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*
Center for Immunology, University of Minnesota, St. Paul, MN 55108; and
Millennium Pharmaceuticals, Cambridge, MA 02139
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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L-selectin, similar to a number of transmembrane proteins, undergoes
ectodomain shedding. Endoproteolysis of L-selectin occurs spontaneously
(2, 3, 4), upon cross-linking or ligand binding
(5, 6, 7), and at increased levels following neutrophil
activation (8, 9). L-selectin is cleaved at an
extracellular site proximal to the membrane (10).
Mutational analysis of amino acids at and proximal to the cleavage site
indicates that the protease activity is relaxed in sequence
specificity, but has spatial and possibly conformational requirements
(2, 3). The protease activity that cleaves L-selectin can
be inhibited by hydroxamic acid-based metalloprotease inhibitors
(6, 11, 12, 13) and is membrane associated (11, 13). Consistent with these earlier findings, a recent study has
shown that lymphocytes from TNF-
converting enzyme
(TACE3/ADAM-17)
null mice are resistant to L-selectin shedding (14).
Unlike the well-characterized cleavage domain of L-selectin, it remains unclear whether the cytoplasmic domain of L-selectin participates in the regulation of L-selectin shedding. Chen et al. (3) showed that partial truncation of the L-selectin cytoplasmic domain did not affect receptor shedding, and proposed that this region was unlikely to regulate endoproteolysis. Recently, it has been shown that calmodulin (CaM) specifically interacts with the cytoplasmic domain of L-selectin in transfected cell lines (4). CaM is a 17-kDa protein that participates in different types of Ca2+-dependent interactions and regulates numerous effectors involved in growth, proliferation, movement, and adhesion (reviewed in Ref. 15). The treatment of human neutrophils with CaM inhibitors such as trifluoperazine (TFP) resulted in L-selectin shedding (4). However, TFP has been shown to induce discrete signaling mechanisms (16), activate membrane matrix metalloproteinases (17), and induce the shedding of various transmembrane proteins (16). Thus, it is unknown whether the action of the CaM inhibitors is at the level of L-selectin itself. To directly investigate whether CaM regulates L-selectin shedding requires mapping the CaM-binding region and mutational analysis of this site. Using an array of synthesized peptides and expressed L-selectin constructs, we demonstrate CaM binding activity in the membrane-proximal region of the cytoplasmic domain, which contains a highly conserved motif. Engineered mutations within this region that disrupt CaM binding significantly increase L-selectin shedding from a transduced hemopoietic cell line. These results provide direct evidence that the cytoplasmic domain of L-selectin participates in modifying the receptor’s susceptibility to proteolysis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The DREG-55 and DREG-200 mAbs (18), directed against the ectodomain of L-selectin, and the CA21 mAb (10), directed against the cytoplasmic domain of L-selectin, have been previously described. The PE-conjugated anti-L-selectin mAb Leu-8 and labeled isotype-negative control mAbs were purchased from BD Immunocytometry Systems (San Jose, CA). The PE-conjugated anti-L-selectin mAb LAM1-116 was purchased from Ancell (Bayport, MN). The EL112 mAb, specific to the ectodomain of E-selectin, was purchased from LigoCyte Pharmaceuticals. (Bozeman, MT) and used as an isotype-negative control mAb. An anti-CaM mAb was purchased from Upstate Biotechnology (Lake Placid, NY). PE-conjugated F(ab')2 goat anti-mouse IgG secondary Ab was purchased from Jackson Immunoresearch (West Grove, PA). HRP-conjugated goat anti-mouse IgG was purchased from Pierce (Rockford, IL).
Mutagenesis
L-selectin mutants were generated using PCR mutagenesis or the
Quik-change Site-directed Mutagenesis kit (Stratagene, San Diego, CA).
Oligonucleotide primers were commercially synthesized (IDT, Coralville,
IA). All successful clones were sequenced for the presence of the
engineered mutations and absence of any spontaneous mutations before
transduction. All L-selectin mutants generated are shown in Table I
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Peripheral blood neutrophils were isolated from normal healthy volunteers by dextran sedimentation and Ficoll-Hypaque centrifugation as previously described (6, 19). The human myeloid cell line K562 (erythroblast) was purchased from American Type Culture Collection (Manassas, VA) and cultured as per the supplier’s instructions. The human kidney epithelial cell line 293GP (generously provided by Dr. S. A. Rosenberg, National Cancer Institute, Bethesda, MD) was maintained in DMEM, 5% FCS, and antibiotics and passaged before confluency. Wild-type and mutant L-selectin cDNAs were ligated into the pDON-AI retroviral vector plasmid (Takara, Shiga, Japan). Virus stocks were generated via transient transfection of 293GP cells with 1 µg retroviral construct and 1 µg pMDG/VSV-G (Dr. S. A. Rosenberg, National Cancer Institute) using Effectene (Qiagen, Valencia, CA). Supernatants containing virus were removed from the cultures 72 h posttransfection, filtered through a 0.45-µm syringe tip filter, and stored at -70°C until used for infection. K562 cells (1 x 106) were pelleted, mixed with 1 ml viral supernatant plus 1 ml fresh K562 medium (RPMI 1640, 10% FCS, and antibiotics), and transferred to a six-well plate (Costar, Corning, NY). Twenty-four hours postinfection, 3 ml K562 medium was added to the cells. Forty-eight hours postinfection, K562 cells were expanded with fresh medium containing 500 µg/ml G418 (Mediatech, Herndon, VA) for selection. The K562 transductants were labeled with anti-L-selectin mAbs and examined by flow cytometry to determine expression efficiency of the L-selectin constructs.
Ab labeling, flow cytometry, and cell sorting
These procedures were performed as previously described (9). Briefly, FcR and nonspecific Ab binding sites were blocked by an initial incubation of the cells with FACS wash buffer (PBS containing 1% goat serum and 5 mM NaN3). Cells were then labeled with a particular mAb at 5 µg/ml diluted in FACS wash buffer. Bound primary Ab was revealed by a PE-conjugated, F(ab')2 goat anti-mouse IgG secondary Ab diluted in FACS wash buffer. Alternatively, cells were labeled with 15 µl of the directly conjugated mAbs. All Ab incubations were performed at 4°C for 30 min, after which the cells were washed twice with FACS wash buffer. For each sample, 10,000 Ab-labeled cells were analyzed by flow cytometry on a FACSCalibur instrument (BD Immunocytometry Systems).
For cell sorting, K562 transductants were labeled with the anti-L-selectin mAb LAM1-116-PE per methods described above, with the exception of sterile PBS plus BSA in place of FACS wash buffer. Cells were sorted using a FACSCalibur instrument.
Immunoprecipitation and immunoblotting
Neutrophils and K562 cell transductants were detergent lysed and immunoprecipitations performed as previously described (4, 19). Eluted samples were resolved on tricine-SDS-polyacrylamide 10–20% gradient gels (Invitrogen, San Diego, CA), then electrotransferred to a nitrocellulose membrane. The membrane was fixed with 0.5% paraformaldehyde and blocked in TBST containing 5% normal goat serum plus 1x Milk Diluent (Kirkegaard & Perry Laboratories, Gaithersburg, MD) for at least 1 h at room temperature. The membrane was probed with various mAbs using a slot blot apparatus (Immunetics, Cambridge, MA), which allowed for the staining of a particular sample with multiple mAbs. Ab reactivity was revealed by HRP-conjugated, goat anti-mouse IgG. The membranes were washed and visualized by addition of SuperSignal chemiluminescent substrate (Pierce) and exposure to film, as per the manufacturer’s instructions. In some SDS-PAGE gels, purified CaM (Upstate Biotechnology) was loaded for control purposes.
ELISA
The L-selectin capture ELISA was performed as previously described with certain modifications (4). Briefly, each K562 transductant or mock-transduced cells were cultured at an initial concentration of 5 x 104 cells/ml in triplicate wells of a 24-well tissue culture plate (Costar). Medium from each well was collected and filtered through a 0.22-µm syringe filter to remove any cell debris. Ninety-six-well Maxisorp plates (Nunc, Roskilde, Denmark) were coated with DREG-55 mAb (2 µg/ml in PBS) or BSA alone (2% in PBS). The plates were washed with PBS and then blocked with 2% BSA. Filtered medium from each well of the 24-well plate was aliquoted in duplicate into the DREG-55- or BSA-coated wells. Trapped L-selectin was detected with biotinylated DREG-200 mAb or an isotype-matched negative control mAb (0.1 µg/ml). The biotinylated molecules were revealed by incubation with a streptavidin-HRP conjugate (Zymed, San Francisco, CA), followed by development with the SuperSignal chemiluminescent substrate. The amount of substrate hydrolyzed was assessed at 300 photomultiplier tube for 260 ms on a Fluoroskan Ascent FL luminometric plate reader (LabSystems, Helsinki, Finland).
Peptide synthesis and solid-phase binding assay
Cellulose-bound peptides were prepared using custom SPOTs
synthesis by Sigma-Genosys (Woodlands, TX), which allowed a maximum
number of 13 aa per peptide. All peptides were synthesized in
triplicate, as indicated in Fig. 2. The cellulose membrane was rinsed
with methanol and washed three times with TBST. To prevent nonspecific
binding, the membranes were treated with blocking buffer (TBST plus 5%
BSA) for 2 h at room temperature. Initially the membrane was
probed with CA21 (5 µg/ml). Ab reactivity was revealed by
HRP-conjugated, goat anti-mouse IgG conjugate, SuperSignal
chemiluminescent substrate, and exposure to film. After each step, the
membrane was extensively washed with TBST. The membrane was then
regenerated per the manufacturer’s instructions, blocked, and probed
with biotinylated CaM (Calbiochem, San Diego, CA). CaM binding was
visualized by HRP-conjugated streptavidin, SuperSignal chemiluminescent
substrate, and exposure to film. After each step, the membrane was
extensively washed with TBST.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It has not been reported whether CaM associates with L-selectin in
primary leukocytes. Using mAbs specific to the cytoplasmic domain
(CA21) or extracellular region (DREG-200) of L-selectin, the receptor
was immunoprecipitated from resting peripheral blood neutrophils. In
both immunoprecipitates, a 17-kDa molecule was detected with an
anti-CaM mAb that comigrated with purified CaM (Fig. 1). The interaction between CaM and a
number of CaM target molecules is Ca2+ dependent.
We found that the presence of EDTA during the immunoprecipitation of
L-selectin abrogated the coprecipitation of CaM (Fig. 1B).
These results indicate a constitutive and
Ca2+-dependent interaction between CaM and
L-selectin in resting neutrophils.
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We have previously demonstrated that a synthesized peptide corresponding to the predicted 17-aa sequence of the L-selectin cytoplasmic domain bound in a direct and specific manner to CaM (4). Additional synthesized peptides were generated to further map the region of CaM binding in the cytoplasmic domain of L-selectin.
A number of L-selectin peptides up to 13 residues in length were
synthesized in triplicate on a cellulose membrane support (Fig. 2
A). Binding activity of the
peptides was initially assessed using the mAb CA21, which has
previously been mapped to the eight COOH-terminal residues of the
L-selectin cytoplasmic domain (10). Consistent with this
finding, CA21 detected peptides that correspond in sequence to the COOH
terminus of the L-selectin cytoplasmic domain and that were greater
than 7 residues in length (Fig. 2B). Next, purified CaM was
used to probe the cellulose membrane. Three 13-aa peptides each
staggered by 2-aa residues were synthesized to span the 17-aa
cytoplasmic domain of L-selectin (Fig. 2A, spots 10–18).
CaM bound to peptides that corresponded to the 13
NH2-terminal residues of the L-selectin
cytoplasmic domain (RRLKKGKKSKRSM) and to peptides shifted toward the
COOH terminus by 2 aa (LKKGKKSKRSMND). However, an additional shift
toward the COOH terminus by 2 aa (KGKKSKRSMNDPY) resulted in an
apparent decrease in the CaM-binding activity of the peptide (Fig. 2C). Reducing this peptide to 6 COOH-terminal residues of
the L-selectin cytoplasmic domain further reduced CaM binding (Fig. 2C). In contrast, reducing the peptide corresponding to the
13 NH2-terminal aa of the L-selectin cytoplasmic
domain to 6 NH2-terminal residues did not
decrease CaM binding (Fig. 2C). In fact, CaM reactivity with
this peptide appeared to be greater than with the extended peptide of
13 residues (Fig. 2C, spots 1–3 vs 10–12), which may be
due to structural differences in the peptides. Similarly, CA21 appears
to have greater reactivity with shorter rather than longer peptides as
well (Fig. 2B, spots 19–21 vs 16–18).
Because structural interactions of CaM with an immobilized peptide may
differ from interactions with L-selectin in an intact cell, we next
generated a series of L-selectin cytoplasmic domain mutants and
expressed them in hemopoietic cell lines. In the first series of
experiments, the predicted 17-aa cytoplasmic domain of L-selectin was
truncated by 8 aa (S
), which retains the conserved region of the
cytoplasmic domain (RRLKKGKKS), or by 16 residues (R) (Table I). The L-selectin constructs were stably
expressed at comparable levels by transduction in the human myeloid
cell line K562 (Fig. 3A),
which does not express endogenous L-selectin. Since these cytoplasmic
tail truncations remove the CA21 epitope, immunoprecipitation of
L-selectin was performed using mAbs directed against the extracellular
region of the receptor. To prevent shedding of the extracellular domain
of L-selectin from confounding the interpretation of these experiments,
wild-type and truncated L-selectin were engineered with a partially
deleted cleavage region (K-Y). This particular mutation has been
shown to abrogate L-selectin shedding (Ref. 2 and see Fig. 5). We found that CaM coprecipitated with L-selectin containing a
full-length or S cytoplasmic domain. In contrast, the
coprecipitation of CaM with L-selectin containing the R cytoplasmic
domain was greatly reduced (Fig. 3B). In combination with
results from the peptide-binding assay, these data indicate that
CaM-binding activity occurs predominantly in the membrane-proximal
region of the L-selectin cytoplasmic domain.
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To directly dissociate CaM from L-selectin, we mutated key amino
acids contributing to the predicted CaM-binding motif of L-selectin.
For instance, leucine-358, which provides a hydrophobic face in the
CaM-binding region, was exchanged for a negatively charged glutamic
acid residue (L/E). To more extensively disrupt the amphiphilic nature
of the CaM-binding motif, leucine-358, lysine-359, and lysine-360 were
substituted by glutamic acid residues (LKK/EEE) or the 17-aa
cytoplasmic domain of L-selectin was truncated by 16 residues (R).
The level of CaM association with the L-selectin mutants was assessed
by immunoprecipitation followed by immunoblot analysis. Again, to
prevent shedding of the extracellular domain of L-selectin, the
constructs were engineered with a partially deleted cleavage region
(K-Y). The L-selectin constructs were expressed in transduced K562
cells. Immunoblot analysis revealed that the mutations L/E (Fig. 4), LKK/EEE (data not shown), and R
(Fig. 3) all abrogated the coprecipitation of CaM.
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K-Y,L/E] L-selectin, or wild-type
L-selectin were stained with a mAb specific to the extracellular region
of the receptor and then sorted by flow cytometry. After which, all
three transductants sustained equivalent levels of intact L-selectin,
as determined by flow cytometry using several mAbs specific to the
ectodomain of L-selectin (Fig. 5A and data not shown). The
transductants were then plated at low density (5 x
104 cells/ml) and the medium supernatant of each
was tested at 24 and 48 h. High cell densities were avoided since
these conditions increased L-selectin shedding. In separate wells, the
transductants were grown under the same conditions and their growth
rate was monitored. For all transductants, cell numbers were equivalent
at all time points (data not shown). ELISA analysis revealed that the
levels of soluble L/E L-selectin were 6.6- and 2.4-fold higher than
soluble, wild-type L-selectin at 24 and 48 h, respectively (Fig. 5B).
Data provided above demonstrates that preventing CaM binding to
L-selectin results in increased L-selectin shedding. Whether CaM can be
dissociated from leukocyte L-selectin has not been determined. This was
examined by comparing the levels of CaM associated with intact
L-selectin and the 6-kDa transmembrane fragment isolated from
neutrophils. L-selectin was immunoprecipitated from equal numbers of
resting and PMA-activated neutrophils using the anti-cytoplasmic
domain mAb CA21, and the levels of coprecipitated CaM were determined
by immunoblot analysis. PMA activation of neutrophils resulted in
essentially complete shedding of L-selectin as evidenced by the
conversion of intact L-selectin into the 6-kDa transmembrane fragment
(Fig. 6). Interestingly, we found that
considerably less CaM was associated with the 6-kDa transmembrane
fragment of L-selectin compared with intact L-selectin (Fig. 6),
indicating a correlation between CaM binding and the state of
L-selectin expression in neutrophils.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We previously showed that various pharmacological inhibitors of CaM induced L-selectin shedding in an apparent activation-independent manner (4). Others have shown that particular CaM inhibitors induce the shedding of multiple transmembrane proteins by a process that is also sensitive to hydroxamic acid-based metalloprotease inhibitors (16, 20). Whether CaM associates with the cytoplasmic domains of these molecules is not well defined. TrKA contains a potential CaM-binding region in its cytoplasmic domain; however, deletion of the TrKA cytoplasmic domain to prevent CaM binding did not inhibit TrKA shedding following the treatment of transfected cells with the CaM inhibitor TFP (16). TFP is basic in nature and has been proposed to disturb the inner phase of the plasma membrane (21), induce discrete signaling mechanisms (16), and even activate membrane type-1 matrix metalloproteinases (17). Thus, it is possible that the action of particular CaM inhibitors may not be at L-selectin itself. Therefore, an approach to more accurately assess the regulatory role of CaM specific to L-selectin would be to directly manipulate the CaM-binding region.
We demonstrate that the exchange of leucine-358 for glutamic acid (L/E)
in the CaM-binding region of L-selectin prevents the coprecipitation of
CaM with L-selectin in transduced K562 cells. This L-selectin construct
was then used to examine the effects of directly dissociating CaM from
L-selectin on shedding. An advantage of exchanging a single amino acid
opposed to a more extensive mutation, such as truncating the
cytoplasmic domain of L-selectin to dissociate CaM, is that other
regions of the cytoplasmic domain are left intact to perhaps mediate
unrelated intermolecular interactions (e.g. -actinin binding
(22)). It was observed that K562 tranductants expressing
L/E L-selectin shed considerably higher levels of L-selectin compared
with transductants expressing wild-type L-selectin. The transductant
expressing cleavage resistant, [K-Y,L/E] L-selectin did not
produce soluble L-selectin above that of the parental K562 cells,
indicating that proteolysis of L/E L-selectin occurred within the
cleavage domain.
The L/E mutation in L-selectin completely prevents CaM binding as
determined by immunoprecipitation and immunoblot analysis. However,
this mutation and even more extensive mutation of the CaM-binding
region (LKK/EEE and R) did not result in complete conversion of
intact L-selectin into the 6-kDa transmembrane fragment (Fig. 5 and
data not shown). It could be inferred from these findings that other
cellular events also participate in regulating L-selectin turnover. For
instance, events induced upon cell activation, including the
up-regulation of TACE protease activity. Both PMA and G protein-coupled
receptors stimulate metalloprotease-dependent L-selectin shedding in
neutrophils (6) by a process that is sensitive to highly
selective protein kinase C inhibitors (9),
indicating similarities in both signaling pathways. We found that the
L/E L-selectin was further down-regulated in expression upon activation
of the K562 transductants with PMA (data not shown). Future studies
will focus on the effects of cell activation events and TACE activity
on L-selectin shedding.
Mutational analysis of L-selectin to determine the effects on
proteolysis have been extensively performed in the receptor’s
extracellular cleavage domain (2, 3), but much less
analysis has been performed in the cytoplasmic domain. Chen et al.
(3) deleted 11 COOH-terminal residues from the predicted
17-aa cytoplasmic domain of L-selectin and observed no significant
change in shedding when compared with wild-type L-selectin. These
findings, however, are not inconsistent with our own. Several lines of
evidence indicate that CaM binding occurs at the
NH2 terminus of the L-selectin cytoplasmic
domain. For instance, synthetic peptides that correspond in sequence to
the 6 NH2-terminal amino acid residues of the
L-selectin cytoplasmic domain demonstrated relatively high reactivity
with CaM (Fig. 2). Also, CaM efficiently coprecipitated with an
L-selectin construct truncated by 8 COOH-terminal residues (Fig. 3).
Finally, the CA21 mAb, which recognizes the COOH-terminal 8 aa residues
of the L-selectin cytoplasmic domain, does not inhibit CaM binding to
L-selectin (4). Of further interest, the membrane-proximal
region of the L-selectin cytoplasmic domain contains a highly conserved
amphiphilic motif, indicating a functional importance
(3).
CaM binding can both activate and inhibit target proteins. The mechanism by which CaM may regulate L-selectin proteolysis is the focus of ongoing studies. Consistent with our data, CaM constitutively associates with L-selectin in resting leukocytes and upon its dissociation L-selectin shedding is enhanced. It is possible that CaM regulates a conformational state of L-selectin and upon its dissociation the cleavage domain changes in position or is enlarged, increasing the receptor’s susceptibility to proteolysis. This may occur in conjunction with predicted structural changes in L-selectin upon leukocyte activation that increase binding activity of the receptor (23).
CaM binding to target molecules can be regulated by
Ca2+ fluctuations and by phosphorylation of the
CaM-binding region (24, 25). Our data indicate the CaM
binding to L-selectin is Ca2+ dependent. However,
intracellular Ca2+ concentrations can promote
different modes of regulation by CaM. For instance, increases in
Ca2+ concentration can either induce or
dissociate CaM-target interactions, e.g., smooth muscle myosin light
chain kinase (26) and neuromodulin (27),
respectively. In addition, the cytoplasmic domain of L-selectin is
phosphorylated on tyrosine (28) and serine
(23) residues following cell activation. Along these
lines, much less CaM was found to be associated with the 6-kDa
transmembrane cleavage fragment of L-selectin from activated
neutrophils compared with intact L-selectin from resting neutrophils.
Thus, changes in intracellular Ca2+
concentrations and/or phosphorylation of L-selectin may dissociate CaM
from L-selectin before proteolysis. Interestingly, a serine residue is
a component of the highly conserved NH2-terminal
region of the L-selectin cytoplasmic domain and the phosphorylation of
L-selectin serine residues has been shown to proceed shedding
(23). It is also possible that shedding of L-selectin in
turn affects CaM binding. Although CaM clearly binds to L-selectin
cytoplasmic domain peptides (Fig. 2), intact L-selectin may better
facilitate CaM binding. Though beyond the scope of this study, it will
be important to determine how CaM binding to L-selectin is
regulated.
In summary, L-selectin shedding is likely to be a natural antiadhesive process, in part, regulating the velocity of leukocyte rolling along the vascular endothelium (6, 7). By better understanding how L-selectin shedding is regulated, it may be possible to produce therapies that manipulate this process.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Bruce Walcheck, Department of Veterinary PathoBiology, University of Minnesota, 295j AS/VM Building, 1988 Fitch Avenue, St. Paul, MN 55108. E-mail address: walch003{at}umn.edu
3 Abbreviations used in this paper: TACE, TNF- converting enzyme; CaM, calmodulin; TFP, trifluoperazine.
Received for publication March 22, 2001. Accepted for publication June 1, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-actinin: receptor positioning in microvilli does not require interaction with -actinin. J. Cell Biol. 129:1155.This article has been cited by other articles:
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  Y. Wang, A. H. Herrera, Y. Li, K. K. Belani, and B. Walcheck Regulation of Mature ADAM17 by Redox Agents for L-Selectin Shedding J. Immunol., February 15, 2009; 182(4): 2449 - 2457. [Abstract] [Full Text] [PDF]
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Y. Furukawa, E. Umemoto, M. H. Jang, K. Tohya, M. Miyasaka, and T. Hirata Identification of Novel Isoforms of Mouse L-selectin with Different Carboxyl-terminal Tails J. Biol. Chem., May 2, 2008; 283(18): 12112 - 12119. [Abstract] [Full Text] [PDF]
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 U. Schaff, P. E. Mattila, S. I. Simon, and B. Walcheck Neutrophil adhesion to E-selectin under shear promotes the redistribution and co-clustering of ADAM17 and its proteolytic substrate L-selectin J. Leukoc. Biol., January 1, 2008; 83(1): 99 - 105. [Abstract] [Full Text] [PDF]
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B. Aktas, M. Pozgajova, W. Bergmeier, S. Sunnarborg, S. Offermanns, D. Lee, D. D. Wagner, and B. Nieswandt Aspirin Induces Platelet Receptor Shedding via ADAM17 (TACE) J. Biol. Chem., December 2, 2005; 280(48): 39716 - 39722. [Abstract] [Full Text] [PDF]
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S. Chattopadhyay, K. R. Santhamma, S. Sengupta, B. McCue, M. Kinter, G. C. Sen, and I. Sen Calmodulin Binds to the Cytoplasmic Domain of Angiotensin-converting Enzyme and Regulates Its Phosphorylation and Cleavage Secretion J. Biol. Chem., October 7, 2005; 280(40): 33847 - 33855. [Abstract] [Full Text] [PDF]
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X. Zou, V. R. Shinde Patil, N. M. Dagia, L. A. Smith, M. J. Wargo, K. A. Interliggi, C. M. Lloyd, D. F. J. Tees, B. Walcheck, M. B. Lawrence, et al. PSGL-1 derived from human neutrophils is a high-efficiency ligand for endothelium-expressed E-selectin under flow Am J Physiol Cell Physiol, August 1, 2005; 289(2): C415 - C424. [Abstract] [Full Text] [PDF]
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M. D. Peterson, R. Jin, S. Hyduk, P. Duchesneau, M. I. Cybulsky, and T. K. Waddell Monocyte Adhesion to Xenogeneic Endothelium during Laminar Flow Is Dependent on {alpha}-Gal-Mediated Monocyte Activation J. Immunol., June 15, 2005; 174(12): 8072 - 8081. [Abstract] [Full Text] [PDF]
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T. Rabie, A. Strehl, A. Ludwig, and B. Nieswandt Evidence for a Role of ADAM17 (TACE) in the Regulation of Platelet Glycoprotein V J. Biol. Chem., April 15, 2005; 280(15): 14462 - 14468. [Abstract] [Full Text] [PDF]
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E. E. Gardiner, J. F. Arthur, M. L. Kahn, M. C. Berndt, and R. K. Andrews Regulation of platelet membrane levels of glycoprotein VI by a platelet-derived metalloproteinase Blood, December 1, 2004; 104(12): 3611 - 3617. [Abstract] [Full Text] [PDF]
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K. Kilian, J. Dernedde, E.-C. Mueller, I. Bahr, and R. Tauber The Interaction of Protein Kinase C Isozymes {alpha}, {iota}, and {theta} with the Cytoplasmic Domain of L-selectin Is Modulated by Phosphorylation of the Receptor J. Biol. Chem., August 13, 2004; 279(33): 34472 - 34480. [Abstract] [Full Text] [PDF]
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A. Ivetic, O. Florey, J. Deka, D. O. Haskard, A. Ager, and A. J. Ridley Mutagenesis of the Ezrin-Radixin-Moesin Binding Domain of L-selectin Tail Affects Shedding, Microvillar Positioning, and Leukocyte Tethering J. Biol. Chem., August 6, 2004; 279(32): 33263 - 33272. [Abstract] [Full Text] [PDF]
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C. A. St. Hill, S. R. Alexander, and B. Walcheck Indirect capture augments leukocyte accumulation on P-selectin in flowing whole blood J. Leukoc. Biol., April 1, 2003; 73(4): 464 - 471. [Abstract] [Full Text] [PDF]
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 L. W. de Rossi, N. A. Horn, W. Buhre, F. Gass, G. Hutschenreuter, and R. Rossaint The Effect of Isoflurane on Neutrophil Selectin and {beta}2-Integrin Activation In Vitro Anesth. Analg., September 1, 2002; 95(3): 583 - 587. [Abstract] [Full Text] [PDF]
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D. HUDRISIER and P. BONGRAND Intercellular transfer of antigen-presenting cell determinants onto T cells: molecular mechanisms and biological significance FASEB J, April 1, 2002; 16(6): 477 - 486. [Abstract] [Full Text] [PDF]
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