidative stress, suggesting active involvement of autophagy in morphologic
remodeling of mitochondria for clearance. To summarize, loss of PINK1 function
elicits oxidative stress and mitochondrial turnover coordinated by the
autophagic and fission/fusion machineries. Furthermore, PINK1 and Parkin may
cooperate through different mechanisms to maintain mitochondrial
homeostasis.Parkinson disease is an age-related neurodegenerative disease that affects
∼1% of the population worldwide. The causes of sporadic cases are unknown,
although mitochondrial or oxidative toxins such as
1-methyl-4-phenylpyridinium, 6-hydroxydopamine
(6-OHDA),3 and
rotenone reproduce features of the disease in animal and cell culture models
(1). Abnormalities in
mitochondrial respiration and increased oxidative stress are observed in cells
and tissues from parkinsonian patients
(2,
3), which also exhibit
increased mitochondrial autophagy
(4). Furthermore, mutations in
parkinsonian genes affect oxidative stress response pathways and mitochondrial
homeostasis (5). Thus,
disruption of mitochondrial homeostasis represents a major factor implicated
in the pathogenesis of sporadic and inherited parkinsonian disorders (PD).The PARK6 locus involved in autosomal recessive and early-onset PD
encodes for PTEN-induced kinase 1 (PINK1)
(6,
7). PINK1 is a cytosolic and
mitochondrially localized 581-amino acid serine/threonine kinase that
possesses an N-terminal mitochondrial targeting sequence
(6,
8). The primary sequence also
includes a putative transmembrane domain important for orientation of the
PINK1 domain (8), a conserved
kinase domain homologous to calcium calmodulin kinases, and a C-terminal
domain that regulates autophosphorylation activity
(9,
10). Overexpression of
wild-type PINK1, but not its PD-associated mutants, protects against several
toxic insults in neuronal cells
(6,
11,
12). Mitochondrial targeting
is necessary for some (13) but
not all of the neuroprotective effects of PINK1
(14), implicating involvement
of cytoplasmic targets that modulate mitochondrial pathobiology
(8). PINK1 catalytic activity
is necessary for its neuroprotective role, because a kinase-deficient K219M
substitution in the ATP binding pocket of PINK1 abrogates its ability to
protect neurons (14). Although
PINK1 mutations do not seem to impair mitochondrial targeting, PD-associated
mutations differentially destabilize the protein, resulting in loss of
neuroprotective activities
(13,
15).Recent studies indicate that PINK1 and Parkin interact genetically
(3,
16-18)
to prevent oxidative stress
(19,
20) and regulate mitochondrial
morphology (21). Primary cells
derived from PINK1 mutant patients exhibit mitochondrial fragmentation with
disorganized cristae, recapitulated by RNA interference studies in HeLa cells
(3).Mitochondria are degraded by macroautophagy, a process involving
sequestration of cytoplasmic cargo into membranous autophagic vacuoles (AVs)
for delivery to lysosomes (22,
23). Interestingly,
mitochondrial fission accompanies autophagic neurodegeneration elicited by the
PD neurotoxin 6-OHDA (24,
25). Moreover, mitochondrial
fragmentation and increased autophagy are observed in neurodegenerative
diseases including Alzheimer and Parkinson diseases
(4,
26-28).
Although inclusion of mitochondria in autophagosomes was once believed to be a
random process, as observed during starvation, studies involving hypoxia,
mitochondrial damage, apoptotic stimuli, or limiting amounts of aerobic
substrates in facultative anaerobes support the concept of selective
mitochondrial autophagy (mitophagy)
(29,
30). In particular,
mitochondrially localized kinases may play an important role in models
involving oxidative mitochondrial injury
(25,
31,
32).Autophagy is involved in the clearance of protein aggregates
(33-35)
and normal regulation of axonal-synaptic morphology
(36). Chronic disruption of
lysosomal function results in accumulation of subtly impaired mitochondria
with decreased calcium buffering capacity
(37), implicating an important
role for autophagy in mitochondrial homeostasis
(37,
38). Recently, Parkin, which
complements the effects of PINK1 deficiency on mitochondrial morphology
(3), was found to promote
autophagy of depolarized mitochondria
(39). Conversely, Beclin
1-independent autophagy/mitophagy contributes to cell death elicited by the PD
toxins 1-methyl-4-phenylpyridinium and 6-OHDA
(25,
28,
31,
32), causing neurite
retraction in cells expressing a PD-linked mutation in leucine-rich repeat
kinase 2 (40). Whereas
properly regulated autophagy plays a homeostatic and neuroprotective role,
excessive or incomplete autophagy creates a condition of “autophagic
stress” that can contribute to neurodegeneration
(28).As mitochondrial fragmentation
(3) and increased mitochondrial
autophagy (4) have been
described in human cells or tissues of PD patients, we investigated whether or
not the engineered loss of PINK1 function could recapitulate these
observations in human neuronal cells (SH-SY5Y). Stable knockdown of endogenous
PINK1 gave rise to mitochondrial fragmentation and increased autophagy and
mitophagy, whereas stable or transient overexpression of PINK1 had the
opposite effect. Autophagy/mitophagy was dependent upon increased
mitochondrial oxidant production and activation of fission. The data indicate
that PINK1 is important for the maintenance of mitochondrial networks,
suggesting that coordinated regulation of mitochondrial dynamics and autophagy
limits cell death associated with loss of PINK1 function. 相似文献
17.
18.
Joey Lai Oliver K. Bernhard Stuart G. Turville Andrew N. Harman John Wilkinson Anthony L. Cunningham 《The Journal of biological chemistry》2009,284(17):11027-11038
C-type lectin receptors expressed on the surface of dendritic cells and
macrophages are able to bind glycoproteins of microbial pathogens via mannose,
fucose, and N-acetylglucosamine. Langerin on Langerhans cells,
dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin
on dendritic cells, and mannose receptor (MR) on dendritic cells and
macrophages bind the human immunodeficiency virus (HIV) envelope protein gp120
principally via high mannose oligosaccharides. These C-type lectin receptors
can also oligomerize to facilitate enhanced ligand binding. This study
examined the effect of oligomerization of MR on its ability to bind to mannan,
monomeric gp120, native trimeric gp140, and HIV type 1 BaL. Mass spectrometry
analysis of cross-linked MR showed homodimerization on the surface of primary
monocyte-derived dendritic cells and macrophages. Both monomeric and dimeric
MR were precipitated by mannan, but only the dimeric form was
co-immunoprecipitated by gp120. These results were confirmed independently by
flow cytometry analysis of soluble monomeric and trimeric HIV envelope and a
cellular HIV virion capture assay. As expected, mannan bound to the
carbohydrate recognition domains of MR dimers mostly in a calcium-dependent
fashion. Unexpectedly, gp120-mediated binding of HIV to dimers on
MR-transfected Rat-6 cells and macrophages was not calcium-dependent, was only
partially blocked by mannan, and was also partially inhibited by
N-acetylgalactosamine 4-sulfate. Thus gp120-mediated HIV binding
occurs via the calcium-dependent, non-calcium-dependent carbohydrate
recognition domains and the cysteine-rich domain at the C terminus of MR
dimers, presenting a much broader target for potential inhibitors of gp120-MR
binding.The mannose receptor
(MR)2 is a C-type
lectin receptor that is expressed on the surface of a variety of cells,
including immature monocyte-derived dendritic cells (MDDC), dermal dendritic
cells, macrophages, and hepatic endothelial cells. It is a multifunctional
protein, involved in antigen recognition and internalization during the early
stages of the innate immune response
(1) as well as physiological
clearance of the endogenous pituitary hormones lutropin and thyrotropin
(2,
3). Recognition of foreign
antigens occurs via mannose, fucose, and GlcNAc residues
(4,
5), which are generally not
found as terminal residues on mammalian glycoproteins but are highly abundant
on surface proteins of pathogens such as the HIV-1 envelope gp120
(6,
7). Once bound, pathogens can
be internalized by endocytosis or phagocytosis, where they are targeted to
lysosomes for proteolytic degradation and presentation on major
histocompatibility complex class II
(8). In immature DCs, soluble
recombinant HIV envelope proteins are processed by this pathway, initially
binding to both dendritic cell-specific intracellular adhesion molecule 3
grabbing non-integrin (DC-SIGN) and MR and ultimately co-localizing with MR
but not DC-SIGN in lysosomes
(9). Furthermore, in immature
DCs and to a greater extent mature DCs, a proportion of intact HIV-1 enters a
unique vesicular compartment that co-localizes with tetraspanin proteins such
as CD81 (10,
11). Recently, this
compartment has been shown to be continuous with the plasma membrane
(11) and does not represent a
continuation of the endolysosomal network. Interestingly, this compartment can
translocate virus from DCs to CD4 T cells, upon the formation of a virological
synapse
(10–12).
Although viral uptake can occur in DCs independent of HIV env
(2), the efficiency of HIV
binding and uptake is greatly enhanced by the presence of C-type lectin-env
interactions. At least initial binding to DC-SIGN (and most likely also MR) is
required for T cell trans-infection
(13).Structurally, the extracellular domain of MR consists of an N-terminal
cysteine-rich domain (Cys-RD), followed by a fibronectin type II domain and
eight carbohydrate recognition domains (CRD) on a single polypeptide backbone
(1). Of the eight CRDs, CRD
4–8 have been shown to be required for high affinity binding of ligands
containing terminal mannose/fucose/GlcNAc residues, with CRD 4 having
demonstrable monosaccharide binding in isolation
(14). Binding and release of
ligand within the low pH environment of the endolysosomal compartment are also
Ca2+-dependent. Acid-induced removal of Ca2+ binding in
CRD 4 and 5 was shown to cause a conformational rearrangement of the domain,
resulting in a loss of carbohydrate binding activity
(15). In contrast, binding of
sulfated carbohydrates to the Cys-RD appears to be Ca2+-independent
as no Ca2+-binding sites were observed in its crystal structure
(2,
16).Oligomerization of CLRs such as DC-SIGN
(17), Langerin
(18), and mannose-binding
protein (19) has been reported
to be essential for binding of oligosaccharide-bearing ligands. Early studies
on MR suggested that it exists solely as a monomeric molecule and that
clustering of multiple CRDs within the single polypeptide backbone was
necessary for high affinity binding of oligosaccharide moieties
(20). However, more recent
studies have shown that dimerization is possible in the presence of
Ca2+ (21) and that
an equilibrium may exist between monomeric and dimeric forms on the cell
surface (22). It is currently
unclear what effect dimerization has on ligand binding to the CRDs; however,
there is evidence that dimerization of MR is required for high affinity
binding of ligands bearing terminal N-acetylgalactosamine 4-sulfate
(GalNAc-4-SO4) such as lutropin and thyrotropin
(22) to the Cys-RD.To date, studies on the oligomerization and ligand binding activity of MR
have used solubilized protein from cell lysates
(20) or purified recombinant
fragments (21). Because the
membrane microenvironment can influence protein associations, soluble forms of
MR may not necessarily be a true model of the quaternary structure and
function of the native protein. Here, we used a well established method of
cross-linking (23) on MDDCs,
monocyte-derived macrophages (MDMs), and MR-transfected Rat-6 cells to
preserve lateral protein-protein interactions between MR on the cell surface
prior to solubilization. Mass spectrometry analysis of affinity-purified
complexes showed they were homo-oligomers, and further resolution of the
complex on a low percentage polyacrylamide gel by SDS-PAGE strongly indicates
that they are dimers. Dimerization of MR was also found to be essential for
binding mannan, monomeric gp120, native trimeric gp140, and HIV-1 viral
particles. Persistence of monomeric gp120 and trimeric gp140 binding to
dimeric MR in the presence of EGTA and various CRD and other inhibitors,
however, suggested that gp120-mediated HIV-1 binding is not
Ca2+-dependent and that at least binding probably occurs to both
Ca2+-dependent and -independent CRDs and also the Cys-RD. 相似文献
19.
Graham H. Diering John Church Masayuki Numata 《The Journal of biological chemistry》2009,284(20):13892-13903
NHE5 is a brain-enriched Na+/H+ exchanger that
dynamically shuttles between the plasma membrane and recycling endosomes,
serving as a mechanism that acutely controls the local pH environment. In the
current study we show that secretory carrier membrane proteins (SCAMPs), a
group of tetraspanning integral membrane proteins that reside in multiple
secretory and endocytic organelles, bind to NHE5 and co-localize predominantly
in the recycling endosomes. In vitro protein-protein interaction
assays revealed that NHE5 directly binds to the N- and C-terminal cytosolic
extensions of SCAMP2. Heterologous expression of SCAMP2 but not SCAMP5
increased cell-surface abundance as well as transporter activity of NHE5
across the plasma membrane. Expression of a deletion mutant lacking the
SCAMP2-specific N-terminal cytosolic domain, and a mini-gene encoding the
N-terminal extension, reduced the transporter activity. Although both Arf6 and
Rab11 positively regulate NHE5 cell-surface targeting and NHE5 activity across
the plasma membrane, SCAMP2-mediated surface targeting of NHE5 was reversed by
dominant-negative Arf6 but not by dominant-negative Rab11. Together, these
results suggest that SCAMP2 regulates NHE5 transit through recycling endosomes
and promotes its surface targeting in an Arf6-dependent manner.Neurons and glial cells in the central and peripheral nervous systems are
especially sensitive to perturbations of pH
(1). Many voltage- and
ligand-gated ion channels that control membrane excitability are sensitive to
changes in cellular pH
(1-3).
Neurotransmitter release and uptake are also influenced by cellular and
organellar pH (4,
5). Moreover, the intra- and
extracellular pH of both neurons and glia are modulated in a highly transient
and localized manner by neuronal activity
(6,
7). Thus, neurons and glia
require sophisticated mechanisms to finely tune ion and pH homeostasis to
maintain their normal functions.Na+/H+ exchangers
(NHEs)3 were
originally identified as a class of plasma membrane-bound ion transporters
that exchange extracellular Na+ for intracellular H+,
and thereby regulate cellular pH and volume. Since the discovery of NHE1 as
the first mammalian NHE (8),
eight additional isoforms (NHE2-9) that share 25-70% amino acid identity have
been isolated in mammals (9,
10). NHE1-5 commonly exhibit
transporter activity across the plasma membrane, whereas NHE6-9 are mostly
found in organelle membranes and are believed to regulate organellar pH in
most cell types at steady state
(11). More recently, NHE10 was
identified in human and mouse osteoclasts
(12,
13). However, the cDNA
encoding NHE10 shares only a low degree of sequence similarity with other
known members of the NHE gene family, raising the possibility that
this sodium-proton exchanger may belong to a separate gene family distantly
related to NHE1-9 (see Ref.
9).NHE gene family members contain 12 putative transmembrane domains
at the N terminus followed by a C-terminal cytosolic extension that plays a
role in regulation of the transporter activity by protein-protein interactions
and phosphorylation. NHEs have been shown to regulate the pH environment of
synaptic nerve terminals and to regulate the release of neurotransmitters from
multiple neuronal populations
(14-16).
The importance of NHEs in brain function is further exemplified by the
findings that spontaneous or directed mutations of the ubiquitously expressed
NHE1 gene lead to the progression of epileptic seizures, ataxia, and
increased mortality in mice
(17,
18). The progression of the
disease phenotype is associated with loss of specific neuron populations and
increased neuronal excitability. However, NHE1-null mice appear to
develop normally until 2 weeks after birth when symptoms begin to appear.
Therefore, other mechanisms may compensate for the loss of NHE1
during early development and play a protective role in the surviving neurons
after the onset of the disease phenotype.NHE5 was identified as a unique member of the NHE gene
family whose mRNA is expressed almost exclusively in the brain
(19,
20), although more recent
studies have suggested that NHE5 might be functional in other cell
types such as sperm (21,
22) and osteosarcoma cells
(23). Curiously, mutations
found in several forms of congenital neurological disorders such as
spinocerebellar ataxia type 4
(24-26)
and autosomal dominant cerebellar ataxia
(27-29)
have been mapped to chromosome 16q22.1, a region containing NHE5.
However, much remains unknown as to the molecular regulation of NHE5 and its
role in brain function.Very few if any proteins work in isolation. Therefore identification and
characterization of binding proteins often reveal novel functions and
regulation mechanisms of the protein of interest. To begin to elucidate the
biological role of NHE5, we have started to explore NHE5-binding proteins.
Previously, β-arrestins, multifunctional scaffold proteins that play a
key role in desensitization of G-protein-coupled receptors, were shown to
directly bind to NHE5 and promote its endocytosis
(30). This study demonstrated
that NHE5 trafficking between endosomes and the plasma membrane is regulated
by protein-protein interactions with scaffold proteins. More recently, we
demonstrated that receptor for activated
C-kinase 1 (RACK1), a scaffold protein that links
signaling molecules such as activated protein kinase C, integrins, and Src
kinase (31), directly
interacts with and activates NHE5 via integrin-dependent and independent
pathways (32). These results
further indicate that NHE5 is partly associated with focal adhesions and that
its targeting to the specialized microdomain of the plasma membrane may be
regulated by various signaling pathways.Secretory carrier membrane proteins (SCAMPs) are a family of evolutionarily
conserved tetra-spanning integral membrane proteins. SCAMPs are found in
multiple organelles such as the Golgi apparatus, trans-Golgi network,
recycling endosomes, synaptic vesicles, and the plasma membrane
(33,
34) and have been shown to
play a role in exocytosis
(35-38)
and endocytosis (39).
Currently, five isoforms of SCAMP have been identified in mammals. The
extended N terminus of SCAMP1-3 contain multiple Asn-Pro-Phe (NPF) repeats,
which may allow these isoforms to participate in clathrin coat assembly and
vesicle budding by binding to Eps15 homology (EH)-domain proteins
(40,
41). Further, SCAMP2 was shown
recently to bind to the small GTPase Arf6
(38), which is believed to
participate in traffic between the recycling endosomes and the cell surface
(42,
43). More recent studies have
suggested that SCAMPs bind to organellar membrane type NHE7
(44) and the serotonin
transporter SERT (45) and
facilitate targeting of these integral membrane proteins to specific
intracellular compartments. We show in the current study that SCAMP2 binds to
NHE5, facilitates the cell-surface targeting of NHE5, and elevates
Na+/H+ exchange activity at the plasma membrane, whereas
expression of a SCAMP2 deletion mutant lacking the N-terminal domain
containing the NPF repeats suppresses the effect. Further we show that this
activity of SCAMP2 requires an active form of a small GTPase Arf6, but not
Rab11. We propose a model in which SCAMPs bind to NHE5 in the endosomal
compartment and control its cell-surface abundance via an Arf6-dependent
pathway. 相似文献
20.
S��bastien Thomas Brigitte Ritter David Verbich Claire Sanson Lyne Bourbonni��re R. Anne McKinney Peter S. McPherson 《The Journal of biological chemistry》2009,284(18):12410-12419
Intersectin-short (intersectin-s) is a multimodule scaffolding protein
functioning in constitutive and regulated forms of endocytosis in non-neuronal
cells and in synaptic vesicle (SV) recycling at the neuromuscular junction of
Drosophila and Caenorhabditis elegans. In vertebrates,
alternative splicing generates a second isoform, intersectin-long
(intersectin-l), that contains additional modular domains providing a guanine
nucleotide exchange factor activity for Cdc42. In mammals, intersectin-s is
expressed in multiple tissues and cells, including glia, but excluded from
neurons, whereas intersectin-l is a neuron-specific isoform. Thus,
intersectin-I may regulate multiple forms of endocytosis in mammalian neurons,
including SV endocytosis. We now report, however, that intersectin-l is
localized to somatodendritic regions of cultured hippocampal neurons, with
some juxtanuclear accumulation, but is excluded from synaptophysin-labeled
axon terminals. Consistently, intersectin-l knockdown (KD) does not affect SV
recycling. Instead intersectin-l co-localizes with clathrin heavy chain and
adaptor protein 2 in the somatodendritic region of neurons, and its KD reduces
the rate of transferrin endocytosis. The protein also co-localizes with
F-actin at dendritic spines, and intersectin-l KD disrupts spine maturation
during development. Our data indicate that intersectin-l is indeed an
important regulator of constitutive endocytosis and neuronal development but
that it is not a prominent player in the regulated endocytosis of SVs.Clathrin-mediated endocytosis
(CME)4 is a
major mechanism by which cells take up nutrients, control the surface levels
of multiple proteins, including ion channels and transporters, and regulate
the coupling of signaling receptors to downstream signaling cascades
(1-5).
In neurons, CME takes on additional specialized roles; it is an important
process regulating synaptic vesicle (SV) availability through endocytosis and
recycling of SV membranes (6,
7), it shapes synaptic
plasticity
(8-10),
and it is crucial in maintaining synaptic membranes and membrane structure
(11).Numerous endocytic accessory proteins participate in CME, interacting with
each other and with core components of the endocytic machinery such as
clathrin heavy chain (CHC) and adaptor protein-2 (AP-2) through specific
modules and peptide motifs
(12). One such module is the
Eps15 homology domain that binds to proteins bearing NPF motifs
(13,
14). Another is the Src
homology 3 (SH3) domain, which binds to proline-rich domains in protein
partners (15). Intersectin is
a multimodule scaffolding protein that interacts with a wide range of
proteins, including several involved in CME
(16). Intersectin has two
N-terminal Eps15 homology domains that are responsible for binding to epsin,
SCAMP1, and numb
(17-19),
a central coil-coiled domain that interacts with Eps15 and SNAP-23 and -25
(17,
20,
21), and five SH3 domains in
its C-terminal region that interact with multiple proline-rich domain
proteins, including synaptojanin, dynamin, N-WASP, CdGAP, and mSOS
(16,
22-25).
The rich binding capability of intersectin has linked it to various functions
from CME (17,
26,
27) and signaling
(22,
28,
29) to mitogenesis
(30,
31) and regulation of the
actin cytoskeleton (23).Intersectin functions in SV recycling at the neuromuscular junction of
Drosophila and C. elegans where it acts as a scaffold,
regulating the synaptic levels of endocytic accessory proteins
(21,
32-34).
In vertebrates, the intersectin gene is subject to alternative splicing, and a
longer isoform (intersectin-l) is generated that is expressed exclusively in
neurons (26,
28,
35,
36). This isoform has all the
binding modules of its short (intersectin-s) counterpart but also has
additional domains: a DH and a PH domain that provide guanine nucleotide
exchange factor (GEF) activity specific for Cdc42
(23,
37) and a C2 domain at the C
terminus. Through its GEF activity and binding to actin regulatory proteins,
including N-WASP, intersectin-l has been implicated in actin regulation and
the development of dendritic spines
(19,
23,
24). In addition, because the
rest of the binding modules are shared between intersectin-s and -l, it is
generally thought that the two intersectin isoforms have the same endocytic
functions. In particular, given the well defined role for the invertebrate
orthologs of intersectin-s in SV endocytosis, it is thought that intersectin-l
performs this role in mammalian neurons, which lack intersectin-s. Defining
the complement of intersectin functional activities in mammalian neurons is
particularly relevant given that the protein is involved in the
pathophysiology of Down syndrome (DS). Specifically, the intersectin gene is
localized on chromosome 21q22.2 and is overexpressed in DS brains
(38). Interestingly,
alterations in endosomal pathways are a hallmark of DS neurons and neurons
from the partial trisomy 16 mouse, Ts65Dn, a model for DS
(39,
40). Thus, an endocytic
trafficking defect may contribute to the DS disease process.Here, the functional roles of intersectin-l were studied in cultured
hippocampal neurons. We find that intersectin-l is localized to the
somatodendritic regions of neurons, where it co-localizes with CHC and AP-2
and regulates the uptake of transferrin. Intersectin-l also co-localizes with
actin at dendritic spines and disrupting intersectin-l function alters
dendritic spine development. In contrast, intersectin-l is absent from
presynaptic terminals and has little or no role in SV recycling. 相似文献