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Johann Schredelseker Anamika Dayal Thorsten Schwerte Clara Franzini-Armstrong Manfred Grabner 《The Journal of biological chemistry》2009,284(2):1242-1251
The paralyzed zebrafish strain relaxed carries a null mutation for
the skeletal muscle dihydropyridine receptor (DHPR) β1a
subunit. Lack of β1a results in (i) reduced membrane
expression of the pore forming DHPR α1S subunit, (ii)
elimination of α1S charge movement, and (iii) impediment of
arrangement of the DHPRs in groups of four (tetrads) opposing the ryanodine
receptor (RyR1), a structural prerequisite for skeletal muscle-type
excitation-contraction (EC) coupling. In this study we used relaxed
larvae and isolated myotubes as expression systems to discriminate specific
functions of β1a from rather general functions of β
isoforms. Zebrafish and mammalian β1a subunits quantitatively
restored α1S triad targeting and charge movement as well as
intracellular Ca2+ release, allowed arrangement of DHPRs in
tetrads, and most strikingly recovered a fully motile phenotype in
relaxed larvae. Interestingly, the cardiac/neuronal
β2a as the phylogenetically closest, and the ancestral
housefly βM as the most distant isoform to β1a
also completely recovered α1S triad expression and charge
movement. However, both revealed drastically impaired intracellular
Ca2+ transients and very limited tetrad formation compared with
β1a. Consequently, larval motility was either only partially
restored (β2a-injected larvae) or not restored at all
(βM). Thus, our results indicate that triad expression and
facilitation of 1,4-dihydropyridine receptor (DHPR) charge movement are common
features of all tested β subunits, whereas the efficient arrangement of
DHPRs in tetrads and thus intact DHPR-RyR1 coupling is only promoted by the
β1a isoform. Consequently, we postulate a model that presents
β1a as an allosteric modifier of α1S
conformation enabling skeletal muscle-type EC coupling.Excitation-contraction
(EC)3 coupling in
skeletal muscle is critically dependent on the close interaction of two
distinct Ca2+ channels. Membrane depolarizations of the myotube are
sensed by the voltage-dependent 1,4-dihydropyridine receptor (DHPR) in the
sarcolemma, leading to a rearrangement of charged amino acids (charge
movement) in the transmembrane segments S4 of the pore-forming DHPR
α1S subunit
(1,
2). This conformational change
induces via protein-protein interaction
(3,
4) the opening of the
sarcoplasmic type-1 ryanodine receptor (RyR1) without need of Ca2+
influx through the DHPR (5).
The release of Ca2+ from the sarcoplasmic reticulum via RyR1
consequently induces muscle contraction. The protein-protein interaction
mechanism between DHPR and RyR1 requires correct ultrastructural targeting of
both channels. In Ca2+ release units (triads and peripheral
couplings) of the skeletal muscle, groups of four DHPRs (tetrads) are coupled
to every other RyR1 and hence are geometrically arranged following the
RyR-specific orthogonal arrays
(6).The skeletal muscle DHPR is a heteromultimeric protein complex, composed of
the voltage-sensing and pore-forming α1S subunit and
auxiliary subunits β1a, α2δ-1, and
γ1 (7). While
gene knock-out of the DHPR γ1 subunit
(8,
9) and small interfering RNA
knockdown of the DHPR α2δ-1 subunit
(10-12)
have indicated that neither subunit is essential for coupling of the DHPR with
RyR1, the lack of the α1S or of the intracellular
β1a subunit is incompatible with EC coupling and accordingly
null model mice die perinatally due to asphyxia
(13,
14). β subunits of
voltage-gated Ca2+ channels were repeatedly shown to be responsible
for the facilitation of α1 membrane insertion and to be
potent modulators of α1 current kinetics and voltage
dependence (15,
16). Whether the loss of EC
coupling in β1-null mice was caused by decreased DHPR membrane
expression or by the lack of a putative specific contribution of the β
subunit to the skeletal muscle EC coupling apparatus
(17,
18) was not clearly resolved.
Recently, other β-functions were identified in skeletal muscle using the
β1-null mutant zebrafish relaxed
(19,
20). Like the
β1-knock-out mouse
(14) zebrafish
relaxed is characterized by complete paralysis of skeletal muscle
(21,
22). While
β1-knock-out mouse pups die immediately after birth due to
respiratory paralysis (14),
larvae of relaxed are able to survive for several days because of
oxygen and metabolite diffusion via the skin
(23). Using highly
differentiated myotubes that are easy to isolate from these larvae, the lack
of EC coupling could be described by quantitative immunocytochemistry as a
moderate ∼50% reduction of α1S membrane expression
although α1S charge movement was nearly absent, and, most
strikingly, as the complete lack of the arrangement of DHPRs in tetrads
(19). Thus, in skeletal muscle
the β subunit enables EC coupling by (i) enhancing α1S
membrane targeting, (ii) facilitating α1S charge movement,
and (iii) enabling the ultrastructural arrangement of DHPRs in tetrads.The question arises, which of these functions are specific for the skeletal
muscle β1a and which ones are rather general properties of
Ca2+ channel β subunits. Previous reconstitution studies made
in the β1-null mouse system
(24,
25) using different β
subunit constructs (26) did
not allow differentiation between β-induced enhancement of non-functional
α1S membrane expression and the facilitation of
α1S charge movement, due to the lack of information on
α1S triad expression levels. Furthermore, the β-induced
arrangement of DHPRs in tetrads was not detected as no ultrastructural
information was obtained.In the present study, we established zebrafish mutant relaxed as
an expression system to test different β subunits for their ability to
restore skeletal muscle EC coupling. Using isolated myotubes for in
vitro experiments (19,
27) and complete larvae for
in vivo expression studies
(28-31)
and freeze-fracture electron microscopy, a clear differentiation between the
major functional roles of β subunits was feasible in the zebrafish
system. The cloned zebrafish β1a and a mammalian (rabbit)
β1a were shown to completely restore all parameters of EC
coupling when expressed in relaxed myotubes and larvae. However, the
phylogenetically closest β subunit to β1a, the
cardiac/neuronal isoform β2a from rat, as well as the
ancestral βM isoform from the housefly (Musca
domestica), could recover functional α1S membrane
insertion, but led to very restricted tetrad formation when compared with
β1a, and thus to impaired DHPR-RyR1 coupling. This impairment
caused drastic changes in skeletal muscle function.The present study shows that the enhancement of functional
α1S membrane expression is a common function of all the
tested β subunits, from β1a to even the most distant
βM, whereas the effective formation of tetrads and thus proper
skeletal muscle EC coupling is an exclusive function of the skeletal muscle
β1a subunit. In context with previous studies, our results
suggest a model according to which β1a acts as an allosteric
modifier of α1S conformation. Only in the presence of
β1a, the α1S subunit is properly folded to
allow RyR1 anchoring and thus skeletal muscle-type EC coupling. 相似文献
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Ting Wang Yatrik M. Shah Tsutomu Matsubara Yueying Zhen Tomotaka Tanabe Tomokazu Nagano Serge Fotso Kristopher W. Krausz T. Mark Zabriskie Jeffrey R. Idle Frank J. Gonzalez 《The Journal of biological chemistry》2010,285(10):7670-7685
A previous study identified the peroxisome proliferator-activated receptor α (PPARα) activation biomarkers 21-steroid carboxylic acids 11β-hydroxy-3,20-dioxopregn-4-en-21-oic acid (HDOPA) and 11β,20-dihydroxy-3-oxo-pregn-4-en-21-oic acid (DHOPA). In the present study, the molecular mechanism and the metabolic pathway of their production were determined. The PPARα-specific time-dependent increases in HDOPA and 20α-DHOPA paralleled the development of adrenal cortex hyperplasia, hypercortisolism, and spleen atrophy, which was attenuated in adrenalectomized mice. Wy-14,643 activation of PPARα induced hepatic FGF21, which caused increased neuropeptide Y and agouti-related protein mRNAs in the hypothalamus, stimulation of the agouti-related protein/neuropeptide Y neurons, and activation of the hypothalamic-pituitary-adrenal (HPA) axis, resulting in increased adrenal cortex hyperplasia and corticosterone production, revealing a link between PPARα and the HPA axis in controlling energy homeostasis and immune regulation. Corticosterone was demonstrated as the precursor of 21-carboxylic acids both in vivo and in vitro. Under PPARα activation, the classic reductive metabolic pathway of corticosterone was suppressed, whereas an alternative oxidative pathway was uncovered that leads to the sequential oxidation on carbon 21 resulting in HDOPA. The latter was then reduced to the end product 20α-DHOPA. Hepatic cytochromes P450, aldehyde dehydrogenase (ALDH3A2), and 21-hydroxysteroid dehydrogenase (AKR1C18) were found to be involved in this pathway. Activation of PPARα resulted in the induction of Aldh3a2 and Akr1c18, both of which were confirmed as target genes through introduction of promoter luciferase reporter constructs into mouse livers in vivo. This study underscores the power of mass spectrometry-based metabolomics combined with genomic and physiologic analyses in identifying downstream metabolic biomarkers and the corresponding upstream molecular mechanisms. 相似文献
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Benoit Pourcet In��s Pineda-Torra Bruno Derudas Bart Staels Corine Glineur 《The Journal of biological chemistry》2010,285(9):5983-5992
The nuclear receptor peroxisome proliferator-activated receptor α (PPARα) is a key regulator of genes implicated in lipid homeostasis and inflammation. PPARα trans-activity is enhanced by recruitment of coactivators such as SRC1 and CBP/p300 and is inhibited by binding of corepressors such as NCoR and SMRT. In addition to ligand binding, PPARα activity is regulated by post-translational modifications such as phosphorylation and ubiquitination. In this report, we demonstrate that hPPARα is SUMOylated by SUMO-1 on lysine 185 in the hinge region. The E2-conjugating enzyme Ubc9 and the SUMO E3- ligase PIASy are implicated in this process. In addition, ligand treatment decreases the SUMOylation rate of hPPARα. Finally, our results demonstrate that SUMO-1 modification of hPPARα down-regulates its trans-activity through the specific recruitment of corepressor NCoR but not SMRT leading to the differential expression of a subset of PPARα target genes. In conclusion, hPPARα SUMOylation on lysine 185 down-regulates its trans-activity through the selective recruitment of NCoR. 相似文献
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Sophie Jamet Jaclyn Bubnell Patrick Pfister Delia Tomoiaga Matthew E. Rogers Paul Feinstein 《PloS one》2015,10(10)
Many G-protein coupled receptors (GPCRs), such as odorant receptors (ORs), cannot be characterized in heterologous cells because of their difficulty in trafficking to the plasma membrane. In contrast, a surrogate OR, the GPCR mouse β2-adrenergic-receptor (mβ2AR), robustly traffics to the plasma membrane. We set out to characterize mβ2AR mutants in vitro for their eventual use in olfactory axon guidance studies. We performed an extensive mutational analysis of mβ2AR using a Green Fluorescent Protein-tagged mβ2AR (mβ2AR::GFP) to easily assess the extent of its plasma membrane localization. In order to characterize mutants for their ability to successfully transduce ligand-initiated signal cascades, we determined the half maximal effective concentrations (EC50) and maximal response to isoprenaline, a known mβ2AR agonist. Our analysis reveals that removal of amino terminal (Nt) N-glycosylation sites and the carboxy terminal (Ct) palmitoylation site of mβ2AR do not affect its plasma membrane localization. By contrast, when both the Nt and Ct of mβ2AR are replaced with those of M71 OR, plasma membrane trafficking is impaired. We further analyze three mβ2AR mutants (RDY, E268A, and C327R) used in olfactory axon guidance studies and are able to decorrelate their plasma membrane trafficking with their capacity to respond to isoprenaline. A deletion of the Ct prevents proper trafficking and abolishes activity, but plasma membrane trafficking can be selectively rescued by a Tyrosine to Alanine mutation in the highly conserved GPCR motif NPxxY. This new loss-of-function mutant argues for a model in which residues located at the end of transmembrane domain 7 can act as a retention signal when unmasked. Additionally, to our surprise, amongst our set of mutations only Ct mutations appear to lower mβ2AR EC50s revealing their critical role in G-protein coupling. We propose that an interaction between the Nt and Ct is necessary for proper folding and/or transport of GPCRs. 相似文献
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Jihee Kim Seungkirl Ahn Keshava Rajagopal Robert J. Lefkowitz 《The Journal of biological chemistry》2009,284(18):11953-11962
Recent studies in receptor-transfected cell lines have demonstrated that
extracellular signal-regulated kinase (ERK) activation by angiotensin type 1A
receptor and other G protein-coupled receptors can be mediated by both G
protein-dependent and β-arrestin-dependent mechanisms. However, few
studies have explored these mechanisms in primary cultured cells expressing
endogenous levels of receptors. Accordingly, here we utilized the
β-arrestin biased agonist for the angiotensin type 1A receptor,
SII-angiotensin (SII), and RNA interference techniques to investigate
angiotensin II (ANG)-activated β-arrestin-mediated mitogenic signaling
pathways in rat vascular smooth muscle cells. Both ANG and SII induced DNA
synthesis via the ERK activation cascade. Even though SII cannot induce
calcium influx (G protein activation) after receptor stimulation, it does
cause ERK activation, although less robustly than ANG. Activation by both
ligands is diminished by depletion of β-arrestin2 by small interfering
RNA, although the effect is more complete with SII. ERK activation at early
time points but not later time points is strongly inhibited by those protein
kinase C inhibitors that can block protein kinase Cζ. Moreover, ANG- and
SII-mediated ERK activation require transactivation of the epidermal growth
factor receptor via metalloprotease 2/9 and Src kinase. β-Arrestin2
facilitates ANG and SII stimulation of Src-mediated phosphorylation of Tyr-845
on the EGFR, a known site for Src phosphorylation. These studies delineate a
convergent mechanism by which G protein-dependent and
β-arrestin-dependent pathways can independently mediate ERK-dependent
transactivation of the EGFR in vascular smooth muscle cells thus controlling
cellular proliferative responses.G protein-coupled receptors, also known as seven transmembrane
(7TM)2 receptors,
control virtually all known physiological processes in mammals
(1). The various functions of
these receptors are mediated and modulated by three families of proteins,
which share the property that they interact virtually universally with the
receptors in a strictly stimulus-dependent way
(1). These three families of
proteins are the heterotrimeric G proteins, the G protein-coupled receptor
kinases (GRKs), and the β-arrestins. Activation of the receptors
stimulates classical G protein-dependent signaling, often involving regulation
of levels of second messengers such as cAMP and diacyglycerol. However, as has
been known for many years, interaction of activated receptors with GRKs
leading to their phosphorylation, and subsequent interaction with
β-arrestins leads to desensitization of G protein signaling.In recent years, however, it has become increasingly clear that the
β-arrestin-GRK system is in fact bifunctional
(2). Thus, even as it
desensitizes G protein signaling by the receptors, it also serves as a signal
transduction system in its own right, activating a growing list of signaling
pathways. These positive signaling functions are often mediated by the ability
of β-arrestin to serve as an adaptor or scaffold molecule, bringing
elements of diverse signaling pathways into proximity with one another and the
receptors and thereby facilitating their activation. This new paradigm for
understanding the previously unrecognized signaling properties of the
β-arrestin-GRK system has been explored in a wide variety of transfected
cultured cell systems.However, to date, relatively little investigation of these novel signaling
pathways has been carried out in primary cell culture systems expressing
endogenous levels of 7TM receptors. In seeking such a system in which to
characterize and compare β-arrestin and G protein-mediated signaling
pathways from a typical 7TM receptor, our attention was drawn to cultured rat
vascular smooth muscle cells (VSMCs). Several features of rat VSMCs suggest
this to be a relevant system for these purposes. Rat VSMCs express a variety
of physiologically important 7TM receptors including the angiotensin II type
1A receptor (AT1R) (3). This
receptor has been the focus of extensive study in transfected cell systems
with respect to its β-arrestin-mediated signaling to a variety of
pathways, most particularly extracellular signal-regulated kinase (ERK).
Moreover, the AT1R mediates the physiologically important effects of
angiotensin II (ANG) on vascular tone as well as on proliferation and
chemotaxis (4,
5). Pathophysiologically, ANG
stimulation of this receptor has been implicated in VSMC proliferation and
chemotaxis, which are thought to play an important role in such important
disease processes as atherosclerosis and restenosis after angioplasty
(6,
7). Moreover, a ligand has been
characterized
[Sar1,Ile4,Ile8](SII)-angiotensin (SII), a
triply mutated angiotensin octapeptide that, in transfected cell systems, acts
as a specific agonist for β-arrestin-mediated signaling, although not
activating G protein-mediated signaling
(8).Accordingly, in the studies described here, we set out to investigate the
characteristics of activation of ERK in rat VSMCs that might be mediated
through G protein as well as β-arrestin signaling. The results not only
demonstrate the importance of β-arrestin-mediated signaling in
ERK-mediated proliferative responses of these cells, but also shed new light
on the molecular mechanisms and interrelationships between the β-arrestin
and classical G protein-mediated activation of these pathways. 相似文献
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Verónica García-Alonso Cristina López-Vicario Esther Titos Eva Morán-Salvador Ana González-Périz Bibiana Rius Marcelina Párrizas Oliver Werz Vicente Arroyo Joan Clària 《The Journal of biological chemistry》2013,288(39):28230-28242
Peroxisome proliferator-activated receptor γ (PPARγ) is a ligand-activated nuclear receptor and a master regulator of adipogenesis. Microsomal prostaglandin E (PGE) synthase-1 (mPGES-1) is an inducible enzyme that couples with cyclooxygenase-2 for the biosynthesis of PGE2. In this study we demonstrate the existence of a coordinate functional interaction between PPARγ and mPGES-1 in controlling the process of pre-adipocyte differentiation in white adipose tissue (WAT). Adipocyte-specific PPARγ knock-out mice carrying an aP2 promoter-driven Cre recombinase transgene showed a blunted response to the adipogenic effects of a high fat diet. Pre-adipocytes from these knock-out mice showed loss of PPARγ and were resistant to rosiglitazone-induced WAT differentiation. In parallel, WAT from these mice showed increased expression of uncoupling protein 1, a mitochondrial enzyme that dissipates chemical energy as heat. Adipose tissue from mice lacking PPARγ also showed mPGES-1 up-regulation and increased PGE2 levels. In turn, PGE2 suppressed PPARγ expression and blocked rosiglitazone-induced pre-adipocyte differentiation toward white adipocytes while directly elevating uncoupling protein 1 expression and pre-adipocyte differentiation into mature beige/brite adipocytes. Consistently, pharmacological mPGES-1 inhibition directed pre-adipocyte differentiation toward white adipocytes while suppressing differentiation into beige/brite adipocytes. This browning effect was reproduced in knockdown experiments using a siRNA directed against mPGES-1. The effects of PGE2 on pre-adipocyte differentiation were not seen in mice lacking PPARγ in adipose tissue and were not mirrored by other eicosanoids (i.e. leukotriene B4). Taken together, these findings identify PGE2 as a key regulator of white-to-brown adipogenesis and suggest the existence of a coordinate regulation of adipogenesis between PPARγ and mPGES-1. 相似文献
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Jason D. Hoffert Chung-Lin Chou Mark A. Knepper 《The Journal of biological chemistry》2009,284(22):14683-14687
Vasopressin controls renal water excretion largely through actions to
regulate the water channel aquaporin-2 in collecting duct principal cells. Our
knowledge of the mechanisms involved has increased markedly in recent years
with the advent of methods for large-scale systems-level profiling such as
protein mass spectrometry, yeast two-hybrid analysis, and oligonucleotide
microarrays. Here we review this progress.Regulation of water excretion by the kidney is one of the most visible
aspects of everyday physiology. An outdoor tennis game on a hot summer day can
result in substantial water losses by sweating, and the kidneys respond by
reducing water excretion. In contrast, excessive intake of water, a frequent
occurrence in everyday life, results in excretion of copious amounts of clear
urine. These responses serve to exact tight control on the tonicity of body
fluids, maintaining serum osmolality in the range of 290–294 mosmol/kg
of H2O through the regulated return of water from the pro-urine in
the renal collecting ducts to the bloodstream.The importance of this process is highlighted when the regulation fails.
For example, polyuria (rapid uncontrolled excretion of water) is a sometimes
devastating consequence of lithium therapy for bipolar disorder. On the other
side of the coin are water balance disorders that result from excessive renal
water retention causing systemic hypo-osmolality or hyponatremia. Hyponatremia
due to excessive water retention can be seen with severe congestive heart
failure, hepatic cirrhosis, and the syndrome of inappropriate
antidiuresis.The chief regulator of water excretion is the peptide hormone
AVP,2 whereas the
chief molecular target for regulation is the water channel AQP2. In this
minireview, we describe new progress in the understanding of the molecular
mechanisms involved in regulation of AQP2 by AVP in collecting duct cells,
with emphasis on new information derived from “systems-level”
approaches involving large-scale profiling and screening techniques such as
oligonucleotide arrays, protein mass spectrometry, and yeast two-hybrid
analysis. Most of the progress with these techniques is in the identification
of individual molecules involved in AVP signaling and binding interactions
with AQP2. Additional related issues are addressed in several recent reviews
(1–4). 相似文献
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