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1.
2.
Tomoya Isaji Yuya Sato Tomohiko Fukuda Jianguo Gu 《The Journal of biological chemistry》2009,284(18):12207-12216
N-Glycosylation of integrin α5β1 plays a crucial role
in cell spreading, cell migration, ligand binding, and dimer formation, but
the detailed mechanisms by which N-glycosylation mediates these
functions remain unclear. In a previous study, we showed that three potential
N-glycosylation sites (α5S3–5) on the β-propeller of
the α5 subunit are essential to the functional expression of the
subunit. In particular, site 5 (α5S5) is the most important for its
expression on the cell surface. In this study, the function of the
N-glycans on the integrin β1 subunit was investigated using
sequential site-directed mutagenesis to remove the combined putative
N-glycosylation sites. Removal of the N-glycosylation sites
on the I-like domain of the β1 subunit (i.e. the Δ4-6
mutant) decreased both the level of expression and heterodimeric formation,
resulting in inhibition of cell spreading. Interestingly, cell spreading was
observed only when the β1 subunit possessed these three
N-glycosylation sites (i.e. the S4-6 mutant). Furthermore,
the S4-6 mutant could form heterodimers with either α5S3-5 or α5S5
mutant of the α5 subunit. Taken together, the results of the present
study reveal for the first time that N-glycosylation of the I-like
domain of the β1 subunit is essential to both the heterodimer formation
and biological function of the subunit. Moreover, because the
α5S3-5/β1S4-6 mutant represents the minimal
N-glycosylation required for functional expression of the β1
subunit, it might also be useful for the study of molecular structures.Integrin is a heterodimeric glycoprotein that consists of both an α
and a β subunit (1). The
interaction between integrin and the extracellular matrix is essential to both
physiologic and pathologic events, such as cell migration, development, cell
viability, immune homeostasis, and tumorigenesis
(2,
3). Among the integrin
superfamily, β1 integrin can combine with 12 distinct α subunits
(α1–11, αv) to form heterodimers, thereby acquiring a wide
variety of ligand specificity
(1,
4). Integrins are thought to be
regulated by inside-out signaling mechanisms that provoke conformational
changes, which modulate the affinity of integrin for the ligand
(5). However, an increasing
body of evidence suggests that cell-surface carbohydrates mediate a variety of
interactions between integrin and its extracellular environment, thereby
affecting integrin activity and possibly tumor metastasis as well
(6–8).Guo et al. (9)
reported that an increase in β1–6-GlcNAc sugar chains on the
integrin β1 subunit stimulated cell migration. In addition, elevated
sialylation of the β1 subunit, because of Ras-induced STGal-I transferase
activity, also induced cell migration
(10,
11). Conversely, cell
migration and spreading were reduced by the addition of a bisecting GlcNAc,
which is a product of N-acetylglucosaminyltransferase III
(GnT-III),2 to the
α5β1 and α3β1 integrins
(12,
13). Alterations of
N-glycans on integrins might also regulate their cis interactions
with membrane-associated proteins, including the epidermal growth factor
receptor, the galectin family, and the tetraspanin family of proteins
(14–19).In addition to the positive and negative regulatory effects of
N-glycan, several research groups have reported that
N-glycans must be present on integrin α5β1 for the
αβ heterodimer formation and proper integrin-matrix interactions.
Consistent with this hypothesis, in the presence of the glycosylation
inhibitor, tunicamycin, normal integrin-substrate binding and transport to the
cell surface are inhibited
(20). Moreover, treatment of
purified integrin with N-glycosidase F blocked both the inherent
association of the subunits and the interaction between integrin and
fibronectin (FN) (21). These
results suggest that N-glycosylation is essential to the functional
expression of α5β1. However, because integrin α5β1
contains 26 potential N-linked glycosylation sites, 14 in the α
subunit and 12 in the β subunit, identification of the sites that are
essential to its biological functions is key to understanding the molecular
mechanisms by which N-glycans alter integrin function. Recently, our
group determined that N-glycosylation of the β-propeller domain
on the α5 subunit is essential to both heterodimerization and biological
functions of the subunit. Furthermore, we determined that sites 3–5 are
the most important sites for α5 subunit-mediated cell spreading and
migration on FN (22). The
purpose of this study was to clarify the roles of N-glycosylation of
the β1 subunit. Therefore, we performed combined substitutions in the
putative N-glycosylation sites by replacement of asparagine residues
with glutamine residues. We subsequently introduced these mutated genes into
β1-deficient epithelial cells (GE11). The results of these mutation
experiments revealed that the N-glycosylation sites on the I-like
domain of the β1 subunit, sites number 4–6 (S4-6), are essential to
both heterodimer formation and biological functions, such as cell
spreading. 相似文献
3.
4.
Ashley S. Williams Li Kang Jenny Zheng Carrie Grueter Deanna P. Bracy Freyja D. James Ambra Pozzi David H. Wasserman 《The Journal of biological chemistry》2015,290(10):6546-6557
Hepatic insulin resistance is associated with increased collagen. Integrin α1β1 is a collagen-binding receptor expressed on hepatocytes. Here, we show that expression of the α1 subunit is increased in hepatocytes isolated from high fat (HF)-fed mice. To determine whether the integrin α1 subunit protects against impairments in hepatic glucose metabolism, we analyzed glucose tolerance and insulin sensitivity in HF-fed integrin α1-null (itga1−/−) and wild-type (itga1+/+) littermates. Using the insulin clamp, we found that insulin-stimulated hepatic glucose production was suppressed by ∼50% in HF-fed itga1+/+ mice. In contrast, it was not suppressed in HF-fed itga1−/− mice, indicating severe hepatic insulin resistance. This was associated with decreased hepatic insulin signaling in HF-fed itga1−/− mice. Interestingly, hepatic triglyceride and diglyceride contents were normalized to chow-fed levels in HF-fed itga1−/− mice. This indicates that hepatic steatosis is dissociated from insulin resistance in HF-fed itga1−/− mice. The decrease in hepatic lipid accumulation in HF-fed itga1−/− mice was associated with altered free fatty acid metabolism. These studies establish a role for integrin signaling in facilitating hepatic insulin action while promoting lipid accumulation in mice challenged with a HF diet. 相似文献
5.
Toru Yoshihara Kazushi Sugihara Yasuhiko Kizuka Shogo Oka Masahide Asano 《The Journal of biological chemistry》2009,284(18):12550-12561
The glycosylation of glycoproteins and glycolipids is important for central
nervous system development and function. Although the roles of several
carbohydrate epitopes in the central nervous system, including polysialic
acid, the human natural killer-1 (HNK-1) carbohydrate, α2,3-sialic acid,
and oligomannosides, have been investigated, those of the glycan backbone
structures, such as Galβ1-4GlcNAc and Galβ1-3GlcNAc, are not fully
examined. Here we report the generation of mice deficient in
β4-galactosyltransferase-II (β4GalT-II). This galactosyltransferase
transfers Gal from UDP-Gal to a nonreducing terminal GlcNAc to synthesize the
Gal β1-4GlcNAc structure, and it is strongly expressed in the central
nervous system. In behavioral tests, the β4GalT-II-/- mice
showed normal spontaneous activity in a novel environment, but impaired
spatial learning/memory and motor coordination/learning. Immunohistochemistry
showed that the amount of HNK-1 carbohydrate was markedly decreased in the
brain of β4GalT-II-/- mice, whereas the expression of
polysialic acid was not affected. Furthermore, mice deficient in
glucuronyltransferase (GlcAT-P), which is responsible for the biosynthesis of
the HNK-1 carbohydrate, also showed impaired spatial learning/memory as
described in our previous report, although their motor coordination/learning
was normal as shown in this study. Histological examination showed abnormal
alignment and reduced number of Purkinje cells in the cerebellum of
β4GalT-II-/- mice. These results suggest that the
Galβ1-4GlcNAc structure in the HNK-1 carbohydrate is mainly synthesized
by β4GalT-II and that the glycans synthesized by β4GalT-II have
essential roles in higher brain functions, including some that are
HNK-1-dependent and some that are not.The glycosylation of glycoproteins, proteoglycans, and glycolipids is
important for their biological activities, stability, transport, and clearance
from circulation, and cell-surface glycans participate in cell-cell and
cell-extracellular matrix interactions. In the central nervous system, several
specific carbohydrate epitopes, including polysialic acid
(PSA),3 the
human natural killer-1 (HNK-1) carbohydrate, α2,3-sialic acid, and
oligomannosides play indispensable roles in neuronal generation, cell
migration, axonal outgrowth, and synaptic plasticity
(1). Functional analyses of the
glycan backbone structures, like lactosamine core (Galβ1-4GlcNAc),
neolactosamine core (Galβ1-3GlcNAc), and polylactosamine
(Galβ1-4GlcNAcβ1-3) have been carried out using gene-deficient mice
in β4-galactosyltransferase-I (β4GalT-I)
(2,
3), β4GalT-V
(4),
β3-N-acetylglucosaminyl-transferase-II (β3GnT-II)
(5), β3GnT-III
(Core1-β3GnT) (6),
β3GnT-V (7), and Core2GnT
(8). However, the roles of
these glycan backbone structures in the nervous system have not been examined
except the olfactory sensory system
(9).β4GalTs synthesize the Galβ1-4GlcNAc structure via the
β4-galactosylation of glycoproteins and glycolipids; the β4GalTs
transfer galactose (Gal) from UDP-Gal to a nonreducing terminal
N-acetylglucosamine (GlcNAc) of N- and O-glycans
with a β-1,4-linkage. The β4GalT family has seven members
(β4GalT-I to VII), of which at least five have similar
Galβ1-4GlcNAc-synthesizing activities
(10,
11). Each β4GalT has a
tissue-specific expression pattern and substrate specificity with overlapping,
suggesting each β4GalT has its own biological role as well as redundant
functions. β4GalT-I and β4GalT-II share the highest identity (52% at
the amino acid level) among the β4GalTs
(12), suggesting these two
galactosyltransferases can compensate for each other. β4GalT-I is
strongly and ubiquitously expressed in various non-neural tissues, whereas
β4GalT-II is strongly expressed in neural tissues
(13,
14). Indeed, the β4GalT
activity in the brain of β4GalT-I-deficient (β4GalT-I-/-)
mice remains as high as 65% of that of wild-type mice, and the expression
levels of PSA and the HNK-1 carbohydrate in the brain of these mice are normal
(15). These results suggest
β4GalTs other than β4GalT-I, like β4GalT-II, are important in
the nervous system.Among the β4GalT family members, only β4GalT-I-/- mice
have been examined extensively; this was done by us and another group. We
reported that glycans synthesized by β4GalT-I play various roles in
epithelial cell growth and differentiation, inflammatory responses, skin wound
healing, and IgA nephropathy development
(2,
16-18).
Another group reported that glycans synthesized by β4GalT-I are involved
in anterior pituitary hormone function and in fertilization
(3,
19). However, no other nervous
system deficits have been reported in these mice, and the role of the
β4-galactosylation of glycoproteins and glycolipids in the nervous system
has not been fully examined.In this study, we generated β4GalT-II-/- mice and examined
them for behavioral abnormalities and biochemical and histological changes in
the central nervous system. β4GalT-II-/- mice were impaired in
spatial learning/memory and motor coordination/learning. The amount of HNK-1
carbohydrate was markedly decreased in the β4GalT-II-/- brain,
but PSA expression was not affected. These results suggest that the
Galβ1-4GlcNAc structure in the HNK-1 carbohydrate is mainly synthesized
by β4GalT-II and that glycans synthesized by β4GalT-II have
essential roles in higher brain functions, including ones that are HNK-1
carbohydrate-dependent and ones that are independent of HNK-1. 相似文献
6.
Nicolas Dzamko Bryce J. W. van Denderen Andrea L. Hevener Sebastian Beck J?rgensen Jane Honeyman Sandra Galic Zhi-Ping Chen Matthew J. Watt Duncan J. Campbell Gregory R. Steinberg Bruce E. Kemp 《The Journal of biological chemistry》2010,285(1):115-122
The AMP-activated protein kinase (AMPK) is an αβγ heterotrimer that regulates appetite and fuel metabolism. We have generated AMPK β1−/− mice on a C57Bl/6 background that are viable, fertile, survived greater than 2 years, and display no visible brain developmental defects. These mice have a 90% reduction in hepatic AMPK activity due to loss of the catalytic α subunits, with modest reductions of activity detected in the hypothalamus and white adipose tissue and no change in skeletal muscle or heart. On a low fat or an obesity-inducing high fat diet, β1−/− mice had reduced food intake, reduced adiposity, and reduced total body mass. Metabolic rate, physical activity, adipose tissue lipolysis, and lipogenesis were similar to wild type littermates. The reduced appetite and body mass of β1−/− mice were associated with protection from high fat diet-induced hyperinsulinemia, hepatic steatosis, and insulin resistance. We demonstrate that the loss of β1 reduces food intake and protects against the deleterious effects of an obesity-inducing diet. 相似文献
7.
Xiaoquan Rao Jixin Zhong Xiaohua Xu Brianna Jordan Santosh Maurya Zachary Braunstein Tse-Yao Wang Wei Huang Sudha Aggarwal Muthu Periasamy Sanjay Rajagopalan Kamal Mehta Qinghua Sun 《PloS one》2013,8(12)
Physical exercise is an important and effective therapy for diabetes. However, its underlying mechanism is not fully understood. Protein kinase Cβ (PKCβ) has been suggested to be involved in the pathogenesis of obesity and insulin resistance, but the role of PKCβ in exercise-induced improvements in insulin resistance is completely unknown. In this study, we evaluated the involvement of PKCβ in exercise-attenuated insulin resistance in high-fat diet (HFD)-fed mice. PKCβ-/- and wild-type mice were fed a HFD with or without exercise training. PKC protein expression, body and tissue weight change, glucose and insulin tolerance, metabolic rate, mitochondria size and number, adipose inflammation, and AKT activation were determined to evaluate insulin sensitivity and metabolic changes after intervention. PKCβ expression decreased in both skeletal muscle and liver tissue after exercise. Exercise and PKCβ deficiency can alleviate HFD-induced insulin resistance, as evidenced by improved insulin tolerance. In addition, fat accumulation and mitochondrial dysfunction induced by HFD were also ameliorated by both exercise and PKCβ deficiency. On the other hand, exercise had little effect on PKCβ-/- mice. Further, our data indicated improved activation of AKT, the downstream signal molecule of insulin, in skeletal muscle and liver of exercised mice, whereas PKCβ deficiency blunted the difference between sedentary and exercised mice. These results suggest that downregulation of PKCβ contributes to exercise-induced improvement of insulin resistance in HFD-fed mice. 相似文献
8.
Yuya Sato Tomoya Isaji Michiko Tajiri Shumi Yoshida-Yamamoto Tsuyoshi Yoshinaka Toshiaki Somehara Tomohiko Fukuda Yoshinao Wada Jianguo Gu 《The Journal of biological chemistry》2009,284(18):11873-11881
Recently we reported that N-glycans on the β-propeller domain
of the integrin α5 subunit (S-3,4,5) are essential for α5β1
heterodimerization, expression, and cell adhesion. Herein to further
investigate which N-glycosylation site is the most important for the
biological function and regulation, we characterized the S-3,4,5 mutants in
detail. We found that site-4 is a key site that can be specifically modified
by N-acetylglucosaminyltransferase III (GnT-III). The introduction of
bisecting GlcNAc into the S-3,4,5 mutant catalyzed by GnT-III decreased cell
adhesion and migration on fibronectin, whereas overexpression of
N-acetylglucosaminyltransferase V (GnT-V) promoted cell migration.
The phenomenon is similar to previous observations that the functions of the
wild-type α5 subunit were positively and negatively regulated by GnT-V
and GnT-III, respectively, suggesting that the α5 subunit could be
duplicated by the S-3,4,5 mutant. Interestingly GnT-III specifically modified
the S-4,5 mutant but not the S-3,5 mutant. This result was confirmed by
erythroagglutinating phytohemagglutinin lectin blot analysis. The reduction in
cell adhesion was consistently observed in the S-4,5 mutant but not in the
S-3,5 mutant cells. Furthermore mutation of site-4 alone resulted in a
substantial decrease in erythroagglutinating phytohemagglutinin lectin
staining and suppression of cell spread induced by GnT-III compared with that
of either the site-3 single mutant or wild-type α5. These results, taken
together, strongly suggest that N-glycosylation of site-4 on the
α5 subunit is the most important site for its biological functions. To
our knowledge, this is the first demonstration that site-specific modification
of N-glycans by a glycosyltransferase results in functional
regulation.Glycosylation is a crucial post-translational modification of most secreted
and cell surface proteins (1).
Glycosylation is involved in a variety of physiological and pathological
events, including cell growth, migration, differentiation, and tumor invasion.
It is well known that glycans play important roles in cell-cell communication,
intracellular signal transduction, protein folding, and stability
(2,
3).Integrins comprise a family of receptors that are important for cell
adhesion. The major function of integrins is to connect cells to the
extracellular matrix, activate intracellular signaling pathways, and regulate
cytoskeletal formation (4).
Integrin α5β1 is well known as a fibronectin
(FN)3 receptor. The
interaction between integrin α5 and FN is essential for cell migration,
cell survival, and development
(5–8).
In addition, integrins are N-glycan carrier proteins. For example,
α5β1 integrin contains 14 and 12 putative N-glycosylation
sites on the α5 and β1 subunits, respectively. Several studies
suggest that N-glycosylation is essential for functional integrin
α5β1. When human fibroblasts were cultured in the presence of
1-deoxymannojirimycin, which prevents N-linked oligosaccharide
processing, immature α5β1 integrin appeared on the cell surface,
and FN-dependent adhesion was greatly reduced
(9). Treatment of purified
integrin α5β1 with N-glycosidase F, which cleaves between
the innermost N-acetylglucosamine (GlcNAc) and asparagine
N-glycan residues of N-linked glycoproteins, prevented the
inherent association between subunits and blocked α5β1 binding to
FN (10).A growing body of evidence indicates that the presence of the appropriate
oligosaccharide can modulate integrin activation.
N-Acetylglucosaminyltransferase III (GnT-III) catalyzes the addition
of GlcNAc to mannose that is β1,4-linked to an underlying
N-acetylglucosamine, producing what is known as a
“bisecting” GlcNAc linkage as shown in
Fig. 1B. GnT-III is
generally regarded as a key glycosyltransferase in N-glycan
biosynthetic pathways and contributes to inhibition of metastasis. The
introduction of a bisecting GlcNAc catalyzed by GnT-III suppresses additional
processing and elongation of N-glycans. These reactions, which are
catalyzed in vitro by other glycosyltransferases, such as
N-acetylglucosaminyltransferase V (GnT-V), which catalyzes the
formation of β1,6 GlcNAc branching structures
(Fig. 1B) and plays
important roles in tumor metastasis, do not proceed because the enzymes cannot
utilize the bisected N-glycans as a substrate. Introduction of the
bisecting GlcNAc to integrin α5 by overexpression of GnT-III resulted in
decreased in ligand binding and down-regulation of cell adhesion and migration
(11–13).
Contrary to the functions of GnT-III, overexpression of GnT-V promoted
integrin α5β1-mediated cell migration on FN
(14). These observations
clearly demonstrate that the alteration of N-glycan structure
affected the biological functions of integrin α5β1. Similarly
characterization of the carbohydrate moieties in integrin α3β1 from
non-metastatic and metastatic human melanoma cell lines showed that expression
of β1,6 GlcNAc branched structures was higher in metastatic cells
compared with non-metastatic cells, confirming the notion that the β1,6
GlcNAc branched structure confers invasive and metastatic properties to cancer
cells. In fact, Partridge et al.
(15) reported that
GnT-V-modified N-glycans containing
poly-N-acetyllactosamine, the preferred ligand for galectin-3, on
surface receptors oppose their constitutive endocytosis, promoting
intracellular signaling and consequently cell migration and tumor
metastasis.Open in a separate windowFIGURE 1.Potential N-glycosylation sites on the α5 subunit and its
modification by GnT-III and GnT-V. A, schematic diagram of
potential N-glycosylation sites on the α5 subunit. Putative
N-glycosylation sites are indicated by triangles, and point
mutations are indicated by crosses (N84Q, N182Q, N297Q, N307Q, N316Q,
N524Q, N530Q, N593Q, N609Q, N675Q, N712Q, N724Q, N773Q, and N868Q).
B, illustration of the reaction catalyzed by GnT-III and GnT-V.
Square, GlcNAc; circle, mannose. TM, transmembrane
domain.In addition, sialylation on the non-reducing terminus of N-glycans
of α5β1 integrin plays an important role in cell adhesion. Colon
adenocarcinomas express elevated levels of α2,6 sialylation and
increased activity of ST6GalI sialyltransferase. Elevated ST6GalI positively
correlated with metastasis and poor survival. Therefore, ST6GalI-mediated
hypersialylation likely plays a role in colorectal tumor invasion
(16,
17). In fact, oncogenic
ras up-regulated ST6GalI and, in turn, increased sialylation of
β1 integrin adhesion receptors in colon epithelial cells
(18). However, this is not
always the case. The expression of hyposialylated integrin α5β1 was
induced by phorbol esterstimulated differentiation in myeloid cells in which
the expression of the ST6GalI was down-regulated by the treatment, increasing
FN binding (19). A similar
phenomenon was also observed in hematopoietic or other epithelial cells. In
these cells, the increased sialylation of the β1 integrin subunit was
correlated with reduced adhesiveness and metastatic potential
(20–22).
In contrast, the enzymatic removal of α2,8-linked oligosialic acids from
the α5 integrin subunit inhibited cell adhesion to FN
(23). Collectively these
findings suggest that the interaction of integrin α5β1 with FN is
dependent on its N-glycosylation and the processing status of
N-glycans.Because integrin α5β1 contains multipotential
N-glycosylation sites, it is important to determine the sites that
are crucial for its biological function and regulation. Recently we found that
N-glycans on the β-propeller domain (sites 3, 4, and 5) of the
integrin α5 subunit are essential for α5β1
heterodimerization, cell surface expression, and biological function
(24). In this study, to
further investigate the underlying molecular mechanism of GnT-III-regulated
biological functions, we characterized the N-glycans on the α5
subunit in detail using genetic and biochemical approaches and found that
site-4 is a key site that can be specifically modified by GnT-III. 相似文献
9.
10.
Norihisa Nishimichi Fumiko Higashikawa Hiromi H. Kinoh Yoshiko Tateishi Haruo Matsuda Yasuyuki Yokosaki 《The Journal of biological chemistry》2009,284(22):14769-14776
Osteopontin (OPN) is a cytokine and ligand for multiple members of the
integrin family. OPN undergoes the in vivo polymerization catalyzed
by cross-linking enzyme transglutaminase 2, which consequently increases the
bioactivity through enhanced interaction with integrins. The integrin
α9β1, highly expressed on neutrophils, binds to the sequence
SVVYGLR only after intact OPN is cleaved by thrombin. The SVVYGLR sequence
appears to be cryptic in intact OPN because α9β1 does not recognize
intact OPN. Because transglutaminase 2-catalyzed polymers change their
physical and chemical properties, we hypothesized that the SVVYGLR site might
also be exposed on polymeric OPN. As expected, α9β1 turned into a
receptor for polymeric OPN, a result obtained by cell adhesion and migration
assays with α9-transfected cells and by detection of direct binding of
recombinant soluble α9β1 with colorimetry and surface plasmon
resonance analysis. Because the N-terminal fragment of thrombin-cleaved OPN, a
ligand for α9β1, has been reported to attract neutrophils, we next
examined migration of neutrophils to polymeric OPN using time-lapse
microscopy. Polymeric OPN showed potent neutrophil chemotactic activity, which
was clearly inhibited by anti-α9β1 antibody. Unexpectedly,
mutagenesis studies showed that α9β1 bound to polymeric OPN
independently of the SVVYGLR sequence, and further, SVVYGLR sequence of
polymeric OPN was cryptic because SVVYGLR-specific antibody did not recognize
polymeric OPN. These results demonstrate that polymerization of OPN generates
a novel α9β1-binding site and that the interaction of this site
with the α9β1 integrin is critical to the neutrophil chemotaxis
induced by polymeric OPN.Acidic phosphorylated secreted glycoprotein osteopontin
(OPN),4 known as a
cytokine, has multiple functions, including roles in tissue remodeling,
fibrosis, mineralization, immunomodulation, inflammation, and tumor metastasis
(1–3).
OPN is also an integrin ligand. At least nine integrins can function as OPN
receptors. α5β1, α8β1, αvβ1, αvβ3,
αvβ5 (1), and
αvβ6 (4) recognize
the linear tripeptide RGD, and α9β1, α4β1, and
α4β7 recognize the sequence, SVVYGLR
(5), adjacent to RGD but only
after OPN has been cleaved by the protease, thrombin
(Fig. 1).Open in a separate windowFIGURE 1.Schematic diagram of OPN. Two integrin-binding sites
(boxed), a thrombin cleavage site (arrow), and a putative
transglutamination site (circled) are shown. The term
thrombin-cleaved nOPN is defined as in the figure.The overlap of receptors for OPN does not necessarily mean that these
integrins play redundant roles in cellular responses to OPN because the
patterns of integrin expression and utilization vary widely among cell types.
In addition, interactions of different integrins with a single ligand can
exert distinct effects on cell behavior in a single cell type. For example, we
have previously reported that signals by ligation of αvβ3,
αvβ6, or α9β1 to a single ligand, tenascin-C,
differently affected cell adhesion, spreading, and proliferation of the colon
cancer cell line, SW480 (6).
Furthermore, intact OPN or thrombin- or matrix metalloproteinase-cleaved OPN
interact with distinct subsets of integrins and exhibit distinct effects on
cell behavior (4,
7,
8). Collectively, some of the
functional diversity of OPN could be attributed to this multiplicity of
receptors and responses. We have recently shown that polymerization of OPN
results in enhanced biological activity
(9). We thus set out to
determine whether polymerized OPN exerts its effects through unique
interactions with integrins.OPN is polymerized by transglutaminase 2 (TG2, EC 2.3.2.13)
(10) that catalyzes formation
of isopeptide cross-links between glutamine and lysine residues in substrate
proteins (11) including OPN.
Polymeric OPN has been identified in vivo in bone
(12) and calcified aorta
(13). We have previously
reported that upon polymerization, OPN displays increased integrin binding
accompanied by enhanced cell adhesion, spreading, migration, and focal contact
formation (9). However, very
little is known about how polymeric OPN induces its biological effects.Integrin α9β1, highly expressed on neutrophils
(14), does not act as a
receptor for intact OPN but does bind to an N-terminal fragment of OPN (nOPN)
that is generated by thrombin cleavage
(15) through the new
C-terminal sequence, SVVYGLR. Protein polymerization can expose otherwise
cryptic domains (16), so we
hypothesized that the SVVYGLR site might be exposed upon polymerization and
serve as a binding site for α9β1. In the present study, we
demonstrate that α9β1 is indeed a receptor for polymeric OPN and
that neutrophil migration induced by polymeric OPN is largely mediated by this
interaction. However, mutational analysis and antibody studies demonstrate
that this interaction does not involve the SVVYGLR site, suggesting the
presence of de novo binding site in polymeric OPN. 相似文献
11.
12.
13.
Lei Zhang Hui Zhao Yu Qiu Horace H. Loh Ping-Yee Law 《The Journal of biological chemistry》2009,284(4):1990-2000
Recent studies have revealed that in G protein-coupled receptor signalings
switching between G protein- and β-arrestin (βArr)-dependent
pathways occurs. In the case of opioid receptors, the signal is switched from
the initial inhibition of adenylyl cyclase (AC) to an increase in AC activity
(AC activation) during prolonged agonist treatment. The mechanism of such AC
activation has been suggested to involve the switching of G proteins activated
by the receptor, phosphorylation of signaling molecules, or receptor-dependent
recruitment of cellular proteins. Using protein kinase inhibitors, dominant
negative mutant studies and mouse embryonic fibroblast cells isolated from Src
kinase knock-out mice, we demonstrated that μ-opioid receptor
(OPRM1)-mediated AC activation requires direct association and activation of
Src kinase by lipid raft-located OPRM1. Such Src activation was independent of
βArr as indicated by the ability of OPRM1 to activate Src and AC after
prolonged agonist treatment in mouse embryonic fibroblast cells lacking both
βArr-1 and -2. Instead the switching of OPRM1 signals was dependent on
the heterotrimeric G protein, specifically Gi2 α-subunit.
Among the Src kinase substrates, OPRM1 was phosphorylated at Tyr336
within NPXXY motif by Src during AC activation. Mutation of this Tyr
residue, together with mutation of Tyr166 within the DRY motif to
Phe, resulted in the complete blunting of AC activation. Thus, the recruitment
and activation of Src kinase by OPRM1 during chronic agonist treatment, which
eventually results in the receptor tyrosine phosphorylation, is the key for
switching the opioid receptor signals from its initial AC inhibition to
subsequent AC activation.Classical G protein-coupled receptor
(GPCR)2 signaling
involves the activation of specific heterotrimeric G proteins and the
subsequent dissociation of α- and βγ-subunits. These G
protein subunits serve as the activators and/or inhibitors of several effector
systems, including adenylyl cyclases, phospholipases, and ion channels
(1). However, recent studies
have shown that GPCR signaling deviates from such a classical linear model.
For example, in kidney and colonic epithelial cells, protease-activated
receptor 1 can transduce its signals through either Gαi/o or
Gαq subunits via inhibition of small GTPase RhoA or
activation of RhoD. Thus, RhoA and RhoD act as molecular switches between the
negative and positive signaling activity of protease-activated receptor 1
(2). Another example is the
ability of β2-adrenergic receptor to switch from
Gs-dependent pathways to non-classical signaling pathways by
coupling to pertussis toxin-sensitive Gi proteins in a
cAMP-dependent protein kinase/protein kinase C phosphorylation-dependent
manner. In this case, the phosphorylation-induced switch in G protein coupling
provides the receptor access to alternative signaling pathways. For
β2-adrenergic receptors, this leads to a
Gi-dependent activation of MAP kinase
(3,
4). Furthermore the involvement
of protein scaffolds, such as β-arrestins in the MAP kinase cascade,
could also alter the GPCR signaling
(5–8).
Hence the formation of “signaling units” or
“receptosomes” would influence the GPCR signaling process and
destination.For opioid receptors, which are members of the rhodopsin GPCR subfamily
receptors, signal switching is also observed. Normally opioid receptors
inhibit AC activity, activate the MAP kinases and Kir3 K+ channels,
inhibit the voltage-dependent Ca2+ channels, and regulate other
effectors such as phospholipase C
(9). However, during prolonged
agonist treatment, not only is there a blunting of these cellular responses
but also a compensatory increase in intracellular cAMP level, which is
particularly significant upon the removal of the agonist or the addition of an
antagonist such as naloxone
(10–12).
This compensatory adenylyl cyclase activation phenomenon has been postulated
to be responsible for the development of drug tolerance and dependence
(13). The observed change from
receptor-mediated AC inhibition to receptor-mediated AC activation reflects
possible receptor signal switching. Although the exact mechanism for such
signal changes has yet to be elucidated, activation of specific protein
kinases and subsequent phosphorylation of AC isoforms
(14,
15) and other signaling
molecules (16) have been
suggested to be the key for observed AC activation. Among all the protein
kinases studied, involvement of protein kinase C, MAP kinase, and Raf-1 has
been implicated in the activation of AC
(17–19).
Alternative mechanisms, such as agonist-induced receptor internalization and
the increase in the constitutive activities of the receptor, also have been
suggested to play a role in increased AC activity after prolonged opioid
agonist treatment (20).
Earlier studies also implicated the switching of the opioid receptor from
Gi/Go to Gs coupling during chronic agonist
treatment (21). Regardless of
the mechanism, the exact molecular events that lead to the switching of opioid
receptor from an inhibitory response to a stimulatory response remain
elusive.Src kinases, which are members of the nonreceptor tyrosine kinase family,
have been implicated in GPCR function because several Src family members such
as cSrc, Fyn, and Yes have been reported to be activated by several GPCRs,
including β2-
(22) and β3
(23)-adrenergic,
M2- (24) and
M3 (25)-muscarinic,
and bradykinin receptors (26).
The GPCRs that are capable of activating Src predominantly couple to
Gi/o family G proteins
(27). Src kinases appear to
associate with, and be activated by, GPCRs themselves either through direct
interaction with intracellular receptor domains or by binding to
GPCR-associated proteins, such as G protein subunits or β-arrestins
(27). Src kinase has been
reported to be activated by κ-
(28) and δ
(29)-opioid receptors and
regulate the c-Jun kinase and MAP kinase activities. Src kinase within the
nucleus accumbens has been implicated in the rewarding effect and
hyperlocomotion induced by morphine in mice
(30). However, it is not clear
whether the Src kinase is activated and involved in the signal transduction in
AC activation after chronic opioid agonist administration.Previously we reported that the lipid raft location of the receptor and the
Gαi2 proteins are two prerequisites for the observed increase
in AC activity during prolonged agonist treatment
(31,
32). Because various protein
kinases including Src kinases and G proteins have been shown to be enriched in
lipid rafts (33), the roles of
these cellular proteins in the eventual switching of opioid receptor signals
from inhibition to stimulation of AC activity were examined in the current
studies. We were able to demonstrate that the association with and subsequent
activation of Src kinase by the μ-opioid receptor (OPRM1), which leads to
eventual tyrosine phosphorylation of OPRM1, are the cellular events required
for the switching of opioid receptor signaling upon chronic agonist
treatment. 相似文献
14.
Joseph Larner 《Experimental diabetes research》2002,3(1):47-60
In this review we discuss the biological significance
of D-chiro-inositol, originally discovered
as a component of a putative mediator of
intracellular insulin action, where as a putative
mediator, it accelerates the dephosphorylation
of glycogen synthase and pyruvate dehydrogenase,
rate limiting enzymes of non-oxidative
and oxidative glucose disposal.Early studies demonstrated a linear relationship
between its decreased urinary excretion
and the degree of insulin resistance present.
When tissue contents, including muscle, of type
2 diabetic subjects were assayed, they demonstrated
a more general body deficiency.
Administration of D-chiro-inositol to diabetic
rats, Rhesus monkeys and now to humans
accelerated glucose disposal and sensitized
insulin action.A defect in vivo in the epimerization of myoinositol to chiro-inositol in insulin sensitive tissues
of the GK type 2 diabetic rat has been elucidated.
Thus, administered D-chiro-inositol
may act to bypass a defective normal epimerization
of myo-inositol to D-chiro-inositol
associated with insulin resistance and act to at
least partially restore insulin sensitivity and glucose
disposal. 相似文献
15.
B��atrice Bailly-Maitre Bengt F. Belgardt Sabine D. Jordan Beatrice Coornaert Miriam John von Freyend Andre Kleinridders Jan Mauer Michael Cuddy Christina L. Kress Diana Willmes Manuela Essig Brigitte Hampel Ulrike Protzer John C. Reed Jens C. Br��ning 《The Journal of biological chemistry》2010,285(9):6198-6207
16.
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
17.
L-isoaspartyl (D-aspartyl) O-methyltransferase deficient mice (Pcmt1−/−) accumulate isomerized aspartyl residues in intracellular proteins until their death due to seizures at approximately 45 days. Previous studies have shown that these mice have constitutively activated insulin signaling in their brains, and that these brains are 20–30% larger than those from age-matched wild-type animals. To determine whether insulin pathway activation and brain enlargement is responsible for the fatal seizures, we administered wortmannin, an inhibitor of the phosphoinositide 3-kinase that catalyzes an early step in the insulin pathway. Oral wortmannin reduced the average brain size in the Pcmt1−/− animals to within 6% of the wild-type DMSO administered controls, and nearly doubled the lifespan of Pcmt1−/− at 60% survival of the original population. Immunoblotting revealed significant decreases in phosphorylation of Akt, PDK1, and mTOR in Pcmt1−/− mice and Akt and PDK1 in wild-type animals upon treatment with wortmannin. These data suggest activation of the insulin pathway and its resulting brain enlargement contributes to the early death of Pcmt1−/− mice, but is not solely responsible for the early death observed in these animals. 相似文献
18.
Karen Vanhoorelbeke Simon F. De Meyer Inge Pareyn Chantal Melchior Sebastien Plan?on Christiane Margue Olivier Pradier Pierre Fondu Nelly Kieffer Timothy A. Springer Hans Deckmyn 《The Journal of biological chemistry》2009,284(22):14914-14920
Three heterozygous mutations were identified in the genes encoding platelet
integrin receptor αIIbβ3 in a patient with an ill defined platelet
disorder: one in the β3 gene (S527F) and two in the αIIb gene
(R512W and L841M). Five stable Chinese hamster ovary cell lines were
constructed expressing recombinant αIIbβ3 receptors bearing the
individual R512W, L841M, or S527F mutation; both the R512W and L841M
mutations; or all three mutations. All receptors were expressed on the cell
surface, and mutations R512W and L841M had no effect on integrin function.
Interestingly, the β3 S527F mutation produced a constitutively active
receptor. Indeed, both fibrinogen and the ligand-mimetic antibody PAC-1 bound
to non-activated αIIbβ3 receptors carrying the S527F mutation,
indicating that the conformation of this receptor was altered and corresponded
to the high affinity ligand binding state. In addition, the conformational
change induced by S527F was evident from basal anti-ligand-induced binding
site antibody binding to the receptor. A molecular model bearing this mutation
was constructed based on the crystal structure of αIIbβ3 and
revealed that the S527F mutation, situated in the third integrin epidermal
growth factor-like (I-EGF3) domain, hindered the αIIbβ3 receptor
from adopting a wild type-like bent conformation. Movement of I-EGF3 into a
cleft in the bent conformation may be hampered both by steric hindrance
between Phe527 in β3 and the calf-1 domain in αIIb and
by decreased flexibility between I-EGF2 and I-EGF3.The platelet receptor αIIbβ3 belongs to the family of integrin
receptors that consist of noncovalently linked α/β-heterodimers.
They are cell-surface receptors that play a role in cell-cell and cell-matrix
interactions. Under resting conditions, integrin receptors adopt the low
affinity conformation and do not interact with their ligands. Inside-out
signaling turns the receptor into a high affinity conformation capable of
ligand binding. Ligand binding itself induces additional conformational
changes resulting in exposure of neoantigenic sites called ligand-induced
binding sites (LIBS)3
and generates in turn outside-in signaling, which triggers a range of
downstream signals (1,
2).Integrin αIIbβ3 is expressed on platelets and megakaryocytes. In
flowing blood under resting conditions, αIIbβ3 does not interact
with its ligand fibrinogen. When a blood vessel is damaged, platelets adhere
at sites of vascular injury and become activated. As a consequence,
αIIbβ3 adopts the high affinity conformation and binds fibrinogen.
This results in platelet aggregation and thrombus formation, which eventually
will stop the bleeding (3).The topology of integrins comprises an extracellular, globular, N-terminal
ligand-binding head domain (the β-propeller domain in the αIIb
chain and the βI domain in the β3 chain) standing on two long legs
or stalks (consisting of thigh, calf-1, and calf-2 domains in the αIIb
chain and hybrid, plexin/semaphorin/integrin (PSI), four integrin endothelial
growth factor-like (I-EGF), and β-tail domains in the β3 chain),
followed by transmembrane and cytoplasmic domains
(1,
2). X-ray crystal structures of
the extracellular domain of non-activated αVβ3 revealed that the
legs are severely bent, putting the head domain next to the membrane-proximal
portions of the legs (4,
5). The bending occurs between
I-EGF1 and I-EGF2 in the β-subunit and between the thigh and calf-1
domains in the α-subunit. This bent conformation represents the low
affinity state of the receptor. The high affinity state of the receptor is
induced by activation and is associated with a large-scale conformational
rearrangement in which the integrin extends with a switchblade-like motion
(2). Recently, the crystal
structure of the entire extracellular domain of αIIbβ3 in its low
affinity conformation was resolved and revealed that this integrin also adopts
the bent conformation under resting conditions
(6). Structural rearrangements
in αIIbβ3 between the bent and extended conformations are similar
to what has been reported for other integrins
(7).We report here that the S527F mutation in the I-EGF3 region of the β3
polypeptide chain of the αIIbβ3 receptor induces a constitutively
active receptor adopting an extended high affinity conformation. This was
evidenced by spontaneous PAC-1, fibrinogen, and anti-LIBS antibody binding.
These data were further corroborated by modeling the replacement of
Ser527 with Phe in the crystal structure of the extracellular
domain of αIIbβ3. In this model, the S527F mutation decreases the
flexibility of I-EGF3 and appears to prevent movement of the lower β-leg
into the cleft between the upper β-leg and the lower α-leg. As a
consequence, formation of the bent conformation of the non-activated receptor
is hampered. 相似文献
19.
Vertebrates produce at least seven distinct β-tubulin isotypes that
coassemble into all cellular microtubules. The functional differences among
these tubulin isoforms are largely unknown, but recent studies indicate that
tubulin composition can affect microtubule properties and cellular
microtubule-dependent behavior. One of the isotypes whose incorporation causes
the largest change in microtubule assembly is β5-tubulin. Overexpression
of this isotype can almost completely destroy the microtubule network, yet it
appears to be required in smaller amounts for normal mitotic progression.
Moderate levels of overexpression can also confer paclitaxel resistance.
Experiments using chimeric constructs and site-directed mutagenesis now
indicate that the hypervariable C-terminal region of β5 plays no role in
these phenotypes. Instead, we demonstrate that two residues found in β5
(Ser-239 and Ser-365) are each sufficient to inhibit microtubule assembly and
confer paclitaxel resistance when introduced into β1-tubulin; yet the
single mutation of residue Ser-239 in β5 eliminates its ability to confer
these phenotypes. Despite the high degree of conservation among β-tubulin
isotypes, mutations affecting residue 365 demonstrate that amino acid
substitutions can be context sensitive; i.e. an amino acid change in
one isotype will not necessarily produce the same phenotype when introduced
into a different isotype. Modeling studies indicate that residue Cys-239 of
β1-tubulin is close to a highly conserved Cys-354 residue suggesting the
possibility that disulfide formation could play a significant role in the
stability of microtubules formed with β1- but not with
β5-tubulin.Microtubules are needed to organize the Golgi apparatus and endoplasmic
reticulum, maintain cell shape, construct ciliary and flagellar axonemes, and
ensure the accurate segregation of genetic material prior to cell division.
These cytoskeletal structures assemble from α- and β-tubulin
heterodimers to form long cylindrical filaments that exist in a state of
dynamic equilibrium characterized by stochastic episodes of slow growth and
rapid shrinkage (1). Impairment
of normal dynamic behavior has serious consequences for cell proliferation and
thus makes microtubules an attractive target for drug development
(2).Vertebrates express multiple β-tubulin genes that produce highly
homologous proteins differing most notably in their C-terminal 15–20
amino acids (3,
4). These variable C-terminal
sequences are conserved across vertebrate species and have been used to
classify β-tubulin genes into distinct isotypes
(5). In mammals, for example,
there are seven known isotypes designated by the numbers I, II, III, IVa, IVb,
V, and VI. The functional significance of the C-terminal sequences is
uncertain, but some studies suggest that they may be involved in binding or
modulating the action of microtubule-interacting proteins
(6–14).
Additional amino acid differences are scattered throughout the primary
sequence, but the functional role of these differences, if any, has not been
elucidated. Although some β-tubulin isotypes are expressed in a
tissue-specific manner (3),
evidence indicates that microtubules incorporate all available isotypes,
including transfected isotypes that are not normally produced in those cells
(5,
15–17).
Genetic experiments designed to test potential functional differences among
the various β-tubulin isotypes have only demonstrated isotype-specific
effects on the assembly of specialized microtubule-containing structures such
as flagellar axonemes in Drosophila or 15-protofilament microtubules
in Caenorhabditis elegans
(18,
19). Thus, the consequences,
if any, of producing multiple β-tubulin isoforms in vertebrate organisms
remain elusive.Our recent work showed that conditional overexpression of isotypes β1,
β2, and β4b has no effect on microtubule assembly or drug
sensitivity in transfected Chinese hamster ovary
(CHO)2 cells
(20). Similarly, expression of
neuronal-specific β4a produced very minor effects on microtubule assembly
but was able to increase sensitivity to paclitaxel, most likely through
increased binding of the drug
(21). On the other hand, high
expression of neuronal-specific β3 reduced microtubule assembly,
conferred low level resistance to paclitaxel, and inhibited cell growth
(22). The most dramatic
effects, however, were seen in cells transfected with β5, a minor but
widely expressed isotype (23).
Even modest overexpression of this isotype reduced microtubule assembly and
conferred paclitaxel resistance, whereas high levels of expression (∼50%
of total tubulin) caused fragmentation and a near complete loss of the
microtubule cytoskeleton (24).
Despite the toxicity associated with β5 overexpression, this isotype was
recently shown to be required for normal mitotic progression and cell
proliferation (25).Because of its importance for cell division, and the extreme phenotype
associated with its overexpression, we sought to identify the structural
differences between β5-tubulin and its more “normal” homolog,
β1. Although there are 40 amino acid differences between the 2 isotypes,
we report that most of the unique properties of β5 can be attributed to
the presence of serine in place of cysteine at residue 239. This residue faces
the colchicine binding pocket and is very close to a highly conserved Cys-354
residue. We propose that Ser-239 found in β5-tubulin may prevent
formation of a disulfide bond that normally stabilizes microtubules. 相似文献
20.
Lu Han Meng-Xiong Tang Yun Ti Zhi-Hao Wang Jia Wang Wen-Yuan Ding Hua Wang Yun Zhang Wei Zhang Ming Zhong 《PloS one》2013,8(11)
STAMP2 is a counterregulator of inflammation and insulin resistance. The aim of this study is to investigate whether activation of STAMP2 improves insulin resistance by regulating macrophage polarization in adipose tissues. The diabetic ApoE−/−/LDLR−/− mouse model was induced by high-fat diet and low-dose streptozotocin. Samples were obtained from epididymal, subcutaneous and brown adipose tissues. Infiltration of M1/M2 macrophages and inflammatory cytokines were investigated by immunohistochemistry. We then used gene overexpression to investigate the effect of STAMP2 on macrophages infiltration and polarization and inflammatory cytokines expression. Our results showed that infiltration of macrophages, the ratio of M1/M2 macrophages and the expression of pro-inflammatory cytokines were enhanced and STAMP2 was downregulated in adipose tissues of diabetic ApoE−/−/LDLR−/− mice compared with control mice. STAMP2 gene overexpression could significantly reduce macrophages infiltration, the ratio of M1/M2 macrophages and the expression of pro-inflammatory cytokines in epididymal and brown adipose tissues, improving insulin resistance. Our results suggested that STAMP2 gene overexpression may improve insulin resistance via regulating macrophage polarization in visceral and brown adipose tissues. 相似文献