共查询到20条相似文献,搜索用时 582 毫秒
1.
2.
3.
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
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.
Christian Rosker Gargi Meur Emily J. A. Taylor Colin W. Taylor 《The Journal of biological chemistry》2009,284(8):5186-5194
Ryanodine receptors (RyR) are Ca2+ channels that mediate
Ca2+ release from intracellular stores in response to diverse
intracellular signals. In RINm5F insulinoma cells, caffeine, and
4-chloro-m-cresol (4CmC), agonists of RyR, stimulated Ca2+
entry that was independent of store-operated Ca2+ entry, and
blocked by prior incubation with a concentration of ryanodine that inactivates
RyR. Patch-clamp recording identified small numbers of large-conductance
(γK = 169 pS) cation channels that were activated by
caffeine, 4CmC or low concentrations of ryanodine. Similar channels were
detected in rat pancreatic β-cells. In RINm5F cells, the channels were
blocked by cytosolic, but not extracellular, ruthenium red. Subcellular
fractionation showed that type 3 IP3 receptors (IP3R3)
were expressed predominantly in endoplasmic reticulum, whereas RyR2 were
present also in plasma membrane fractions. Using RNAi selectively to reduce
expression of RyR1, RyR2, or IP3R3, we showed that RyR2 mediates
both the Ca2+ entry and the plasma membrane currents evoked by
agonists of RyR. We conclude that small numbers of RyR2 are selectively
expressed in the plasma membrane of RINm5F pancreatic β-cells, where they
mediate Ca2+ entry.Ryanodine receptors
(RyR)3 and inositol
1,4,5-trisphosphate receptors (IP3R)
(1,
2) are the archetypal
intracellular Ca2+ channels. Both are widely expressed, although
RyR are more restricted in their expression than IP3R
(3,
4). In common with many cells,
pancreatic β-cells and insulin-secreting cell lines express both
IP3R (predominantly IP3R3)
(5,
6) and RyR (predominantly RyR2)
(7). Both RyR and
IP3R are expressed mostly within membranes of the endoplasmic (ER),
where they mediate release of Ca2+. Functional RyR are also
expressed in the secretory vesicles
(8,
9) or, and perhaps more likely,
in the endosomes of β-cells
(10). Despite earlier
suggestions (11),
IP3R are probably not present in the secretory vesicles of
β-cells (8,
12,
13).All three subtypes of IP3R are stimulated by IP3 with
Ca2+ (1), and the
three subtypes of RyR are each directly regulated by Ca2+. However,
RyR differ in whether their most important physiological stimulus is
depolarization of the plasma membrane (RyR1), Ca2+ (RyR2) or
additional intracellular messengers like cyclic ADP-ribose. The latter
stimulates both Ca2+ release and insulin secretion in β-cells
(8,
14). The activities of both
families of intracellular Ca2+ channels are also modulated by many
additional signals that act directly or via phosphorylation
(15,
16). Although they commonly
mediate release of Ca2+ from the ER, both IP3R and RyR
select rather poorly between Ca2+ and other cations (permeability
ratio, PCa/PK ∼7)
(1,
17). This may allow
electrogenic Ca2+ release from the ER to be rapidly compensated by
uptake of K+ (18),
and where RyR or IP3R are expressed in other membranes it may allow
them to affect membrane potential.Both Ca2+ entry and release of Ca2+ from
intracellular stores contribute to the oscillatory increases in cytosolic
Ca2+ concentration ([Ca2+]i) that
stimulate exocytosis of insulin-containing vesicles in pancreatic β-cells
(7). Glucose rapidly
equilibrates across the plasma membrane (PM) of β-cells and its oxidative
metabolism by mitochondria increases the cytosolic ATP/ADP ratio, causing
KATP channels to close
(19). This allows an
unidentified leak current to depolarize the PM
(20) and activate
voltage-gated Ca2+ channels, predominantly L-type Ca2+
channels (21). The resulting
Ca2+ entry is amplified by Ca2+-induced Ca2+
release from intracellular stores
(7), triggering exocytotic
release of insulin-containing dense-core vesicles
(22). The importance of this
sequence is clear from the widespread use of sulfonylurea drugs, which close
KATP channels, in the treatment of type 2 diabetes. Ca2+
uptake by mitochondria beneath the PM further stimulates ATP production,
amplifying the initial response to glucose and perhaps thereby contributing to
the sustained phase of insulin release
(23). However, neither the
increase in [Ca2+]i nor the insulin release
evoked by glucose or other nutrients is entirely dependent on Ca2+
entry (7,
24) or closure of
KATP channels (25).
This suggests that glucose metabolism may also more directly activate RyR
(7,
26) and/or IP3R
(27) to cause release of
Ca2+ from intracellular stores. A change in the ATP/ADP ratio is
one means whereby nutrient metabolism may be linked to opening of
intracellular Ca2+ channels because both RyR
(28) and IP3R
(1) are stimulated by ATP.The other major physiological regulators of insulin release are the
incretins: glucagon-like peptide-1 and glucose-dependent insulinotropic
hormone (29). These hormones,
released by cells in the small intestine, stimulate synthesis of cAMP in
β-cells and thereby potentiate glucose-evoked insulin release
(30). These pathways are also
targets of drugs used successfully to treat type 2 diabetes
(29). The responses of
β-cells to cAMP involve both cAMP-dependent protein kinase and epacs
(exchange factors activated by cAMP)
(31,
32). The effects of the latter
are, at least partly, due to release of Ca2+ from intracellular
stores via RyR
(33–35)
and perhaps also via IP3R
(36). The interplays between
Ca2+ and cAMP signaling generate oscillatory changes in the
concentrations of both messengers
(37). RyR and IP3R
are thus implicated in mediating responses to each of the major physiological
regulators of insulin secretion: glucose and incretins.Here we report that in addition to expression in intracellular stores,
which probably include both the ER and secretory vesicles and/or endosomes,
functional RyR2 are also expressed in small numbers in the PM of RINm5F
insulinoma cells and rat pancreatic β-cells. 相似文献
6.
Hongjie Yuan Katie M. Vance Candice E. Junge Matthew T. Geballe James P. Snyder John R. Hepler Manuel Yepes Chian-Ming Low Stephen F. Traynelis 《The Journal of biological chemistry》2009,284(19):12862-12873
Zinc is hypothesized to be co-released with glutamate at synapses of the
central nervous system. Zinc binds to NR1/NR2A
N-methyl-d-aspartate (NMDA) receptors with high affinity
and inhibits NMDAR function in a voltage-independent manner. The serine
protease plasmin can cleave a number of substrates, including
protease-activated receptors, and may play an important role in several
disorders of the central nervous system, including ischemia and spinal cord
injury. Here, we demonstrate that plasmin can cleave the native NR2A
amino-terminal domain (NR2AATD), removing the functional high
affinity Zn2+ binding site. Plasmin also cleaves recombinant
NR2AATD at lysine 317 (Lys317), thereby producing a
∼40-kDa fragment, consistent with plasmin-induced NR2A cleavage fragments
observed in rat brain membrane preparations. A homology model of the
NR2AATD predicts that Lys317 is near the surface of the
protein and is accessible to plasmin. Recombinant expression of NR2A with an
amino-terminal deletion at Lys317 is functional and Zn2+
insensitive. Whole cell voltage-clamp recordings show that Zn2+
inhibition of agonist-evoked NMDA receptor currents of NR1/NR2A-transfected
HEK 293 cells and cultured cortical neurons is significantly reduced by
plasmin treatment. Mutating the plasmin cleavage site Lys317 on
NR2A to alanine blocks the effect of plasmin on Zn2+ inhibition.
The relief of Zn2+ inhibition by plasmin occurs in
PAR1-/- cortical neurons and thus is independent of interaction
with protease-activated receptors. These results suggest that plasmin can
directly interact with NMDA receptors, and plasmin may increase NMDA receptor
responses through disruption or removal of the amino-terminal domain and
relief of Zn2+ inhibition.N-Methyl-d-aspartate
(NMDA)2 receptors are
one of three types of ionotropic glutamate receptors that play critical roles
in excitatory neurotransmission, synaptic plasticity, and neuronal death
(1–3).
NMDA receptors are comprised of glycine-binding NR1 subunits in combination
with at least one type of glutamate-binding NR2 subunit
(1,
4). Each subunit contains three
transmembrane domains, one cytoplasmic re-entrant membrane loop, one bi-lobed
domain that forms the ligand binding site, and one bi-lobed amino-terminal
domain (ATD), thought to share structural homology to periplasmic amino
acid-binding proteins
(4–6).
Activation of NMDA receptors requires combined stimulation by glutamate and
the co-agonist glycine in addition to membrane depolarization to overcome
voltage-dependent Mg2+ block of the ion channel
(7). The activity of NMDA
receptors is negatively modulated by a variety of extracellular ions,
including Mg2+, polyamines, protons, and Zn2+ ions,
which can exert tonic inhibition under physiological conditions
(1,
4). Several extracellular
modulators such as Zn2+ and ifenprodil are thought to act at the
ATD of the NMDA receptor
(8–14).Zinc is a transition metal that plays key roles in both catalytic and
structural capacities in all mammalian cells
(15). Zinc is required for
normal growth and survival of cells. In addition, neuronal death in
hypoxia-ischemia and epilepsy has been associated with Zn2+
(16–18).
Abnormal metabolism of zinc may contribute to induction of cytotoxicity in
neurodegenerative diseases, such as Alzheimer''s disease, Parkinson''s disease,
and amyotrophic lateral sclerosis
(19). Zinc is co-released with
glutamate at excitatory presynaptic terminals and inhibits native NMDA
receptor activation (20,
21). Zn2+ inhibits
NMDA receptor function through a dual mechanism, which includes
voltage-dependent block and voltage-independent inhibition
(22–24).
Voltage-independent Zn2+ inhibition at low nanomolar concentrations
(IC50, 20 nm) is observed for NR2A-containing NMDA
receptors
(25–28).
Evidence has accumulated that the amino-terminal domain of the NR2A subunit
controls high-affinity Zn2+ inhibition of NMDA receptors, and
several histidine residues in this region may constitute part of an
NR2A-specific Zn2+ binding site
(8,
9,
11,
12). For the NR2A subunit,
several lines of evidence suggest that Zn2+ acts by enhancing
proton inhibition (8,
11,
29,
30).Serine proteases present in the circulation, mast cells, and elsewhere
signal directly to cells by cleaving protease-activated receptors (PARs),
members of a subfamily of G-protein-coupled receptors. Cleavage exposes a
tethered ligand domain that binds to and activates the cleaved receptors
(31,
32). Protease receptor
activation has been studied extensively in relation to coagulation and
thrombolysis (33). In addition
to their circulation in the bloodstream, some serine proteases and PARs are
expressed in the central nervous system, and have been suggested to play roles
in physiological conditions (e.g. long-term potentiation or memory)
and pathophysiological states such as glial scarring, edema, seizure, and
neuronal death (31,
34–36).Functional interactions between proteases and NMDA receptors have
previously been suggested. Earlier studies reported that the blood-derived
serine protease thrombin potentiates NMDA receptor response more than 2-fold
through activation of PAR1
(37). Plasmin, another serine
protease, similarly potentiates NMDA receptor response
(38). Tissue-plasminogen
activator (tPA), which catalyzes the conversion of the zymogen precursor
plasminogen to plasmin and results in PAR1 activation, also interacts with and
cleaves the ATD of the NR1 subunit of the NMDA receptor
(39,
40). This raises the
possibility that plasmin may also interact directly with the NMDA receptor
subunits to modulate receptor response. We therefore investigated the ability
of plasmin to cleave the NR2A NMDA receptor subunit. We found that nanomolar
concentrations of plasmin can cleave within the ATD, a region that mediates
tonic voltage-independent Zn2+ inhibition of NR2A-containing NMDA
receptors. We hypothesized that plasmin cleavage reduces the
Zn2+-mediated inhibition of NMDA receptors by removing the
Zn2+ binding domain. In the present study, we have demonstrated
that Zn2+ inhibition of agonist-evoked NMDA currents is decreased
significantly by plasmin treatment in recombinant NR1/NR2A-transfected HEK 293
cells and cultured cortical neurons. These concentrations of plasmin may be
pathophysiologically relevant in situations in which the blood-brain barrier
is compromised, which could allow blood-derived plasmin to enter brain
parenchyma at concentrations in excess of these that can cleave NR2A. Thus,
ability of plasmin to potentiate NMDA function through the relief of the
Zn2+ inhibition could exacerbate the harmful actions of NMDA
receptor overactivation in pathological situations. In addition, if newly
cleaved NR2AATD enters the bloodstream during ischemic injury, it
could serve as a biomarker of central nervous system injury. 相似文献
7.
Neeliyath A. Ramakrishnan Marian J. Drescher Dennis G. Drescher 《The Journal of biological chemistry》2009,284(3):1364-1372
The molecular mechanisms underlying synaptic exocytosis in the hair cell,
the auditory and vestibular receptor cell, are not well understood. Otoferlin,
a C2 domain-containing Ca2+-binding protein, has been implicated as
having a role in vesicular release. Mutations in the OTOF gene cause
nonsyndromic deafness in humans, and OTOF knock-out mice are deaf. In
the present study, we generated otoferlin fusion proteins containing two of
the same amino acid substitutions detected in DFNB9 patients (P1825A in C2F
and L1011P in C2D). The native otoferlin C2F domain bound syntaxin 1A and
SNAP-25 in a Ca2+-dependent manner (with optimal 61
μm free Ca2+ required for binding). These
interactions were greatly diminished for C2F with the P1825A mutation,
possibly because of a reduction in tertiary structural change, induced by
Ca2+, for the mutated C2F compared with the native C2F. The
otoferlin C2D domain also bound syntaxin 1A, but with weaker affinity
(Kd = 1.7 × 10–5 m) than
for the C2F interaction (Kd = 2.6 ×
10–9 m). In contrast, it was the otoferlin C2D
domain that bound the Cav1.3 II-III loop, in a
Ca2+-dependent manner. The L1011P mutation in C2D rendered this
binding insensitive to Ca2+ and considerably diminished. Overall,
we demonstrated that otoferlin interacts with two main target-SNARE proteins
of the hair-cell synaptic complex, syntaxin 1A and SNAP-25, as well as the
calcium channel, with the otoferlin C2F and C2D domains of central importance
for binding. Because mutations in the otoferlin C2 domains that cause deafness
in humans impair the ability of otoferlin to bind syntaxin, SNAP-25, and the
Cav1.3 calcium channel, it is these interactions that may mediate
regulation by otoferlin of hair cell synaptic exocytosis critical to inner ear
hair cell function.Calcium is a key regulator of synaptic vesicle fusion (reviewed in Ref.
1). In mechanosensory hair
cells, calcium microdomains (2)
and possibly nanodomains (3)
are formed when voltage-gated calcium channels open upon depolarization.
Calcium at these sites is thought to activate protein interactions, leading to
vesicle fusion. Some of the key players in this process are the
target-SNARE2
proteins, syntaxin 1A and SNAP-25, and the vesicle-SNARE, synaptobrevin
(4). Vesicle-SNARE
synaptotagmin 1 plays a crucial role as a calcium sensor at the neuronal
synapse, modulating calcium channels and vesicle release by a
Ca2+-dependent interaction with other SNARE proteins in the
presence of lipid molecules
(4–6).
However, in vertebrate mechanosensory hair cells, synaptotagmin 1 is not
detected (7). Instead, fast
neurotransmitter release in auditory and vestibular hair cells, facilitated
largely by an L-type voltagegated calcium channel, Cav1.3
(8,
9), is thought to be modulated
by a newly discovered protein, otoferlin, acting as the Ca2+ sensor
and vesicle-binding protein. When mutated, otoferlin causes DFNB9 nonsyndromic
deafness (10). Gene sequences
of different deaf families show that the OTOF gene can undergo
mutation at multiple locations
(11–13).
Recently, it has been demonstrated that otoferlin is necessary for synaptic
exocytosis from hair cells
(14). Further, an engineered
mutation in the C2B domain of otoferlin has been shown to cause deafness in
mice (15). However, the
precise function of otoferlin as a synaptic protein is not well
understood.Specific mutations in the otoferlin C2F (P1825A) or C2D (L1011P) domains in
humans have been documented to cause DFNB9 deafness
(11,
12). Previous studies
suggested that a region of otoferlin containing all three C2 domains, D, E,
and F, binds directly to the t-SNARE molecules syntaxin 1A and SNAP-25 in
response to an increase in Ca2+ concentration
(14). However, it is not
understood how a single amino acid substitution in one domain of otoferlin,
such as C2F (11) or C2D
(12), might independently lead
to deafness. Here, we examine the role of otoferlin as a Ca2+
sensor as well as a facilitator of vesicle fusion, as indicated by
protein-protein interactions and their [Ca2+] dependence. 相似文献
8.
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
9.
10.
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
11.
Mette Laursen Maike Bublitz Karine Moncoq Claus Olesen Jesper Vuust M?ller Howard S. Young Poul Nissen J. Preben Morth 《The Journal of biological chemistry》2009,284(20):13513-13518
We have determined the structure of the sarco(endo)plasmic reticulum
Ca2+-ATPase (SERCA) in an E2·Pi-like form
stabilized as a complex with , an
ATP analog, adenosine 5′-(β,γ-methylene)triphosphate
(AMPPCP), and cyclopiazonic acid (CPA). The structure determined at 2.5Å
resolution leads to a significantly revised model of CPA binding when compared
with earlier reports. It shows that a divalent metal ion is required for CPA
binding through coordination of the tetramic acid moiety at a characteristic
kink of the M1 helix found in all P-type ATPase structures, which is expected
to be part of the cytoplasmic cation access pathway. Our model is consistent
with the biochemical data on CPA function and provides new measures in
structure-based drug design targeting Ca2+-ATPases, e.g.
from pathogens. We also present an extended structural basis of ATP modulation
pinpointing key residues at or near the ATP binding site. A structural
comparison to the Na+,K+-ATPase reveals that the
Phe93 side chain occupies the equivalent binding pocket of the CPA
site in SERCA, suggesting an important role of this residue in stabilization
of the potassium-occluded E2 state of Na+,K+-ATPase.The Ca2+-ATPase from sarco(endo)plasmic reticulum of rabbit
skeletal muscle
(SERCA,5 isoform 1a)
is a thoroughly studied member of the P-type ATPase family
(1). SERCA possesses 10
transmembrane helices (M1 through M10) with both the N terminus and the C
terminus facing the cytoplasmic side and three cytoplasmic domains, inserted
in loops between M2 and M3 (A-domain) and between M4 and M5 (P- and N-domain)
(2). The enzyme mediates the
uptake of Ca2+ ions into the lumen of the sarcoplasmic reticulum
(SR) after their release into the cytoplasm through calcium release channels
during muscle contraction (3).
SERCA, plasma membrane Ca2+-ATPase, and a third, Golgi-located
secretory pathway Ca2+-ATPase are important factors in calcium and
manganese homeostasis, transport, signaling, and regulation
(4,
5).Crystal structures of all major states in the reaction cycle of SERCA have
been determined. These include the Ca2E1·ATP
state (6,
7) with high affinity
Ca2+ binding sites accessible from the cytoplasmic side of the SR
membrane, the calcium-occluded
transition state (6), the open
E2P state with luminal facing ion binding sites that have low affinity for
Ca2+ and high affinity for protons
(8) and the proton-occluded
H2–3E2[ATP] state with a bound modulatory ATP
(9). This considerable amount
of structural information has turned the Ca2+-ATPase into a
valuable model system for studies on structural rearrangements that take place
during the catalytic cycle of P-type ATPases. SERCA is considered a promising
drug target in medical research, with a particular focus on prostate cancer
and infectious diseases. Several compounds have already been shown to bind and
inhibit SERCA by stabilizing the enzyme in a particular conformational state.
Thapsigargin (TG), cyclopiazonic acid (CPA), and 2,5-di-(tert-butyl)
hydroquinone (BHQ) stabilize an E2-like state, and 1,3-dibromo-2,4,6-tri
(methylisothiouronium)benzene stabilizes an E1-P-like conformation
(10–13).
CPA is a toxic indole tetramic acid first isolated from Penicillium
cyclopium (14) and later
found to be produced by Aspergillus versicolor and Aspergillus
flavus. Like TG, CPA specifically binds to and inhibits SERCA with
nanomolar affinity (15).
Indeed, CPA is widely used in biochemical and physiological studies on
Ca2+ signaling and muscle function, where it causes Ca2+
store depletion due to specific inhibition of Ca2+ reuptake by
SERCA. CPA and TG were originally proposed to bind to similar sites on SERCA
(16), but recent crystal
structures have shown a distinct site of interaction
(17,
18). Despite these structural
insights, a previously demonstrated magnesium dependence of CPA binding
(19) remained unexplained, and
opposing CPA binding modes were observed (see below).Tetramic acids are synthesized naturally, and more than 150 natural
derivatives have been isolated from bacterial and fungal species (reviewed in
Ref. 20). Tetramic acids
possessing a 3-acyl group have the ability to chelate divalent metal ions. For
instance, tenuazonic acid from the fungus Phoma sorghina has been
shown to form complexes with Ca2+ and Mg2+
(21), as well as heavier
metals such as Cu(II), Ni(II), and Fe(III)
(22).Previously published crystallographic structures of the SERCA·CPA
complex (PDB ID 2O9J and 2EAS) demonstrated that CPA binds within the proposed
calcium access channel of SERCA. However, the structures did not reveal a role
for magnesium, and the orientation of CPA within this binding site differed in
the two studies (17,
18). To address these
ambiguities, we have determined the crystal structure of SERCA in complex with
, AMPPCP (an ATP analog), and
Mn2+·CPA. The structure reveals novel insight into CPA
binding, which we find to be mediated by a divalent cation, as demonstrated by
means of the anomalous scattering properties of Mn2+. Further and
improved refinement using previously deposited data (PDB ID 2O9J and 2OA0), in
light of our new findings, also revealed a strong plausibility for a magnesium
ion bound at this site. Furthermore, we find a new configuration of the bound
AMPPCP nucleotide, addressing the modulatory role of ATP binding to the
E2·Pi occluded conformation of SERCA. 相似文献
12.
J. Shawn Goodwin Gaynor A. Larson Jarod Swant Namita Sen Jonathan A. Javitch Nancy R. Zahniser Louis J. De Felice Habibeh Khoshbouei 《The Journal of biological chemistry》2009,284(5):2978-2989
The psychostimulants d-amphetamine (AMPH) and methamphetamine
(METH) release excess dopamine (DA) into the synaptic clefts of dopaminergic
neurons. Abnormal DA release is thought to occur by reverse transport through
the DA transporter (DAT), and it is believed to underlie the severe behavioral
effects of these drugs. Here we compare structurally similar AMPH and METH on
DAT function in a heterologous expression system and in an animal model. In
the in vitro expression system, DAT-mediated whole-cell currents were
greater for METH stimulation than for AMPH. At the same voltage and
concentration, METH released five times more DA than AMPH and did so at
physiological membrane potentials. At maximally effective concentrations, METH
released twice as much [Ca2+]i from internal
stores compared with AMPH. [Ca2+]i responses to
both drugs were independent of membrane voltage but inhibited by DAT
antagonists. Intact phosphorylation sites in the N-terminal domain of DAT were
required for the AMPH- and METH-induced increase in
[Ca2+]i and for the enhanced effects of METH on
[Ca2+]i elevation. Calmodulin-dependent protein
kinase II and protein kinase C inhibitors alone or in combination also blocked
AMPH- or METH-induced Ca2+ responses. Finally, in the rat nucleus
accumbens, in vivo voltammetry showed that systemic application of
METH inhibited DAT-mediated DA clearance more efficiently than AMPH, resulting
in excess external DA. Together these data demonstrate that METH has a
stronger effect on DAT-mediated cell physiology than AMPH, which may
contribute to the euphoric and addictive properties of METH compared with
AMPH.The dopamine transporter
(DAT)3 is a main
target for psychostimulants, such as d-amphetamine (AMPH),
methamphetamine (METH), cocaine (COC), and methylphenidate (Ritalin®). DAT
is the major clearance mechanism for synaptic dopamine (DA)
(1) and thereby regulates the
strength and duration of dopaminergic signaling. AMPH and METH are substrates
for DAT and competitively inhibit DA uptake
(2,
3) and release DA through
reverse transport
(4–9).
AMPH- and METH-induced elevations in extracellular DA result in complex
neurochemical changes and profound psychiatric effects
(2,
10–16).
Despite their structural and pharmacokinetic similarities, a recent National
Institute on Drug Abuse report describes METH as a more potent stimulant than
AMPH with longer lasting effects at comparable doses
(17). Although the route of
METH administration and its availability must contribute to the almost four
times higher lifetime nonmedical use of METH compared with AMPH
(18), there may also be
differences in the mechanisms that underlie the actions of these two drugs on
the dopamine transporter.Recent studies by Joyce et al.
(19) have shown that compared
with d-AMPH alone, the combination of d- and
l-AMPH in Adderall® significantly prolonged the time course of
extracellular DA in vivo. These experiments demonstrate that subtle
structural features of AMPH, such as chirality, can affect its action on
dopamine transporters. Here we investigate whether METH, a more lipophilic
analog of AMPH, affects DAT differently than AMPH, particularly in regard to
stimulated DA efflux.METH and AMPH have been reported as equally effective in increasing
extracellular DA levels in rodent dorsal striatum (dSTR), nucleus accumbens
(NAc) (10,
14,
20), striatal synaptosomes,
and DAT-expressing cells in vitro
(3,
6). John and Jones
(21), however, have recently
shown in mouse striatal and substantia nigra slices, that AMPH is a more
potent inhibitor of DA uptake than METH. On the other hand, in synaptosomes
METH inhibits DA uptake three times more effectively than AMPH
(14), and in DAT-expressing
COS-7 cells, METH releases DA more potently than AMPH (EC50 = 0.2
μm for METH versus EC50 = 1.7
μm for AMPH) (5).
However, these differences do not hold up under all conditions. For example,
in a study utilizing C6 cells, the disparity between AMPH and METH was not
found (12).The variations in AMPH and METH data extend to animal models. AMPH- and
METH-mediated behavior has been reported as similar
(22), lower
(20), or higher
(23) for AMPH compared with
METH. Furthermore, although the maximal locomotor activation response was less
for METH than for AMPH at a lower dose (2 mg/kg, intraperitoneal), both drugs
decreased locomotor activity at a higher dose (4 mg/kg)
(20). In contrast, in the
presence of a salient stimuli, METH is more potent in increasing the overall
magnitude of locomotor activity in rats yet is equipotent with AMPH in the
absence of these stimuli
(23).The simultaneous regulation of DA uptake and efflux by DAT substrates such
as AMPH and METH, as well as the voltage dependence of DAT
(24), may confound the
interpretation of existing data describing the action of these drugs. Our
biophysical approaches allowed us to significantly decrease the contribution
of DA uptake and more accurately determine DAT-mediated DA efflux with
millisecond time resolution. We have thus exploited time-resolved, whole-cell
voltage clamp in combination with in vitro and in vivo
microamperometry and Ca2+ imaging to compare the impact of METH and
AMPH on DAT function and determine the consequence of these interactions on
cell physiology.We find that near the resting potential, METH is more effective than AMPH
in stimulating DAT to release DA. In addition, at efficacious concentrations
METH generates more current, greater DA efflux, and higher Ca2+
release from internal stores than AMPH. Both METH-induced or the lesser
AMPH-induced increase in intracellular Ca2+ are independent of
membrane potential. The additional Ca2+ response induced by METH
requires intact phosphorylation sites in the N-terminal domain of DAT.
Finally, our in vivo voltammetry data indicate that METH inhibits
clearance of locally applied DA more effectively than AMPH in the rat nucleus
accumbens, which plays an important role in reward and addiction, but not in
the dorsal striatum, which is involved in a variety of cognitive functions.
Taken together these data imply that AMPH and METH have distinguishable
effects on DAT that can be shown both at the molecular level and in
vivo, and are likely to be implicated in the relative euphoric and
addictive properties of these two psychostimulants. 相似文献
13.
Oliver Trentmann Benjamin Jung Horst Ekkehard Neuhaus Ilka Haferkamp 《The Journal of biological chemistry》2008,283(52):36486-36493
Chlamydiales and Rickettsiales as metabolically impaired,
intracellular pathogenic bacteria essentially rely on “energy
parasitism” by the help of nucleotide transporters (NTTs). Also in plant
plastids NTT-type carriers catalyze ATP/ADP exchange to fuel metabolic
processes. The uptake of ATP4-, followed by energy consumption and
the release of ADP3-, would lead to a metabolically disadvantageous
accumulation of negative charges in form of inorganic phosphate
(Pi) in the bacterium or organelle if no interacting Pi
export system exists. We identified that Pi is a third substrate of
several NTT-type ATP/ADP transporters. During adenine nucleotide
hetero-exchange, Pi is cotransported with ADP in a one-to-one
stoichiometry. Additionally, Pi can be transported in exchange with
solely Pi. This Pi homo-exchange depends on the presence
of ADP and provides a first indication for only one binding center involved in
import and export. Furthermore, analyses of mutant proteins revealed that
Pi interacts with the same amino acid residue as the
γ-phosphate of ATP. Import of ATP in exchange with ADP plus
Pi is obviously an efficient way to couple energy provision with
the export of the two metabolic products (ADP plus Pi) and to
maintain cellular phosphate homeostasis in intracellular living “energy
parasites” and plant plastids. The additional Pi transport
capacity of NTT-type ATP/ADP transporters makes the existence of an
interacting Pi exporter dispensable and might explain why a
corresponding protein so far has not been identified.Most organisms possess the capacity to resynthesize the fundamental energy
currency ATP by fusion of ADP and Pi. Generally, in eukaryotes the
major part of energy is produced in specialized organelles, the mitochondria.
Mitochondrial ADP/ATP carriers
(AACs)2 mediate the
export of newly synthesized ATP in strict counter-exchange with cytosolic ADP
and therefore provide energy to the cellular metabolism
(1). Plants additionally
generate high amounts of ATP during photosynthesis in chloroplasts. However,
under conditions of limiting or missing photosynthetic activity, plant
plastids depend on external energy supply
(2–4).
Specific nucleotide transporters (NTTs) located in the inner plastid envelope
membrane mediate the required energy import
(5). These transporters
structurally, functionally, and phylogenetically differ from mitochondrial
AACs. They catalyze the import of cytosolic ATP in exchange with stromal ADP,
are monomers consisting of 12 predicted transmembrane helices, and are related
to the functionally heterogeneous group of bacterial NTTs
(5).Although most prokaryotic organisms are able to regenerate ATP and
therefore are considered as energetically self-sustaining, the obligate
intracellular living bacterial orders Chlamydiales and
Rickettsiales are impaired in energy and nucleotide synthesis or even
completely lost the corresponding pathways
(6–8).
Therefore, these bacteria, which comprise important human pathogens
(9,
10), essentially rely on
nucleotide and energy import. Bacterial NTTs catalyze the required import of a
broad range of nucleotides and NAD or facilitate the counter-exchange of ATP
and ADP (5,
11–15).
The latter process has been termed “energy parasitism” and
obviously is of high importance for the survival of rickettsial and chlamydial
cells (5,
16–18).Although import measurements on intact Escherichia coli cells
expressing the corresponding proteins allowed characterization of many
bacterial and plastidial NTTs
(12–15,
19–24),
a very important physiological question is still not clarified. The uptake of
ATP4- in exchange with ADP3- in absence of a concerted
Pi export would result in a charge difference and a phosphate
imbalance in the bacterial cell. In mitochondria, phosphate carriers
metabolically cooperate with AACs because they provide Pi for ATP
synthesis (25). Similarly, it
was assumed that NTT-type ATP/ADP transporters cooperate with phosphate
exporters to guarantee phosphate homeostasis in the bacterium or plastid.
However, a Pi exporter interacting with ATP/ADP transporters is not
known in “energy parasites” or plant plastids. Bacterial and plant
phosphate transport systems rather facilitate Pi import or the
counter-exchange of Pi and phosphorylated compounds and therefore
do not allow net Pi export
(26–29).
Furthermore, the newly identified plastidial (proton-driven) phosphate
transporters are not preferentially expressed under conditions or in tissues
that require ATP provision to the plastid
(30,
31).Recently, we succeeded in the purification of the first recombinant NTT
from Protochlamydia amoebophila (PamNTT1), a parachlamydial
endosymbiont of the protist Acantamoeba
(32). The functional
reconstitution of the highly pure PamNTT1 into artificial lipid
vesicles for the first time allowed the biochemical characterization of a
representative nonmitochondrial ATP/ADP transporter unaffected by the complex
metabolic situation of the bacterial cell. We demonstrated that in contrast to
mitochondrial AACs, PamNTT1 catalyzes a membrane potential
independent, electroneutral adenine nucleotide hetero-exchange
(32,
33). The latter could argue
for a cotransport of a counterion compensating for the electrogenic
ATP4-/ADP3- exchange.Here, we investigated possible ions accompanying ATP or ADP transport.
Interestingly, we uncovered that PamNTT1 and also rickettsial and
plastidial ATP/ADP transporters accept an additional important substrate,
which is Pi. We performed a comprehensive characterization of the
Pi transport and gained new insights into the transport properties
of ATP/ADP transporters. 相似文献
14.
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
15.
Christian Grimm Simone J?rs Stefan Heller 《The Journal of biological chemistry》2009,284(20):13823-13831
The varitint-waddler mutation A419P renders TRPML3 constitutively active,
resulting in cationic overload, particularly in sustained influx of
Ca2+. TRPML3 is expressed by inner ear sensory hair cells, and we
were intrigued by the fact that hair cells are able to cope with expressing
the TRPML3(A419P) isoform for weeks before they ultimately die. We
hypothesized that the survival of varitint-waddler hair cells is linked to
their ability to deal with Ca2+ loads due to the abundance of
plasma membrane calcium ATPases (PMCAs). Here, we show that PMCA2
significantly reduced [Ca2+]i increase and
apoptosis in HEK293 cells expressing TRPML3(A419P). The deaf-waddler isoform
of PMCA2, operating at 30% efficacy, showed a significantly decreased ability
to rescue the Ca2+ loading of cells expressing TRPML3(A419P). When
we combined mice heterozygous for the varitint-waddler mutant allele with mice
heterozygous for the deaf-waddler mutant allele, we found severe hair bundle
defects as well as increased hair cell loss compared with mice heterozygous
for each mutant allele alone. Furthermore, 3-week-old double mutant mice
lacked auditory brainstem responses, which were present in their respective
littermates containing single mutant alleles. Likewise, heterozygous double
mutant mice exhibited severe circling behavior, which was not observed in mice
heterozygous for TRPML3(A419P) or PMCA2(G283S) alone. Our results provide a
molecular rationale for the delayed hair cell loss in varitint-waddler mice.
They also show that hair cells are able to survive for weeks with sustained
Ca2+ loading, which implies that Ca2+ loading is an
unlikely primary cause of hair cell death in ototoxic stress situations.Varitint-waddler (Va) mice express a mutant isoform (A419P) of the
transient receptor potential channel TRPML3 (murine gene symbol,
Mcoln3) that results in profound hearing loss, vestibular defects
(circling behavior, imbalance, head bobbing, waddling), pigmentation
deficiencies, sterility, and perinatal lethality in homozygous animals
(1). A second Mcoln3
variant (VaJ) that arose in the Va background
carries two mutations (I362T and A419P) and shows a phenotype with reduced
severity, particularly in heterozygous animals
(1). The A419P mutation in
Va and VaJ mice is located in
transmembrane-spanning domain
5(TM5)3 of TRPML3,
where it leads to a constitutively open channel, resulting in highly elevated
[Ca2+]i
(2-5).
In contrast to the effect of the A419P mutation on TRPML3 channel activity,
the single I362T mutation does not appear to affect
[Ca2+]i
(3,
5). When combined with the
A419P mutation, as found in VaJ mice, the constitutive
activity of this mutant TRPML3 isoform is comparable with that of A419P alone
(2-5).Here, we show that HEK293 cells expressing TRPML3-(A419P) or
TRPML3(I362T/A419P) undergo rapid apoptosis. This apoptosis is suppressed by
coexpression of plasma membrane calcium ATPase type 2 (PMCA2). In
varitint-waddler mice, sensory hair cells survive for weeks after birth
(6), which raised the question
of whether this survival could be the result of the hair cells'' ability to
deal with normally transient and localized Ca2+ influx, a feature
that is centered around the high levels of mobile Ca2+ buffers and
PMCA isoforms found in sensory hair cells
(7-10).
We decided to test this hypothesis in vivo by utilizing deaf-waddler
mice that carry a mutation (G283S) in the Atp2b2 gene encoding mutant
PMCA2. Mice homozygous for PMCA2(G283S)
(Atp2b2dfw/dfw) are deaf and have poor
balance (11). Compared with
Atp2b2 knock-out mice, deaf-waddler mice display a milder phenotype
because PMCA2(G283S) retains 30% of its biological activity compared with the
wild-type isoform (12). We
found that sensory hair cell loss, hearing loss, and vestibular dysfunction
were aggravated in mice carrying varitint-waddler and deaf-waddler alleles
compared with animals carrying the single mutant alleles. Our results reveal
that the Ca2+-buffering and Ca2+ extrusion abilities of
hair cells are powerful enough to prevent cell death for weeks, even in the
presence of constitutively active TRPML3(A419P), which is able to induce rapid
apoptosis in other cells. 相似文献
16.
17.
Michaela Kudrnac Stanislav Beyl Annette Hohaus Anna Stary Thomas Peterbauer Eugen Timin Steffen Hering 《The Journal of biological chemistry》2009,284(18):12276-12284
Voltage dependence and kinetics of CaV1.2 activation are
affected by structural changes in pore-lining S6 segments of the
α1-subunit. Significant effects are induced by either proline
or threonine substitutions in the lower third of segment IIS6 (“bundle
crossing region”), where S6 segments are likely to seal the channel in
the closed conformation (Hohaus, A., Beyl, S., Kudrnac, M., Berjukow, S.,
Timin, E. N., Marksteiner, R., Maw, M. A., and Hering, S. (2005) J. Biol.
Chem. 280, 38471–38477). Here we report that S435P in IS6 results
in a large shift of the activation curve (-25.9 ± 1.2 mV) and slower
current kinetics. Threonine substitutions at positions Leu-429 and Leu-434
induced a similar kinetic phenotype with shifted activation curves (L429T by
-6.6 ± 1.2 and L434T by -12.1 ± 1.7 mV). Inactivation curves of
all mutants were shifted to comparable extents as the activation curves.
Interdependence of IS6 and IIS6 mutations was analyzed by means of mutant
cycle analysis. Double mutations in segments IS6 and IIS6 induce either
additive (L429T/I781T, -34.1 ± 1.4 mV; L434T/I781T, -40.4 ± 1.3
mV; L429T/L779T, -12.6 ± 1.3 mV; and L434T/L779T, -22.4 ± 1.3
mV) or nonadditive shifts of the activation curves along the voltage axis
(S435P/I781T, -33.8 ± 1.4 mV). Mutant cycle analysis revealed energetic
coupling between residues Ser-435 and Ile-781, whereas other paired mutations
in segments IS6 and IIS6 had independent effects on activation gating.Ca2+ current through CaV1.2 channels initiates muscle
contraction, release of hormones and neurotransmitters, and affects
physiological processes such as vision, hearing, and gene expression
(1). Their pore-forming
α1-subunit is composed of four homologous domains formed by
six transmembrane segments (S1–S6)
(2). The signal of the
voltage-sensing machinery, consisting of multiple charged amino acids (located
in segments S4 and adjacent structures of each domain), is transmitted to the
pore region (3). Conformational
changes in pore lining S6 and adjacent segments finally lead to pore openings
(activation) and closures (inactivation).Our understanding of how CaV1.2 channels open and close is
largely based on extrapolations of structural information from potassium
channels. The crystal structures of the closed conformation of two bacterial
potassium channels (KcsA and MlotiK)
(4,
5) show a gate located at the
intracellular channel mouth formed by tightly packed S6 helices. The crystal
structure of the open conformation of Kv1.2
(6,
7) revealed a bent S6 with the
highly conserved PXP motif apparently acting as a hinge (see
8). The activation mechanism
proposed for MthK channels involves helix bending at a highly conserved
glycine at position 83 (see Ref.
9, “glycine gating
hinge” hypothesis).Compared with potassium channels, the pore of CaV is asymmetric,
and none of the four S6 segments has a putative helix-bending PXP
motif. Furthermore, the conserved glycine (corresponding to position 83 in
MthK, see Ref. 10) is only
present in segments IS6 and IIS6 (for review see Ref.
11). We have shown that
substituting proline for this glycine in IIS6 of CaV1.2 does not
significantly affect gating
(12).Zhen et al. (13)
investigated the pore lining S6 segments of CaV2.1 using the
substituted cysteine accessibility method. The accessibility of cysteines was
changed by opening and closing the channel, consistent with the gate being on
the intracellular side. The general picture of a channel gate close to the
inner channel mouth of CaV1.2 was recently supported by
pharmacological studies
(14).Substitution of hydrophilic residues in the lower third of segment IIS6 of
CaV1.2 (LAIA motif, 779–784, see Ref.
12) induces pronounced changes
in channel gating as follows: a shift in the voltage dependence of activation
accompanied by a slowing of the activation kinetics near the footstep of the
m∞(V) curve and a slowing of deactivation
at all potentials. Interestingly, these changes in channel gating resemble the
effects of proline substitution of Gly-219 in the bacterial sodium channel
from Bacillus halodurans (“Gly-219 gating hinge,” see
Ref. 15).The strongest shifts of the activation curve reported so far were observed
for proline substitutions
(12). As prolines in an
α-helix cause a rigid kink with an angle of about 26°
(16), we hypothesized that
these mutants were causing a kink in helix IIS6 similar to a bend that would
normally occur flexibly during the activation process
(12).Here we extend our previous study by systematically substituting residues
in segment IS6 of CaV1.2 by proline or the small and polar
threonine. Several functional IS6 mutants with shifted activation and
inactivation characteristics were identified (S435P, L429T, and L434T), and
the interdependence of IS6 and IIS6 mutations was analyzed. Mutant cycle
analysis revealed both mutually independent and energetically coupled
contributions of IS6 and IIS6 residues on activation gating. 相似文献
18.
Roger W. Hunter Carol MacKintosh Ingeborg Hers 《The Journal of biological chemistry》2009,284(18):12339-12348
The elevation of [cAMP]i is an important mechanism of
platelet inhibition and is regulated by the opposing activity of adenylyl
cyclase and phosphodiesterase (PDE). In this study, we demonstrate that a
variety of platelet agonists, including thrombin, significantly enhance the
activity of PDE3A in a phosphorylation-dependent manner. Stimulation of
platelets with the PAR-1 agonist SFLLRN resulted in rapid and transient
phosphorylation of PDE3A on Ser312, Ser428,
Ser438, Ser465, and Ser492, in parallel with
the PKC (protein kinase C) substrate, pleckstrin. Furthermore, phosphorylation
and activation of PDE3A required the activation of PKC, but not of PI3K/PKB,
mTOR/p70S6K, or ERK/RSK. Activation of PKC by phorbol esters also resulted in
phosphorylation of the same PDE3A sites in a PKC-dependent, PKB-independent
manner. This was further supported by the finding that IGF-1, which strongly
activates PI3K/PKB, but not PKC, did not regulate PDE3A. Platelet activation
also led to a PKC-dependent association between PDE3A and 14-3-3 proteins. In
contrast, cAMP-elevating agents such as PGE1 and forskolin-induced
phosphorylation of Ser312 and increased PDE3A activity, but did not
stimulate 14-3-3 binding. Finally, complete antagonism of
PGE1-evoked cAMP accumulation by thrombin required both
Gi and PKC activation. Together, these results demonstrate that
platelet activation stimulates PKC-dependent phosphorylation of PDE3A on
Ser312, Ser428, Ser438, Ser465,
and Ser492 leading to a subsequent increase in cAMP hydrolysis and
14-3-3 binding.Upon vascular injury, platelets adhere to the newly exposed subintimal
collagen and undergo activation leading to platelet spreading to cover the
damaged region and release of thrombogenic factors such as ADP and thromboxane
A2. In addition, platelets are activated by thrombin, which is
generated as a result of activation of the coagulation pathway, and stimulates
platelets by cleaving the protease-activated receptors
(PAR),2
PAR-1 and PAR-4. The final common pathway is the exposure of fibrinogen
binding sites on integrin αIIbβ3 resulting in
platelet aggregation and thrombus formation.Thrombin-mediated cleavage of PARs leads to activation of phospholipase C
β (PLC), hydrolysis of phosphatidylinositol (PI) 4,5-bisphosphate and a
subsequent increase in [Ca2+]i and activation
of protein kinase C (PKC). Protein kinase C contributes to platelet activation
both directly, through affinity regulation of the fibrinogen receptor,
integrin αIIbβ3
(1), and indirectly by
enhancing degranulation (2).
Thrombin also stimulates activation of PI 3-kinases and subsequent generation
of PI (3,
4,
5) trisphosphate and PI
(3,
4) bisphosphate
(3), which recruit protein
kinase B (PKB) to the plasma membrane where it becomes phosphorylated and
activated.Platelet activation is opposed by agents that raise intracellular
3′-5′-cyclic adenosine monophosphate
([cAMP]i). cAMP is a powerful inhibitory second messenger
that down-regulates platelet function by interfering with Ca2+
homeostasis, degranulation and integrin activation
(4). Synthesis of cAMP is
stimulated by mediators such as prostaglandin I2 (PGI2),
which bind to Gs-coupled receptors leading to activation of
adenylate cyclase (AC). This inhibitory pathway is opposed by thrombin, which
inhibits the elevation of cAMP indirectly via autocrine activation of the
Gi-coupled ADP receptor P2Y12. cAMP signaling is
terminated by hydrolysis to biologically inert 5′-AMP by
3′-phosphodiesterases. Platelets express two cAMP phosphodiesterase
isoforms, cGMP-stimulated PDE2 and cGMP-inhibited PDE3A. PDE3A is the most
abundant isoform in platelets and has a ∼250-fold lower
Km for cAMP than PDE2
(4). As a consequence of these
properties, PDE3A exerts a greater influence on cAMP homeostasis, particularly
at resting levels. The importance of PDE3A in platelet function is further
emphasized by the finding that the PDE3A inhibitors cilostamide and milrinone
raise basal cAMP levels and strongly inhibit thrombin-induced platelet
activation (5). Furthermore,
PDE3A-/- mice demonstrate increased resting levels of platelet cAMP
and are protected against a model of pulmonary thrombosis
(6). In contrast, the PDE2
inhibitor EHNA has no significant effect on cAMP levels and platelet
aggregation (7,
8). The activity of PDE3A is
therefore essential to maintain low equilibrium levels of cAMP and determine
the threshold for platelet activation
(7).Like its paralogue PDE3B, it has recently become clear that PDE3A activity
can be positively regulated by phosphorylation in platelets and human oocytes
(9,
10). There is some evidence
that PKB may be involved in this regulation, although the phosphorylation
sites are poorly characterized. In contrast, phosphorylation of PDE3A in HeLa
cells was stimulated by phorbol esters and blocked by inhibitors of PKC
(11). In this study, we aimed
to identify the signaling pathways and phosphorylation sites that are involved
in regulation of platelet PDE3A. Here, we show strong evidence that PKC, and
not PKB, is involved in agonist-stimulated PDE3A phosphorylation on
Ser312, Ser428, Ser438, Ser465,
and Ser492, leading to an increase in PDE3A activity, 14-3-3
binding and modulation of intracellular cAMP levels. 相似文献
19.
20.
Yoon Namkung Concetta Dipace Jonathan A. Javitch David R. Sibley 《The Journal of biological chemistry》2009,284(22):15038-15051
We investigated the role of G protein-coupled receptor kinase
(GRK)-mediated phosphorylation in agonist-induced desensitization, arrestin
association, endocytosis, and intracellular trafficking of the D2
dopamine receptor (DAR). Agonist activation of D2 DARs results in
rapid and sustained receptor phosphorylation that is solely mediated by GRKs.
A survey of GRKs revealed that only GRK2 or GRK3 promotes D2 DAR
phosphorylation. Mutational analyses resulted in the identification of eight
serine/threonine residues within the third cytoplasmic loop of the receptor
that are phosphorylated by GRK2/3. Simultaneous mutation of these eight
residues results in a receptor construct, GRK(-), that is completely devoid of
agonist-promoted GRK-mediated receptor phosphorylation. We found that both
wild-type (WT) and GRK(-) receptors underwent a similar degree of
agonist-induced desensitization as assessed using [35S]GTPγS
binding assays. Similarly, both receptor constructs internalized to the same
extent in response to agonist treatment. Furthermore, using bioluminescence
resonance energy transfer assays to directly assess receptor association with
arrestin3, we found no differences between the WT and GRK(-) receptors. Thus,
phosphorylation is not required for arrestin-receptor association or
agonist-induced desensitization or internalization. In contrast, when we
examined recycling of the D2 DARs to the cell surface, subsequent
to agonist-induced endocytosis, the GRK(-) construct exhibited less recycling
in comparison with the WT receptor. This impairment appears to be due to a
greater propensity of the GRK(-) receptors to down-regulate once internalized.
In contrast, if the receptor is highly phosphorylated, then receptor recycling
is promoted. These results reveal a novel role for GRK-mediated
phosphorylation in regulating the post-endocytic trafficking of a G
protein-coupled receptor.Dopamine receptors
(DARs)3 are members of
the GPCR superfamily and consist of five structurally distinct subtypes
(1,
2). These can be divided into
two subfamilies on the basis of their structure and pharmacological and
transductional properties (3).
The “D1-like” subfamily includes the D1 and
D5 receptors, which couple to the heterotrimeric G proteins
GS or GOLF to stimulate adenylyl cyclase activity and
raise intracellular levels of cAMP. The D2-like subfamily includes
the D2, D3, and D4 receptors, which couple to
inhibitory Gi/o proteins to reduce adenylyl cyclase activity as
well as modulate voltage-gated K+ or Ca2+ channels.
Within the central nervous system, these receptors modulate movement, learning
and memory, reward and addiction, cognition, and certain neurendocrine
functions. As with other GPCRs, the DARs are subject to a wide variety of
regulatory mechanisms, which can either positively or negatively modulate
their expression and functional activity
(4).Upon agonist activation, most GPCRs undergo desensitization, a homeostatic
process that results in a waning of receptor response despite continued
agonist stimulation (5,
6). Desensitization is believed
to involve the phosphorylation of receptors by either G protein-coupled
receptor kinases (GRKs) and/or second messenger-activated kinases such as PKA
or PKC. Homologous forms of desensitization involve only agonist-activated
receptors and appear to be primarily mediated by GRKs. In many cases,
GRK-mediated phosphorylation has been shown to decrease receptor/G protein
interactions and also initiate arrestin binding, which further promotes
endocytosis of the receptor through clathrin-coated pits
(7–9).
Once internalized, GPCRs can engage additional signaling pathways
(10), be sorted for recycling
to the plasma membrane, or targeted for degradation
(7–9).
Among the DARs, the D2 receptor is arguably one of the most
validated drug targets in neurology and psychiatry. For instance, all
receptor-based anti-parkinsonian drugs work via stimulating the D2
DAR (11), whereas all Food and
Drug Administration-approved antipsychotic agents are antagonists of this
receptor subtype (12,
13). The D2 DAR is
also therapeutically targeted in other disorders such as restless legs
syndrome, tardive dyskinesia, Tourette syndrome, and hyperprolactinemia. As
such, more knowledge concerning the regulation of the D2 DAR could
be helpful in improving current therapies or devising new treatment
strategies.In comparison with other GPCRs, however, detailed mechanistic information
concerning regulation of the D2 DAR is mostly lacking, although
some progress has recently been made. For instance, we
(14) and others
(15) have found that
PKC-mediated phosphorylation can regulate both D2 receptor
desensitization and trafficking. In our PKC study, we mapped out all of the
PKC phosphorylation sites within the third intracellular loop (IC3) of the
receptor, and we determined the existence of two PKC phosphorylation domains.
Both of these domains were found to regulate receptor sequestration, whereas
only one domain regulated functional uncoupling in response to PKC activation
(14). In response to agonist
activation, the D2 DAR has also been shown to undergo functional
desensitization (4), although
this has not been intensively investigated. More thoroughly examined is the
observation that agonist stimulation of the D2 DAR promotes its
sequestration from the cell surface into vesicular compartments that appear
distinct from those harboring internalized D1 DARs or
β-adrenergic receptors
(16–21).
In addition to uncertainty over the endocytic pathway involved, controversy
also exists as to whether or not D2 DAR internalization is
dynamin-dependent and whether the internalized receptors can partially or
completely recycle to the cell surface or, alternatively, if they undergo
degradation (19,
21–24).
The D2 DAR does appear to undergo GRK-mediated phosphorylation upon
agonist activation, which has been suggested to promote arrestin association
and receptor sequestration
(16,
19,
25), although this process has
not been studied in detail and its relationship to functional
receptor desensitization is unknown.In this study, we have further characterized GRK-mediated phosphorylation
of the D2 DAR and determined its role in agonist-induced receptor
desensitization, internalization, and recycling. Using site-directed
mutagenesis, we have mapped out all of the GRK phosphorylation sites within
the D2 DAR and determined that these are distinct from the PKC
phosphorylation sites. Using a GRK phosphorylation-null mutant receptor, we
found, surprisingly, that GRK-mediated phosphorylation is not actually
required for agonist-induced receptor desensitization, arrestin association,
or internalization. In contrast, we found that the GRK phosphorylation-null
receptor was impaired in its ability to recycle to the cell surface subsequent
to internalization and was degraded to a greater extent in comparison with the
wild-type receptor. These results suggest that GRK-mediated phosphorylation of
the D2 DAR regulates its intracellular trafficking or sorting once
internalized, a novel mechanism for GRK-mediated regulation of GPCR
function. 相似文献