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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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Saija Kiljunen Neeta Datta Svetlana V. Dentovskaya Andrey P. Anisimov Yuriy A. Knirel Jos�� A. Bengoechea Otto Holst Mikael Skurnik 《Journal of bacteriology》2011,193(18):4963-4972
φA1122 is a T7-related bacteriophage infecting most isolates of Yersinia pestis, the etiologic agent of plague, and used by the CDC in the identification of Y. pestis. φA1122 infects Y. pestis grown both at 20°C and at 37°C. Wild-type Yersinia pseudotuberculosis strains are also infected but only when grown at 37°C. Since Y. pestis expresses rough lipopolysaccharide (LPS) missing the O-polysaccharide (O-PS) and expression of Y. pseudotuberculosis O-PS is largely suppressed at temperatures above 30°C, it has been assumed that the phage receptor is rough LPS. We present here several lines of evidence to support this. First, a rough derivative of Y. pseudotuberculosis was also φA1122 sensitive when grown at 22°C. Second, periodate treatment of bacteria, but not proteinase K treatment, inhibited the phage binding. Third, spontaneous φA1122 receptor mutants of Y. pestis and rough Y. pseudotuberculosis could not be isolated, indicating that the receptor was essential for bacterial growth under the applied experimental conditions. Fourth, heterologous expression of the Yersinia enterocolitica O:3 LPS outer core hexasaccharide in both Y. pestis and rough Y. pseudotuberculosis effectively blocked the phage adsorption. Fifth, a gradual truncation of the core oligosaccharide into the Hep/Glc (l-glycero-d-manno-heptose/d-glucopyranose)-Kdo/Ko (3-deoxy-d-manno-oct-2-ulopyranosonic acid/d-glycero-d-talo-oct-2-ulopyranosonic acid) region in a series of LPS mutants was accompanied by a decrease in phage adsorption, and finally, a waaA mutant expressing only lipid A, i.e., also missing the Kdo/Ko region, was fully φA1122 resistant. Our data thus conclusively demonstrated that the φA1122 receptor is the Hep/Glc-Kdo/Ko region of the LPS core, a common structure in Y. pestis and Y. pseudotuberculosis. 相似文献
5.
6.
Skeletal muscle is a post-mitotic tissue maintained by repair and regeneration through a population of stem cell-like satellite cells. Following muscle injury, satellite cell proliferation is mediated by local signals ensuring sufficient progeny for tissue repair. Age–related changes in satellite cells as well as to the local and systemic environment potentially impact on the capacity of satellite cells to generate sufficient progeny in an ageing organism resulting in diminished regeneration. ‘Rejuvenation’ of satellite cell progeny and regenerative capacity by environmental stimuli effectors suggest that a subset of age-dependent satellite cell changes may be reversible. Epigenetic regulation of satellite stem cells that include DNA methylation and histone modifications which regulate gene expression are potential mechanisms for such reversible changes and have been shown to control organismal longevity. The area of health and ageing that is likely to benefit soonest from advances in the biology of adult stem cells is the emerging field of regenerative medicine. Further studies are needed to elucidate the mechanisms by which epigenetic modifications regulate satellite stem cell function and will require an increased understanding of stem-cell biology, the environment of the aged tissue and the interaction between the two. 相似文献
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The DSM-IV major depression "bereavement exclusion" (BE), which recognizes that depressive symptoms are sometimes normal in recently bereaved individuals, is proposed for elimination in DSM-5. Evidence cited for the BE's invalidity comes from two 2007 reviews purporting to show that bereavement-related depression is similar to other depression across various validators, and a 2010 review of subsequent research. We examined whether the 2007 and 2010 reviews and subsequent relevant literature support the BE's invalidity. Findings were: a) studies included in the 2007 reviews sampled bereavement-related depression groups most of whom were not BE-excluded, making them irrelevant for evaluating BE validity; b) three subsequent studies cited by the 2010 review as supporting BE elimination did examine BE-excluded cases but were in fact inconclusive; and c) two more recent articles comparing recurrence of BE-excluded and other major depressive disorder cases both support the BE's validity. We conclude that the claimed evidence for the BE's invalidity does not exist. The evidence in fact supports the BE's validity and its retention in DSM-5 to prevent false positive diagnoses. We suggest some improvements to increase validity and mitigate risk of false negatives. 相似文献
9.
10.
Chiara Ferrario Giovanni Ricci Christian Milani Gabriele Andrea Lugli Marco Ventura Giovanni Eraclio Francesca Borgo Maria Grazia Fortina 《PloS one》2013,8(12)
The population structure and diversity of Lactococcus garvieae, an emerging pathogen of increasing clinical significance, was determined at both gene and genome level. Selected lactococcal isolates of various origins were analyzed by a multi locus sequence typing (MLST). This gene-based analysis was compared to genomic characteristics, estimated through the complete genome sequences available in database. The MLST identified two branches containing the majority of the strains and two branches bearing one strain each. One strain was particularly differentiated from the other L. garvieae strains, showing a significant genetic distance. The genomic characteristics, correlated to the MLST-based phylogeny, indicated that this “separated strain” appeared first and could be considered the evolutionary intermediate between Lactococcus lactis and L. garvieae main clusters. A preliminary genome analysis of L. garvieae indicated a pan-genome constituted of about 4100 genes, which included 1341 core genes and 2760 genes belonging to the dispensable genome. A total of 1491 Clusters of Orthologous Genes (COGs) were found to be specific to the 11 L. garvieae genomes, with the genome of the “separated strain” showing the highest presence of unique genes. 相似文献
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Nelli Mnatsakanyan Arathianand M. Krishnakumar Toshiharu Suzuki Joachim Weber 《The Journal of biological chemistry》2009,284(17):11336-11345
ATP synthase uses a unique rotational mechanism to convert chemical energy
into mechanical energy and back into chemical energy. The helix-turn-helix
motif, termed “DELSEED-loop,” in the C-terminal domain of the
β subunit was suggested to be involved in coupling between catalysis and
rotation. Here, the role of the DELSEED-loop was investigated by functional
analysis of mutants of Bacillus PS3 ATP synthase that had 3–7
amino acids within the loop deleted. All mutants were able to catalyze ATP
hydrolysis, some at rates several times higher than the wild-type enzyme. In
most cases ATP hydrolysis in membrane vesicles generated a transmembrane
proton gradient, indicating that hydrolysis occurred via the normal rotational
mechanism. Except for two mutants that showed low activity and low abundance
in the membrane preparations, the deletion mutants were able to catalyze ATP
synthesis. In general, the mutants seemed less well coupled than the wild-type
enzyme, to a varying degree. Arrhenius analysis demonstrated that in the
mutants fewer bonds had to be rearranged during the rate-limiting catalytic
step; the extent of this effect was dependent on the size of the deletion. The
results support the idea of a significant involvement of the DELSEED-loop in
mechanochemical coupling in ATP synthase. In addition, for two deletion
mutants it was possible to prepare an
α3β3γ subcomplex and measure nucleotide
binding to the catalytic sites. Interestingly, both mutants showed a severely
reduced affinity for MgATP at the high affinity site.F1F0-ATP synthase catalyzes the final step of
oxidative phosphorylation and photophosphorylation, the synthesis of ATP from
ADP and inorganic phosphate. F1F0-ATP synthase consists
of the membrane-embedded F0 subcomplex, with, in most bacteria, a
subunit composition of ab2c10, and the peripheral
F1 subcomplex, with a subunit composition of
α3β3γδε. The energy
necessary for ATP synthesis is derived from an electrochemical transmembrane
proton (or, in some organisms, a sodium ion) gradient. Proton flow down the
gradient through F0 is coupled to ATP synthesis on F1 by
a unique rotary mechanism. The protons flow through (half) channels at the
interface of the a and c subunits, which drives rotation of the ring of c
subunits. The c10 ring, together with F1 subunits
γ and ε, forms the rotor. Rotation of γ leads to
conformational changes in the catalytic nucleotide binding sites on the β
subunits, where ADP and Pi are bound. The conformational changes
result in the formation and release of ATP. Thus, ATP synthase converts
electrochemical energy, the proton gradient, into mechanical energy in the
form of subunit rotation and back into chemical energy as ATP. In bacteria,
under certain physiological conditions, the process runs in reverse. ATP is
hydrolyzed to generate a transmembrane proton gradient, which the bacterium
requires for such functions as nutrient import and locomotion (for reviews,
see Refs.
1–6).F1 (or F1-ATPase) has three catalytic nucleotide
binding sites located on the β subunits at the interface to the adjacent
α subunit. The catalytic sites have pronounced differences in their
nucleotide binding affinity. During rotational catalysis, the sites switch
their affinities in a synchronized manner; the position of γ determines
which catalytic site is the high affinity site
(Kd1 in the nanomolar range), which site is the
medium affinity site (Kd2 ≈ 1
μm), and which site is the low affinity site
(Kd3 ≈ 30–100 μm; see
Refs. 7 and
8). In the original crystal
structure of bovine mitochondrial F1
(9), one of the three catalytic
sites, was filled with the ATP analog
AMP-PNP,2 a second was
filled with ADP (plus azide) (see Ref.
10), and the third site was
empty. Hence, the β subunits are referred to as βTP,
βDP, and βE. The occupied β subunits,
βTP and βDP, were in a closed conformation,
and the empty βE subunit was in an open conformation. The main
difference between these two conformations is found in the C-terminal domain.
Here, the “DELSEED-loop,” a helix-turn-helix structure containing
the conserved DELSEED motif, is in an “up” position when the
catalytic site on the respective β subunit is filled with nucleotide and
in a “down” position when the site is empty
(Fig. 1A). When all
three catalytic sites are occupied by nucleotide, the previously open
βE subunit assumes an intermediate, half-closed
(βHC) conformation. It cannot close completely because of
steric clashes with γ
(11).Open in a separate windowFIGURE 1.The βDELSEED-loop. A, interaction of the
βTP and βE subunits with theγ
subunit.β subunits are shown in yellow andγ in
blue. The DELSEED-loop (shown in orange, with the DELSEED
motif itself in green)of βTP interacts with the
C-terminal helixγ and the short helix that runs nearly perpendicular to
the rotation axis. The DELSEED-loop of βE makes contact with
the convex portion of γ, formed mainly by the N-terminal helix. A
nucleotide molecule (shown in stick representation) occupies the catalytic
site of βTP, and the subunit is in the closed conformation.
The catalytic site on βE is empty, and the subunit is in the
open conformation. This figure is based on Protein Data Bank file 1e79
(32). B, deletions in
the βDELSEED-loop. The loop was “mutated” in silico
to represent the PS3 ATP synthase. The 3–4-residue segments that are
removed in the deletion mutants are color-coded as follows:
380LQDI383, pink;
384IAIL387, green;
388GMDE391, yellow;
392LSD394, cyan;
395EDKL398, orange;
399VVHR402, blue. Residues that are the most
involved in contacts with γ are labeled. All figures were generated
using the program PyMOL (DeLano Scientific, San Carlos, CA).The DELSEED-loop of each of the three β subunits makes contact with
the γ subunit. In some cases, these contacts consist of hydrogen bonds
or salt bridges between the negatively charged residues of the DELSEED motif
and positively charged residues on γ. The interactions of the
DELSEED-loop with γ, its movement during catalysis, the conservation of
the DELSEED motif (see 12–14).
Thus, the finding that an AALSAAA mutant in the
α3β3γ complex of ATP synthase from the
thermophilic Bacillus PS3, where several hydrogen bonds/salt bridges
to γ are removed simultaneously, could drive rotation of γ with
the same torque as the wild-type enzyme
(14) came as a surprise. On
the other hand, it seems possible that it is the bulk of the DELSEED-loop,
more so than individual interactions, that drives rotation of γ.
According to a model favored by several authors
(6,
15,
16) (see also Refs.
17–19),
binding of ATP (or, more precisely, MgATP) to the low affinity catalytic site
on βE and the subsequent closure of this site, accompanied by
its conversion into the high affinity site, are responsible for driving the
large (80–90°) rotation substep during ATP hydrolysis, with the
DELSEED-loop acting as a “pushrod.” A recent molecular dynamics
(20) study supports this model
and implicates mainly the region around several hydrophobic residues upstream
of the DELSEED motif (specifically βI386 and
βL387)3 as being
responsible for making contact with γ during the large rotation
substep.
TABLE 1
Conservation of residues in the DELSEED-loop Amino acids found in selected species in the turn region of the DELSEED-loop. Listed are all positions subjected to deletions in the present study. Residue numbers refer to the PS3 enzyme. Consensus annotation: p, polar residue; s, small residue; h, hydrophobic residue; –, negatively charged residue; +, positively charged residue.Open in a separate windowIn the present study, we investigated the function of the DELSEED-loop using an approach less focused on individual residues, by deleting stretches of 3–7 amino acids between positions β380 and β402 of ATP synthase from the thermophilic Bacillus PS3. We analyzed the functional properties of the deletion mutants after expression in Escherichia coli. The mutants showed ATPase activities, which were in some cases surprisingly high, severalfold higher than the activity of the wild-type control. On the other hand, in all cases where ATP synthesis could be measured, the rates where below or equal to those of the wild-type enzyme. In Arrhenius plots, the hydrolysis rates of the mutants were less temperature-dependent than those of wild-type ATP synthase. In those cases where nucleotide binding to the catalytic sites could be tested, the deletion mutants had a much reduced affinity for MgATP at high affinity site 1. The functional role of the DELSEED-loop will be discussed in light of the new information. 相似文献13.
14.
Lee Suyeon Ha Ji-Hye Moon Duk-Soo Youn Soyoung Kim Changnam Park Boram Kim Myong-Jae Kim Hyo-Won Chung Seockhoon 《Sleep and biological rhythms》2019,17(3):277-285
Sleep and Biological Rhythms - We evaluated the sleep environment factors that influence children’s sleep, and the relationship between co-sleeping and parenting stress and parents’... 相似文献
15.
Guo-Dong Li David C. Chiara Jonathan B. Cohen Richard W. Olsen 《The Journal of biological chemistry》2009,284(18):11771-11775
Photoaffinity labeling of γ-aminobutyric acid type A
(GABAA)-receptors (GABAAR) with an etomidate analog and
mutational analyses of direct activation of GABAAR by neurosteroids
have each led to the proposal that these structurally distinct general
anesthetics bind to sites in GABAARs in the transmembrane domain at
the interface between the β and α subunits. We tested whether the
two ligand binding sites might overlap by examining whether neuroactive
steroids inhibited etomidate analog photolabeling. We previously identified
(Li, G. D., Chiara, D. C., Sawyer, G. W., Husain, S. S., Olsen, R. W., and
Cohen, J. B. (2006) J. Neurosci. 26, 11599–11605) azietomidate
photolabeling of GABAAR α1Met-236 and βMet-286 (in
αM1 and βM3). Positioning these two photolabeled amino acids in a
single type of binding site at the interface of β and α subunits
(two copies per pentamer) is consistent with a GABAAR homology
model based upon the structure of the nicotinic acetylcholine receptor and
with recent αM1 to βM3 cross-linking data. Biologically active
neurosteroids enhance rather than inhibit azietomidate photolabeling, as
assayed at the level of GABAAR subunits on analytical SDS-PAGE, and
protein microsequencing establishes that the GABAAR-modulating
neurosteroids do not inhibit photolabeling of GABAAR
α1Met-236 or βMet-286 but enhance labeling of α1Met-236. Thus
modulatory steroids do not bind at the same site as etomidate, and neither of
the amino acids identified as neurosteroid activation determinants (Hosie, A.
M., Wilkins, M. E., da Silva, H. M., and Smart, T. G. (2006) Nature
444, 486–489) are located at the subunit interface defined by our
etomidate site model.GABAA3
receptors (GABAAR) are major mediators of brain inhibitory
neurotransmission and participate in most circuits and behavioral pathways
relevant to normal and pathological function
(1). GABAAR are
subject to modulation by endogenous neurosteroids, as well as myriad
clinically important central nervous system drugs including general
anesthetics, benzodiazepines, and possibly ethanol
(1,
2). The mechanism of
GABAAR modulation by these different classes of drugs is of major
interest, including identification of the receptor amino acid residues
involved in binding and action of the drugs.In the absence of high resolution crystal structures of drug-receptor
complexes, the locations of anesthetic binding sites in GABAARs
have been predicted based upon analyses of functional properties of point
mutant receptors, which identified residues in the α and β subunit
M1–M4 transmembrane helices important for modulation by volatile
anesthetics (primarily α subunit) and by intravenous agents, including
etomidate and propofol (β subunit)
(3–5).
Position βM2–15, numbered relative to the N terminus of the helix,
functions as a major determinant of etomidate and propofol potency as GABA
modulators in vitro and in vivo
(6–8).
By contrast, this residue is not implicated for modulation by the
neurosteroids, potent endogenous modulators of GABAAR
(9).Photoaffinity labeling, which allows the identification of residues in
proximity to drug binding sites
(10,
11), has been used to identify
two GABAAR amino acids covalently modified by the etomidate analog
[3H]azietomidate
(12): α1Met-236 within
αM1 and βMet-286 within βM3. Photolabeling of these residues
was inhibited equally by nonradioactive etomidate and enhanced proportionately
by GABA present in the assay, consistent with the presence of these two
residues in a common drug binding pocket that would be located at the
interface between the β and α subunits in the transmembrane domain
(12). Mutational analyses
identify these positions as etomidate and propofol sensitivity determinants
(13–15).A recent mutagenesis study
(16) identified two other
residues in GABAAR αM1 and βM3 as critical for direct
activation by neurosteroids, αThr-236 (rat numbering, corresponding to
α1Thr-237, bovine numbering used here and by Li et al.
(12))4
and βTyr-284. These residues were also proposed to contribute to a
neurosteroid binding pocket in the transmembrane domain at the interface
between β and α subunits, based upon their location in an
alternative GABAAR structural model that positioned those amino
acids, and not α1Met-236 or βMet-286, at the subunit interface. For
GABAARs and other members of the Cys-loop superfamily of
neurotransmitter-gated ion channels, the transmembrane domain of each subunit
is made up of a loose bundle of four α helices (M1–M4), with M2
from each subunit contributing to the lumen of the ion channel and M4
positioned peripherally in greatest contact with lipid, as seen in the
structures of the Torpedo nicotinic acetylcholine receptor (nAChR)
(17) and in distantly related
prokaryote homologs (18).
However, uncertainties in the alignment of GABAAR subunit sequences
relative to those of the nAChR result in alternative GABAAR
homology models (12,
19,
20) that differ in the
location of amino acids in the M3 and M4 membrane-spanning helices and in the
M1 helix in some models (16,
21).If etomidate and neurosteroids both bind at the same β/α
interface in the GABAAR transmembrane domain, the limited space
available for ligand binding suggests that their binding pockets might overlap
and that ligand binding would be mutually exclusive. To address this question,
we photolabeled purified bovine brain GABAAR with
[3H]azietomidate in the presence of different neuroactive steroids
and determined by protein microsequencing whether active neurosteroids
inhibited labeling of α1Met-236 and βMet-286, as expected for
mutually exclusive binding, or resulted in [3H]azietomidate
photolabeling of other amino acids, a possible consequence of allosteric
interactions. Active steroids failed to inhibit labeling and enhanced labeling
of α1Met-236, clearly indicating that the neurosteroid and the etomidate
sites are distinct. Our GABAAR homology model that positions
α1Met-236 and βMet-286 at the β/α interface, but not
that of Hosie et al.
(16), is also consistent with
cysteine substitution cross-linking studies
(20,
22), which define the
proximity relations between amino acids in the αM1, αM2,
αM3, and βM3 helices, and these results support the interpretation
that the two residues photolabeled by [3H]azietomidate are part of
a single subunit interface binding pocket, whereas the steroid sensitivity
determinants identified by mutagenesis neither are at the β/α
subunit interface nor are contributors to a common binding pocket. 相似文献
16.
Bleecker AB 《Current opinion in plant biology》1998,1(1):73-78
Recent studies on the differential expression of genes associated with leaf senescence support the long-standing interpretation of plant senescence as an organized, genetically controlled process. Sequence identities of genes that are differentially expressed in senescing leaves indicate roles in the salvage of nutrients. By considering this salvage function as the selected trait and the degeneration and death of the tissue a pleiotropic consequence of nutrient redistribution, the process of leaf senescence can be reconciled with evolutionary theories on the origins of senescence in animals. 相似文献
17.
18.
Yuya Sato Toshihiko Uemura Keisuke Morimitsu Ryoko Sato-Nishiuchi Ri-ichiroh Manabe Junichi Takagi Masashi Yamada Kiyotoshi Sekiguchi 《The Journal of biological chemistry》2009,284(21):14524-14536
Integrin α8β1 interacts with a variety of Arg-Gly-Asp
(RGD)-containing ligands in the extracellular matrix. Here, we examined the
binding activities of α8β1 integrin toward a panel of
RGD-containing ligands. Integrin α8β1 bound specifically to
nephronectin with an apparent dissociation constant of 0.28 ± 0.01
nm, but showed only marginal affinities for fibronectin and other
RGD-containing ligands. The high-affinity binding to α8β1 integrin
was fully reproduced with a recombinant nephronectin fragment derived from the
RGD-containing central “linker” segment. A series of deletion
mutants of the recombinant fragment identified the LFEIFEIER sequence on the
C-terminal side of the RGD motif as an auxiliary site required for
high-affinity binding to α8β1 integrin. Alanine scanning
mutagenesis within the LFEIFEIER sequence defined the EIE sequence as a
critical motif ensuring the high-affinity integrin-ligand interaction.
Although a synthetic LFEIFEIER peptide failed to inhibit the binding of
α8β1 integrin to nephronectin, a longer peptide containing both the
RGD motif and the LFEIFEIER sequence was strongly inhibitory, and was
∼2,000-fold more potent than a peptide containing only the RGD motif.
Furthermore, trans-complementation assays using recombinant fragments
containing either the RGD motif or LFEIFEIER sequence revealed a clear
synergism in the binding to α8β1 integrin. Taken together, these
results indicate that the specific high-affinity binding of nephronectin to
α8β1 integrin is achieved by bipartite interaction of the integrin
with the RGD motif and LFEIFEIER sequence, with the latter serving as a
synergy site that greatly potentiates the RGD-driven integrin-ligand
interaction but has only marginal activity to secure the interaction by
itself.Integrins are a family of adhesion receptors that interact with a variety
of extracellular ligands, typically cell-adhesive proteins in the
extracellular matrix
(ECM).2 They play
mandatory roles in embryonic development and the maintenance of tissue
architectures by providing essential links between cells and the ECM
(1). Integrins are composed of
two non-covalently associated subunits, termed α and β. In mammals,
18 α and 8 β subunits have been identified, and combinations of
these subunits give rise to at least 24 distinct integrin heterodimers. Based
on their ligand-binding specificities, ECM-binding integrins are classified
into three groups, namely laminin-, collagen- and RGD-binding integrins
(2,
3), of which the RGD-binding
integrins have been most extensively investigated. The RGD-binding integrins
include α5β1, α8β1, αIIbβ3, and
αV-containing integrins, and have been shown to interact with a variety
of ECM ligands, such as fibronectin and vitronectin, with distinct binding
specificities.The α8 integrin subunit was originally identified in chick nerves
(4). Integrin α8β1
is expressed in the metanephric mesenchyme and plays a crucial role in
epithelial-mesenchymal interactions during the early stages of kidney
morphogenesis. Disruption of the α8 gene in mice was found to be
associated with severe defects in kidney morphogenesis
(5) and stereocilia development
(6). To date, α8β1
integrin has been shown to bind to fibronectin, vitronectin, osteopontin,
latency-associated peptide of transforming growth factor-β1, tenascin-W,
and nephronectin (also named POEM)
(7–13),
among which nephronectin is believed to be an α8β1 integrin ligand
involved in kidney development
(10).Nephronectin is one of the basement membrane proteins whose expression and
localization patterns are restricted in a tissue-specific and developmentally
regulated manner (10,
11). Nephronectin consists of
five epidermal growth factor-like repeats, a linker segment containing the RGD
cell-adhesive motif (designated RGD-linker) and a meprin-A5 protein-receptor
protein-tyrosine phosphatase μ (MAM) domain (see
Fig. 3A). Although the
physiological functions of nephronectin remain only poorly understood, it is
thought to play a role in epithelial-mesenchymal interactions through binding
to α8β1 integrin, thereby transmitting signals from the epithelium
to the mesenchyme across the basement membrane
(10). Recently, mice deficient
in nephronectin expression were produced by homologous recombination
(14). These
nephronectin-deficient mice frequently displayed kidney agenesis, a phenotype
reminiscent of α8 integrin knock-out mice
(14), despite the fact that
other RGD-containing ligands, including fibronectin and osteopontin, were
expressed in the embryonic kidneys
(9,
15). The failure of the other
RGD-containing ligands to compensate for the deficiency of nephronectin in the
developing kidneys suggests that nephronectin is an indispensable
α8β1 ligand that plays a mandatory role in epithelial-mesenchymal
interactions during kidney development.Open in a separate windowFIGURE 3.Binding activities of α8β1 integrin to nephronectin and its
fragments. A, schematic diagrams of full-length nephronectin
(NN) and its fragments. RGD-linker and RGD-linker
(GST), the central RGD-containing linker segments expressed in
mammalian and bacterial expression systems, respectively; PRGDV, a
short RGD-containing peptide modeled after nephronectin and expressed as a GST
fusion protein (see Fig.
4A for the peptide sequence). The arrowheads
indicate the positions of the RGD motif. B, purified recombinant
proteins were analyzed by SDS-PAGE in 7–15% gradient (left and
center panels) and 12% (right panels) gels, followed by
Coomassie Brilliant Blue (CBB) staining, immunoblotting with an
anti-FLAG mAb, or lectin blotting with PNA. The quantities of proteins loaded
were: 0.5 μg (for Coomassie Brilliant Blue staining) and 0.1 μg (for
blotting with anti-FLAG and PNA) in the left and center
panels;1 μg in the right panel. C, recombinant proteins (10
nm) were coated on microtiter plates and assessed for their binding
activities toward α8β1 integrin (10 nm) in the presence
of 1 mm Mn2+. The backgrounds were subtracted as
described in the legend to Fig.
2. The results represent the mean ± S.D. of triplicate
determinations. D, titration curves of α8β1 integrin bound
to full-length nephronectin (NN, closed squares), the RGD-linker
segments expressed in 293F cells (RGD-linker, closed triangles) and
E. coli (RGD-linker (GST), open
triangles), the MAM domain (MAM, closed diamonds), and the PRGDV
peptide expressed as a GST fusion protein in E. coli (PRGDV
(GST), open circles). The assays were performed as described
in the legend to Fig.
2B. The results represent the means of duplicate
determinations.Although ligand recognition by RGD-binding integrins is primarily
determined by the RGD motif in the ligands, it is the residues outside the RGD
motif that define the binding specificities and affinities toward individual
integrins (16,
17). For example,
α5β1 integrin specifically binds to fibronectin among the many
RGD-containing ligands, and requires not only the RGD motif in the 10th type
III repeat but also the so-called “synergy site” within the
preceding 9th type III repeat for fibronectin recognition
(18). Recently, DiCara et
al. (19) demonstrated
that the high-affinity binding of αVβ6 integrin to its natural
ligands, e.g. foot-and-mouth disease virus, requires the RGD motif
immediately followed by a Leu-Xaa-Xaa-Leu/Ile sequence, which forms a helix to
align the two conserved hydrophobic residues along the length of the helix.
Given the presence of many naturally occurring RGD-containing ligands, it is
conceivable that the specificities of the RGD-binding integrins are dictated
by the sequences flanking the RGD motif or those in neighboring domains that
come into close proximity with the RGD motif in the intact ligand proteins.
However, the preferences of α8β1 integrin for RGD-containing
ligands and how it secures its high-affinity binding toward its preferred
ligands remain unknown.In the present study, we investigated the binding specificities of
α8β1 integrin toward a panel of RGD-containing cell-adhesive
proteins. Our data reveal that nephronectin is a preferred ligand for
α8β1 integrin, and that a LFEIFEIER sequence on the C-terminal side
of its RGD motif serves as a synergy site to ensure the specific high-affinity
binding of nephronectin to α8β1 integrin. 相似文献
19.