THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 280, NO. 41, pp. 34447–34457, October 14, 2005
© 2005 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
Identification of the Surfactant Protein A Receptor 210 as the
Unconventional Myosin 18A*
Received for publication, May 12, 2005, and in revised form, July 11, 2005 Published, JBC Papers in Press, August 8, 2005, DOI 10.1074/jbc.M505229200
Ching-Hui Yang‡, Jacek Szeliga‡, Jeremy Jordan‡, Shawn Faske§, Zvjezdana Sever-Chroneos‡, Bre Dorsett‡,
Robert E. Christian¶, Robert E. Settlage¶, Jeffrey Shabanowitz¶, Donald F. Hunt储, Jeffrey A. Whitsett§,
and Zissis C. Chroneos‡1
From the ‡Center of Biomedical Research, University of Texas Health Center, Tyler, Texas 75708-3154, §Section of Neonatology,
Perinatal and Pulmonary Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio 45229, the ¶Chemistry
Department and the 储Departments of Chemistry and Pathology, University of Virginia, Charlottesville, Virginia 22904
Mass spectrometric characterization of the surfactant protein A
(SP-A) receptor 210 (SP-R210) led to the identification of myosin
(Myo) XVIIIA and nonmuscle myosin IIA. Antibodies generated
against the unique C-terminal tail of MyoXVIIIA revealed that
MyoXVIIIA, MyoIIA, and SP-R210 have overlapping tissue distribution, all being highly expressed in myeloid cells, bone marrow,
spleen, lymph nodes, and lung. Western blot analysis of COS-1 cells
stably transfected with either MyoXVIIIA or MyoIIA indicated that
SP-R210 antibodies recognize MyoXVIIIA. Furthermore, MyoXVIIIA but not MyoIIA localized to the surface of COS-1 cells, and
most importantly, expression of MyoXVIIIA in COS-1 cells conferred SP-A binding. Western analysis of recombinant MyoXVIIIA
domains expressed in bacteria mapped the epitopes of previously
derived SP-R210 antibodies to the neck region of MyoXVIIIA. Antibodies raised against the neck domain of MyoXVIIIA blocked the
binding of SP-A to macrophages. Together, these findings indicate
that MyoXVIIIA constitutes a novel receptor for SP-A.
Surfactant protein A (SP-A)2 is a lipid-associated lung collectin playing an important role in binding and clearance of pathogens and the
regulation of inflammatory responses in the lung (1, 2). Studies in vitro
have demonstrated that SP-A is involved in both innate and adaptive
host defense through its ability to regulate both pro- and anti-inflammatory activities of macrophages (3–12), induce phagocytosis of
microbes (13–21) and apoptotic cells (22, 23), stimulate chemotaxis
(24 –26), inhibit lymphocyte proliferation (27–29), and inhibit dendritic
cell differentiation (30). Studies in SP-A null mutant mice support roles
* This work was supported by NHLBI Grants HL068127 (to Z. C. C.) and GM37537 (to
D. F. H.) and NHLBI SCOR Grant HL56387 (to J. A. W.) from the National Institutes of
Health and a Parker Francis Fellowship grant (to Z. C. C.). The costs of publication of
this article were defrayed in part by the payment of page charges. This article must
therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI
Data Bank with accession number(s) AAV80770, AY692137, AY692138, and AY692139.
1
To whom correspondence should be addressed: University of Texas Health Center,
Center of Biomedical Research, 11937 U. S. Highway 271, Tyler, TX 75708-3154. Tel.:
903-877-7941; Fax: 903-877-5876; E-mail: zissis.chroneos@uthct.edu.
2
The abbreviations used are: SP-A, surfactant protein A; SP-R210, surfactant protein A
receptor 210; MyoXVIIIA, myosin 18A; MyoIIA, nonmuscle myosin 2A; SP-R210S and
SP-R210L, short and long SP-R210 isoforms of MyoXVIIIA, respectively; MyoXVIIIAn,
MyoXVIIIA neck domain; MyoXVIIIAct, MyoXVIIIA C-terminal domain; LRP, lipoprotein
receptor-related protein; BSA, bovine serum albumin; UTR, untranslated region;
mAM, murine alveolar monocytes; IP, immunoprecipitation; PBS, phosphate-buffered saline; MALDI, matrix-assisted laser desorption ionization; TOF, time of flight;
HPLC, high performance liquid chromatography; MS, mass spectrometry; CHAPS,
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; MES, 4-morpholineethanesulfonic acid; DMEM, Dulbecco’s modified Eagle’s medium; NHS,
N-hydroxysuccinimide;PE, phycoerythrin; ER, endoplasmic reticulum.
OCTOBER 14, 2005 • VOLUME 280 • NUMBER 41
of SP-A in microbial clearance (31–35), inflammation (5, 8, 32, 33,
35– 40), and adaptive immunity (41).
The mechanisms that mediate a plethora of SP-A functions in mucosal host defense of the lung are not characterized in detail. A number of
cell-surface molecules have been implicated to mediate SP-A function.
For example, SP-A alters the binding of lipopolysaccharide to CD14,
while modulating peptidoglycan and zymosan-induced inflammation in
macrophages through TLR-2 (3, 6, 42). Guillot et al. (4) showed that
SP-A can stimulate macrophages through TLR-4. More recently, it was
shown that macropinocytosis of apoptotic cells by SP-A is mediated by
the binding of SP-A to a complex of calreticulin and CD91 (43) on
macrophages. In addition, binding of SP-A to SIRP␣ suppressed macrophage inflammation in alveolar macrophages (23, 43). Most interestingly, Kuroki and co-workers (13) and Schlesinger and co-workers (19)
have shown that SP-A binding can induce phagocytosis indirectly
through the up-regulation of the scavenger and mannose receptors,
respectively. Earlier, it was demonstrated that the binding of SP-A to the
high affinity SP-A receptor SP-R210 (44) coordinates macrophage activation and phagocytosis of mycobacteria (17, 45), suppresses the proliferation of T lymphocytes (46), and alters the metabolic activity of alveolar type II epithelial cells (44). Therefore, it was crucial to determine
the molecular identity of the SP-R210 receptor.
In the present study, we used a variety of biochemical, molecular, and
cellular experiments to determine that cell-surface MyoXVIIIA is the
high affinity SP-R210 receptor. Together our findings establish the first
physical link between SP-A-mediated functions and the unconventional
cell-surface MyoXVIIIA.
EXPERIMENTAL PROCEDURES
Materials—Chemicals were from Sigma, unless noted otherwise.
United States Department of Agriculture tested fetal bovine serum was
purchased from either Sigma or Hyclone (Logan, UT) and heat-inactivated at 56 °C. CHAPS detergent was from Calbiochem. Whatman
0.2-␮ PES filters, Millipore Centricon-Plus filtration units, Greiner tissue culture dishes, and Cell-Gro DMEM and RPMI tissue culture media
were purchased from Fisher. Sulfo-NHS-biotin, streptavidin-agarose,
and protein G-Sepharose were from Pierce. HiTrap protein G-Sepharose, PD-10-Sepharose pre-packed columns, and NHS-activated
Sepharose were from Amersham Biosciences. Na125I and ECL chemiluminescence kit were from PerkinElmer Life Sciences. Polyclonal antihuman platelet MyoIIA was from BTI Biomedical Technologies
(Stoughton, MA). PE-conjugated goat anti-rabbit IgG and PE-conjugated streptavidin were from BD Biosciences. Most electrophoresis supplies and molecular weight standards were from Bio-Rad. NOVEX
3– 8% Tris acetate gels, cell dissociation medium, ExpressHyb buffer,
Trizol, RadPrime random primer labeling kit, and pcDNA3.1 expres-
JOURNAL OF BIOLOGICAL CHEMISTRY
34447
SP-A Receptor SP-R210
sion vector were from Invitrogen. The Improm-II reverse transcription
system, Wizard SV miniprep DNA purification system, and TAQ
polymerase were from Promega (Madison, WI). The GeneJuice transfection reagent was from Novagen (San Diego, CA). Sequence grade
trypsin was from Roche Applied Science. Restriction enzymes, T4
ligase, and Vent威-polymerase were from New England Biolabs (Beverly,
MA). The COS-1, U937, and THP-1 cell lines were from the American
Tissue Culture Collection (Manassas, VA). The MyoXVIIIA cDNA
KIAA0216 clone ha04661 was obtained from the Kazusa DNA Research
Institute (Chiba, Japan). The human MyoIIA cDNA cloned into the
pCMV-XL6 expression vector was purchased from Origene (Rockville,
MD). The mouse and human cDNA for LRP-5 and LRP-6 were obtained
from Dr. J. F. Hess, Merck (47, 48). Therapeutic lung lavage obtained
from alveolar proteinosis patients was a gift of Dr. Francis McCormack,
University of Cincinnati College of Medicine (Cincinnati, OH).
Animals—Wild type C57BL/6 pathogen-free mice, 4 – 6 weeks of age,
were obtained from The Jackson Laboratories (Bar Harbor, ME). Pathogen-free Sprague-Dawley rats of 200 –250 g weight were obtained from
Charles River Breeding Laboratories (Worcester, MA). Animals were
used in accordance with institutional animal care and use committee
protocols.
Purification of SP-A—Human SP-A was purified from frozen alveolar
proteinosis lavage by modification of a procedure described previously
(49). Surfactant aggregate was concentrated by centrifugation at
5,000 ⫻ g and suspended in 50 ml of PBS, and 5-ml aliquots were stored
frozen at ⫺20 °C. Surfactant lipid was extracted by a dropwise addition
of concentrated lavage to isobutyl alcohol (1:5 volume ratio). Delipidated protein was centrifuged at 20,000 ⫻ g, partially dried under nitrogen gas, completely dried in a lyophilizer, and then rehydrated in extraction buffer overnight (EB: 25 mM Tris, pH 7.5, 0.15 M NaCl, and 20 mM
octyl-␤-D-glucoside). Rehydrated surfactant was extracted three times
by vigorous vortexing and centrifugation at 20,000 ⫻ g for 20 min in EB.
Insoluble SP-A was then dialyzed for 48 h against four changes of solubilization buffer (SB: 5 mM HEPES, pH 7.5, 0.02% azide). Insoluble protein was removed by centrifugation at 50,000 ⫻ g. The supernatant was
adjusted to 20 mM in CaCl2 and 1 M in NaCl by addition of 0.5 M CaCl2
and NaCl crystals to re-precipitate SP-A. SP-A was then collected at
20,000 ⫻ g for 20 min and washed three times in 5 mM HEPES, pH 7.5,
20 mM CaCl2, 1 M NaCl. The precipitated SP-A was solubilized in 5 mM
HEPES, 5 mM EDTA, pH 7.5, at a concentration of 1 mg/ml, and dialyzed extensively against SB to remove EDTA. The purified protein was
sterilized over a Whatman 0.2-␮ PES filter, and 1-mg aliquots were
lyophilized and stored frozen in an anhydrous environment at ⫺20 °C.
This procedure removed contaminating IgG and albumin.
Noncovalent Immobilization of SP-A—Purified SP-A (1 mg/ml) was
dialyzed against MES, pH 6.5, and incubated for 2 h on ice with 0.1
volume of fresh 1 mg/ml sulfo-NHS-biotin to label SP-A at its N terminus. Biotinylated SP-A was purified on PD-10-Sepharose pre-equilibrated in 5 mM HEPES, pH 7.5. Biotinylated SP-A (0.5 mg/ml) was then
added to 1.5 ml of packed streptavidin-agarose beads equilibrated in a
modified detergent extraction buffer containing CHAPS (50) (DEB: 20
mM Tris, pH 7.5, 1% CHAPS, 5 mM MgCl2, 5 mM CaCl2). Biotinylated
SP-A and streptavidin beads were rotated for 2 h at 4 °C and then
washed with DEB to remove unbound SP-A. Approximately 75% of
biotinylated SP-A bound to the streptavidin-agarose beads under these
conditions.
Cell Culture—All cell lines were cultured in DMEM or RPMI supplemented with 10% fetal bovine serum and maintained in a humidified
tissue culture incubator under an atmosphere of 95% air, 10% CO2 for
DMEM grown cells and 5% CO2 for RPMI grown cells.
34448 JOURNAL OF BIOLOGICAL CHEMISTRY
Isolation of SP-R210—Rat SP-R210 was obtained by detergent and
salt extraction of rat lung membranes as described previously (44).
Mouse SP-R210 was isolated from murine alveolar monocytes (mAM)
(51). Frozen stocks of mAM cells were seeded in 10 150-mm2 tissue
culture flasks and cultured to confluence. Confluent adherent cells were
lifted in DMEM containing 0.05% trypsin and 5 mM EDTA, and cells
were reseeded into 10 roller bottles containing 200 ml of DMEM. The
cells were cultured 4 days in a humidified roller bottle incubator to a
density of 300 million cells per flask. The cells in each flask were washed
twice in ice-cold PBS and then lysed in 20 ml of DEB buffer containing
a protease inhibitor mix (44). Post-nuclear supernatants were obtained
at 600 ⫻ g and allowed to stand overnight. Insoluble protein aggregates
were then removed by centrifugation at 100,000 ⫻ g for 1 h. Clarified
supernatants were concentrated 10-fold over 30,000 Mr cut-off Centricon-Plus filtration units. The concentrated extracts were pooled and
pre-adsorbed with 10 ml of streptavidin-agarose beads. Pre-adsorbed
extracts were obtained by centrifugation at 25,000 ⫻ g and rotated overnight at 4 °C with biotinylated SP-A䡠streptavidin-agarose beads. Bound
proteins were washed in a 1 ⫻ 5-cm glass column with 25 ml each of
DEB buffer and then with detergent free-DEB buffer. SP-A-bound proteins were eluted in 0.15-ml fractions using DEB containing 10 mM
EDTA and 1 M NaCl. Eluted proteins were visualized on silver-stained
SDS-polyacrylamide gels, and SP-R210 was monitored on Western
blots using SP-R210 antibodies (44). Appropriate fractions were pooled
and concentrated by centrifugation to a final volume of 20 ␮l over a
100,000 Mr cut-off Microcon-100 filtration unit.
Mass Spectrometry—The rat membrane SP-R210 was separated by
SDS-PAGE as described previously and visualized using colloidal blue
(Bio-Rad). The SP-R210 band was excised and in-gel digested with trypsin, and iodoacetamide-treated peptides were extracted according to a
standard procedure (52). Matrix-assisted laser desorption ionization
(MALDI) mass spectra were obtained on a MALDI-TOF SPEC SE mass
spectrometer in reflectron mode (Waters, Milford, MA). Peptides were
searched against Swiss-Prot and TREMBL data bases using the Peptident tool (53). The search was restricted to mammalian or rodent species for proteins between 160 and 240 kDa and pI 5–7. The pI restriction
was based on two-dimensional gel electrophoresis results indicating a
heterogeneous SP-R210 band within the 5–7 pI range. The mass tolerance was set at ⫾0.1– 0.5 Da. For peptide sequence analysis, mAM
SP-R210 was isolated as described above, fractionated by SDS-PAGE on
a Tris acetate gel (3– 8%), and visualized by silver staining according to
the procedure of Sanchez et al. (54). The silver-stained SP-R210 band
was excised and subjected to in-gel digestion with trypsin. The resulting
peptides were analyzed by nanoflow HPLC interfaced to electrospray
ionization on a quadrupole ion mass spectrometer (LCQ) (ThermoFinnigan, San Jose, CA) (55). MS/MS spectra were searched against
a nonredundant protein data base using SEQUEST (56). Sequence
assignments were verified by manual interpretation of the corresponding MS/MS spectra.
Primary Structure Analysis—Domain identification was accomplished using Pfam (57). Transmembrane segments were evaluated
using TMbase (58). Short sequence motifs were determined using the
eukaryotic linear motif resource (59). Sequence alignment was performed using MacVector software.
Expression of MyoXVIIIA and MyoIIA in COS-1 Cells—A blunt-end
BsaAI cDNA fragment of human MyoXVIIIA from KIAA0216 cDNA
clone hs04661 was inserted into the EcoRV site of the pcDNA3.1
expression vector. This cDNA fragment encompasses the predicted
open reading frame with flanking 68 and 250 bp of 5⬘- and 3⬘-UTR. The
protein encoded by this cDNA is designated as MyoXVIIIA␤/SP-R210S
VOLUME 280 • NUMBER 41 • OCTOBER 14, 2005
SP-A Receptor SP-R210
TABLE ONE
Identification of mouse Myosin XVIIIA in rat lung SP-R210
Following detergent and salt extraction of rat lung membranes, gel-purified SP-R210 was excised and in-gel trypsin-digested, and peptides were fractionated by
MALDI-assisted mass spectrometry and searched against mammalian proteins in Swiss-Prot and TREMBL data bases using the PeptIdent tool. The search was
restricted for iodoacetamide-modified peptides against proteins in the 5–7 pI and 160 –240-kDa mass range and a mass tolerance of a ⫾0.1– 0.5 Da. A total of 31
peptides ranging in mass between 666.56 and 3499.25 was measured. One or two missed cleavages were allowed.
Peptide
Measured mass
Calculated mass
⌬Mass
1
2
3
4
5
6
7
8
9
910.490
1168.550
1500.980
1608.930
1861.820
1972.090
2159.980
2426.470
2466.060
910.427
1168.597
1500.766
1608.933
1861.938
1972.009
2159.984
2426.446
2466.288
0.064
⫺0.046
0.215
⫺0.001
0.119
⫺0.093
0.003
⫺0.024
0.228
(see “Results”). Expression plasmids containing MyoXVIIIA␤/SPR210S in the sense or antisense orientations were produced in Escherichia coli, purified by standard procedures, and transfected into COS-1
cells using GeneJuice. Stable transfectants with plasmid in the sense
orientation (MyoXVIIIA␤/SP-R210S-COS-1 cells) or antisense orientation (control-COS-1 cells) were selected in the presence of 300 ␮g/ml
neomycin. The entire 7.5-kb MyoIIA cDNA was subcloned into the
NotI restriction site of the pcDNA3.1 vector.
Polyclonal Antibodies—Polyclonal antibodies against gel-purified rat
SP-R210 were described previously (44). The C-terminal domains
(MyoXVIIIAct) and the neck (MyoXVIIIAn) domains of MyoXVIIIA
were expressed in bacteria (60), and polyclonal antibodies were generated commercially in rabbits (CoCalico Biological Laboratories, Reamstown, PA) by a standard protocol using TiterMax (Sigma) as primary
adjuvant. Antigen boost was in incomplete Freund’s adjuvant. The
MyoXVIIIAct antigen was supplied as a lyophilized powder. Immunoaffinity-purified MyoXVIIIAct antibody was obtained by affinity chromatography using recombinant MyoXVIIIAct covalently attached to
NHS-activated Sepharose. The MyoXVIIIAn antigen was rendered
insoluble by dialysis in PBS and supplied as a lyophilized powder. Total
polyclonal IgG was purified by affinity chromatography on a HiTrap
protein G-Sepharose column.
Northern Blot Hybridization—Total RNA from indicated tissues and
cells was isolated using Trizol reagent according to the manufacturer’s
directions. For Northern hybridization, 10 ␮g of total RNA was separated using 1.2% agarose denaturing gels. The membranes were blocked
for 1 h in ExpressHyb buffer and hybridized in the same buffer with
indicated 32P-labeled cDNA probes for 1 h at 65 °C. Membranes were
washed three times for 30 min each in 3, 2, and 1⫻ SSC in the presence
of 0.1% SDS at 65 °C.
Western Blot Analysis—Total protein was isolated from the indicated
tissues using Trizol reagent after the isolation of total RNA according
to the manufacturer’s directions. Protein concentration was measured
using the BCA assay and BSA as standard. Protein was used immediately
after isolation. Proteins, 20 – 40 ␮g, were separated on SDS-polyacrylamide gels, transferred to nitrocellulose, and probed first with
indicated primary and then horseradish peroxidase-conjugated
secondary antibodies. Bound antibodies were visualized by enhanced
chemiluminescence.
Immunoprecipitation—mAM cells, seeded at 4 ⫻ 106 cells, were
grown for 48 h to 90% confluency in 150-mm2 tissue culture flasks
(Greiner). Next, the cells were washed in PBS, pH 7.4, lifted from tissue
culture flasks in enzyme-free cell dissociation medium (Invitrogen), and
washed again in PBS. The cells were then lysed in a modified immuno-
OCTOBER 14, 2005 • VOLUME 280 • NUMBER 41
Peptide location in mouse MyoXVIIIA
1643
DFESEKR1649
LLDAMRMYR1105
1971
NKGPSKAPSDDGSLK1985
305
LKVQPIPELSELSR318
1512
VVSLEAELQDISSQESK1528
698
AAYLLGCSLEELSSAIFK715
1351
AAEINGEVDDDDAGGEWRLK1370
1014
KKSLCIQIKLQVDALIDTIKR1034
1428
CQRLTAELQDTKLHLEGQQVR1448
1097
precipitation buffer (50) (IP: 50 mM HEPES, pH 7.5, 1% CHAPS, 0.1 M
NaCl, 5 mM CaCl2, 5 mM MgCl2, supplemented with DEB protease
inhibitor mixture (44)) at a concentration of 1 ⫻ 107 cells per ml at 4 °C
for 30 min with constant rotation. Post-nuclear lysates obtained at
14,000 ⫻ g were rotated at 4 °C for 1 h in the presence of 0.1 mg/ml
control IgG. 50 ␮l of a 30% protein G-Sepharose suspension in IP buffer
was then added, and the extracts were rotated for an additional hour at
4 °C. Pre-adsorbed extracts were then incubated 2 h to overnight with
the indicated antibodies. To capture immune complexes, lysates were
incubated with 50 ␮l of protein G-Sepharose beads for 2 h at 4 °C. The
beads were then washed three times in IP buffer, two times in detergentfree IP buffer containing 0.5 M NaCl, and an additional two times in IP
buffer. Protein G-bound proteins were eluted in 2⫻ Laemmli buffer.
Denatured proteins were then separated by 7% SDS-PAGE and analyzed
on Western blots as indicated.
SP-A Binding—Human SP-A was radiolabeled with 125I and used in
radioligand binding assays according to a procedure described previously (44). Binding assays were also performed using N-terminally biotinylated SP-A. Bound SP-A was visualized by flow cytometry using phycoerythrin-conjugated streptavidin.
Flow Cytometry—Cells were washed with PBS, lifted from the tissue
culture dishes in cell dissociation medium, and placed in FACS blocking
buffer (PBS supplemented with 5% heat-inactivated goat serum, 0.5%
BSA) at a concentration of 5–10 million cells per ml. Next, the cells were
incubated with 1 ␮g of rabbit control or immune antibody for 30 min,
washed twice in binding buffer, and then incubated for 30 min with 0.5
␮g of PE-conjugated goat anti-rabbit IgG. To measure the binding of
SP-A, mAM cells at 2 ⫻ 106 cells/ml were incubated with 25 ␮g/ml
biotinylated SP-A on ice for 2 h in 25 mM HEPES, pH 7.5, 1.5 mM CaCl2,
0.2 mM MgCl2, 1% BSA (44) and then washed twice in binding buffer
and incubated for 30 min with 0.5 ␮g of PE-conjugated streptavidin.
Control or MyoXVIIIAn antibodies at 50 ␮g/ml were added together
with SP-A. Labeled samples were analyzed by flow cytometry using a
Coulter Epics Elite instrument and Expo32 software.
RESULTS
Peptide Sequencing of SP-R210—The identity of SP-R210 was confirmed by two distinct approaches. Rat SP-R210 was obtained after
extensive extraction of detergent-insoluble membranes with potassium
chloride to deplete MyoIIA (44). Then peptide fingerprints were
obtained by MALDI-assisted mass spectrometry following in-gel trypsin digestion of rat SP-R210. This analysis resulted in peptide fingerprints that matched the unconventional mouse and human myosin
MyoXVIIIA (61– 63) (TABLE ONE). Only fragments of the rat homo-
JOURNAL OF BIOLOGICAL CHEMISTRY
34449
SP-A Receptor SP-R210
FIGURE 1. Purification of mouse SP-R210. Mouse SP-R210 was affinity-purified from
mAM cells using immobilized biotinylated SP-A as described under “Experimental Procedures.” A, detection of SP-R210 by Western analysis in unprocessed cell lysate and the
flow-through eluent after affinity chromatography. B, Western analysis of SP-R210 fractions eluted by 1 M NaCl, 10 mM EDTA (lanes 10 –17). Lane R indicates SP-R210 retained on
the column. C, silver staining of concentrated SP-R210 from the size-fractionated pooled
fractions. For Western analysis proteins were fractionated on 8 –10% SDS-polyacrylamide gels. Purified SP-R210 was electrophoresed on a 3– 8% Tris acetate gel.
log are currently reported in the NCBI data base. The SP-R210 band had
a more complex composition with an additional fingerprint matching
mouse, rat, and human rho-dependent citron kinase (64) and the
lipoprotein receptor-related protein LRP-5 (47, 48) in both mouse and
human. We expressed mouse and human LRP-5 and its close homolog
LRP-6 in COS-1 cells, but these receptors were not recognized by SPR210 antibodies and did not confer SP-A binding, suggesting that they
may have an indirect role in SP-A function (not shown). Because a single
receptor could not be identified in the SP-R210 band, we next sought to
purify SP-R210 in soluble form by affinity chromatography in order to
sequence its components by electrospray ionization mass spectrometry.
By timing the length in culture, we arrived at conditions where most
immunoreactive SP-R210 could be released from insoluble cytoskeletal
proteins of mAM cells (51) by using a modified extraction buffer (50)
containing CHAPS detergent instead of Triton X-100 (see “Experimental Procedures”). Solubilized SP-R210 was captured by affinity chromatography using N-terminally immobilized SP-A. Fig. 1A illustrates that
most immunoreactive SP-R210 in mAM extracts bound to noncovalently immobilized SP-A as less than 5% of immunoreactive SP-R210
was found in the flow-through (Fig. 1A), indicating that solubilized
mAM SP-R210 represents the same protein recovered in insoluble form
in rat lung membranes. SP-R210 was eluted slowly over several fractions
in the presence of 1 M NaCl and 10 mM EDTA (Fig. 1B, lanes 10 –17).
Most interestingly, a significant fraction of SP-R210 remained bound to
the SP-A affinity beads (Fig. 1B, lane R), suggesting a tight association
between immobilized SP-A and SP-R210. The salt/EDTA-resistant
SP-A䡠SP-R210 complex was completely eluted in the presence of 10 mM
dithiothreitol, but in this case the SP-R210 band was contaminated with
SP-A as judged by Western blot analysis (data not shown). To obtain
sequence information, the silver-stained pooled SP-R210 (see “Experi-
34450 JOURNAL OF BIOLOGICAL CHEMISTRY
mental Procedures”) (Fig. 1, B and C, lanes 11–16) was excised and
digested with trypsin, and the resulting digest was then analyzed by
nanoflow HPLC interfaced to electrospray ionization on an LCQ mass
spectrometer. Sequences of 26 peptides were obtained in this experiment. As shown in TABLE TWO, XVIII of the sequenced peptides
derived from two members of the myosin family of proteins. Thus, 16
peptides were identified as cellular MyoIIA, and two peptides were identified as MyoXVIIIA. The identification of MyoXVIIIA is consistent
with the results of TABLE ONE. The sequence of peptides 1 and 2 is
conserved in both mouse and human MyoXVIIIA (TABLE TWO). The
sequence of 10 MyoIIA peptides is identical in both mouse and human
homologs of MyoIIA (TABLE TWO). Peptides 6 and 14 are identical to
mouse MyoIIA, differing by a single amino acid residue from the human
homolog. Both peptides 7 and 9 are homologous to residues 342–355 of
the human MyoIIA peptide, but the alanine at position 353 indicates
that these peptides are derived from mouse MyoIIA. However, peptide
7 lacks leucine at position 349 and contains an alanine insertion at
position 343. This novel peptide has not been reported in the current
annotation of the C57BL/6 mouse genome. Peptide 9 contains valine at
position 343 instead of isoleucine. Peptide 9 is identical to a MyoIIA
variant in Xenopus laevis (GenBankTM accession A59282), suggesting
the presence of an additional variant of MyoIIA in mAM cells or polymorphism in this area of the molecule. Similarly, peptide 15 contains
methionine at position 869 instead of threonine in both reported mouse
and human MyoIIA sequences, but the leucine at position 874 matches
human MyoIIA. Of the eight additional peptides that were sequenced
(not shown on TABLE TWO), four were identical to human SP-A,
indicating that some SP-A leaked from the column during the high salt
wash. In addition, there were three peptides identical to ferritin light
chain, and one identical to ferritin heavy chain. The finding of 22-kDa
ferritin subunits in the SP-R210 band suggests a stable association
between ferritin and SP-A and a potential role of SP-A in local iron
availability or transport (65). Because MyoXVIIIA was identified by
both fingerprint analysis and direct peptide sequencing, we next
hypothesized that MyoXVIIIA is a valid candidate SP-A receptor molecule. To further focus on characterization of MyoXVIIIA as the SPR210 receptor, we first had to gain better understanding of the MyoXVIIIA structure.
Analysis of MyoXVIIIA Structure—Fig. 2A illustrates the domain
organization of the two major isoforms of MyoXVIIIA. The classification of this myosin is based on the sequence of the myosin motor
domain (63). In addition, this myosin has a typical IQ motif and a
dimeric coiled-coil domain as shown in Fig. 2A. The C-terminal 126 –
140 amino acids is unique to MyoXVIIIA (Fig. 2A, MyoXVIIIAct). The
amino acid sequence of the long and short variants of MyoXVIIIA is
shown in Fig. 2B, Underlined in lowercase letters are the locations of
identified peptides 1 and 2 (TABLE TWO). The long variants of this
myosin isoform are distinguished by the presence of an N-terminal PDZ
protein interaction domain and are shown on Fig. 2A as MyoXVIIIA␣/
MysPDZ␣/SP-R210L. The short variants of MyoXVIIIA lacking the
PDZ and KE sequence are shown on Fig. 2A as MyoXVIIIA␤/
MysPDZ␤/SP-R210S. Obinata and co-workers (61) identified the
MysPDZ␣ variant of MyoXVIIIA in stromal fibroblasts as a novel myosin having the PDZ protein interaction domain and a KE-rich sequence
at the amino terminus, and subsequently, Mori et al. (62) reported the
characterization of the MysPDZ␤ in spleen. These MyoXVIIIA variants
are generated by alternative RNA splicing. Northern analysis using a
cDNA probe representing the C-terminal coding sequence indicates the
expression of multiple tissue and cell-specific MyoXVIIIA mRNA species (Fig. 3A). Thus, spleen, liver, and kidney express a 7.5-kb mRNA as
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SP-A Receptor SP-R210
TABLE TWO
Identification of myosin XVIIIA and cellular myosin IIA in mouse SP-R210
Mouse SP-R210 was purified from mAM cells by affinity chromatography and size exclusion as described under “Experimental Procedures.” The excised
silver-stained SP-R210 band was in-gel trypsin-digested, and the resulting peptides were analyzed by nanoflow HPLC interfaced to electrospray ionization on a
quadrupole ion trap mass spectrometer (52). Sequenced SP-R210 peptides are aligned to the corresponding mouse (Ms) and human (Hm) MyoXVIIIA and
MyoIIA peptides. Underlined boldface letters are single amino acid substitutions in the corresponding HmMyoIIA sequence.
A) Myo XVIIIA
Peptide 1 AGSATVLSGSIAGLEGGSQLALR
MsMyoXVIIIA 973RAGSATVLSGSIAGLEGGSQLALR996
HuMyoXVIIIA 977RAGSATVLSGSIAGLEGGSQLALR1000
B) Nonmuscle myosin IIA
Peptide 3 EDQSILCTGESGAGK
MsMYOIIA 165REDQSILCTGESGAGK180
HuMYOIIA 165REDQSILCTGESGAGK180
Peptide 4
MsMYOIIA
HuMYOIIA
Peptide 5 QLLQANPILEAFGNAK
MsMYOIIA 209RQLLQANPILEAFGNAK225
HuMYOIIA 209RQLLQANPILEAFGNAK225
Peptide 6
MsMYOIIA
HuMYOIIA
Peptide 7 VIASGVLQGNIAFK
MsMYOIIA 341RVISGVLQLGNIAFK355
HuMYOIIA 341RVI SGVLQ LGNIVFK355
Peptide 8
MsMYOIIA
HuMYOIIA
Peptide 9 VVSAVLQLGNIAFK
MsMYOIIA 341RVISGVLQLGNIAFK355
HuMYOIIA 341RVISGVLQLGNIVFK355
Peptide 11 VVFQEFR
MsMYOIIA 711RVVFQEFR718
HuMYOIIA 711RVVFQEFR718
Peptide 10
MsMYOIIA
HuMYOIIA
Peptide 12
MsMYOIIA
HuMYOIIA
Peptide 13 ALELDSNLYR
MsMYOIIA 745KALELDSNLYR755
HuMYOIIA 745KALELDSNLYR755
Peptide 14
MsMYOIIA
HuMYOIIA
Peptide 15 LMEMET LQSQLMAEK
MsMYOIIA 867RLTEMETMQSQLMAEK882
HuMYOIIA 867RLTEMETLQSQLMAEK882
Peptide 16
MsMYOIIA
HuMYOIIA
Peptide 17 ELEDATETADAMNR
MsMYOIIA 1898RELEDATETADAMNR1912
HuMYOIIA 1898RELEDATETADAMNR1912
Peptide 18
MsMYOIIA
HuMYOIIA
reported by Furusawa et al. (61) for MysPDZ␣. However, the message
expressed by mAM and bone marrow cells is slightly smaller at 7.0 kb in
length, hence the designation of SP-R210L on Fig. 2A. On this note,
RT-PCR analysis of MyoXVIIIA mRNA supports the expression of
more N-terminal variants of MyoXVIIIA in mAM cells.3 Muscle and
heart express larger messages of 8.0 –9.0 kb. In addition to the 7.0 –
7.5-kb MyoXVIIIA messages, spleen and bone marrow also express
shorter 6.0 – 6.5-kb variants (Fig. 3A), reflecting the expression of
MyoXVIIIA␤/MysPDZ␤/SP-R210S (Fig. 4). These shorter mRNAs do
not hybridize with probes representing the PDZ domain (data not
shown) consistent with previous studies (62). The short message is
strongly expressed in the monocytic THP-1 and promonocytic U937
cell lines (Fig. 4A) and was found in lung and mAM cells on long exposure of the Northern blots (not shown). Two short variants of MyoXVIIIA have been identified. The sequence alignment in Fig. 3B compares
the 5⬘-UTR mRNA sequence of the MysPDZ␤ expressed in mouse
spleen cells (62) to the mRNA of MyoXVIIIA␤ produced by human
KG-1 myelocytes (62). The latter mRNA is studied further in the present report and is termed as MyoXVIIIA␤/SP-R210S to reflect the role of
this isoform in SP-A binding (see below). The protein sequence of
MyoXVIIIA␤/SP-R210S is shown in boldface letters in Fig. 2B. The pre3
Peptide 2
MsMyoXVIIIA
HuMyoXVIIIA
C-H. Yang and Z. C. Chroneos, unpublished data.
OCTOBER 14, 2005 • VOLUME 280 • NUMBER 41
ELQTQYDALKK
KELQTQYDALKK1334
1327
KELQTQYDALKK1338
1323
QTLENERGELANEVK
KQTLENERGELANEVK1234
1219
KQTLENERGELANEVK1234
1219
LQVELDSVTGLLSQSDSK
KLQVELDSVTGLLSQSDSK1295
1277
KLQVELDNVTGLLSQSDSK1295
1277
DFSALESQLQDTQELLQEENR
KDFSALESQLQDTQELLQEENR1322
1301
KDFSALESQLQDTQELLQEENR1322
1301
ALEQQVEEMK
RALEQQVEEMK1539
1529
RALEQQVEEMK1539
LEVNLQAMK
1557
RLEVNLQAMKA1566
1557
RLEVNLQAMKA1566
1529
DLEAHIDTANK
KDLEAHIDTANK1631
1620
KDLEAHIDSANK1631
1620
QLEEAEEEAQR
RQLEEAEEEAQR1888
1877
RQLEEAEEEAQR1888
1877
IIGLDQVAGMSETALPGAFK
RIIGLDQVAGMSETALPGAFK637
617
RIIGLDQVAGMSETALPGAFK637
617
dicted start codon in MyoXVIIIA␤/SP-R210S is at position 485 bp
downstream from the start codon of MysPDZ␤ (arrows in Fig. 3B). A
69-nucleotide insertion in the human MyoXVIIIA␤/SP-R210S splice
variant introduces two in-frame stop codons (underlined in Fig. 3B).
Although an equivalent MyoXVIIIA␤/SP-R210S splice variant in mouse
remains to be established, it is also possible that MysPDZ␤ may express
more than one variant by alternative start codon usage.
Identification of SP-R210 as MyoXVIIIA—To determine the tissue
distribution of MyoXVIIIA variants, we generated polyclonal antibodies
against the common C terminus of MyoXVIIIA/MysPDZ/SP-R210
(MyoXVIIIAct on Fig. 2A) isoforms. Given the identification of MyoIIA
shown in TABLE TWO, we compared the tissue distribution of MyoXVIIIA, SP-R210, and MyoIIA by Western blot analysis. The results of
Fig. 4 demonstrate that MyoXVIIIAct antibodies recognize short 210 –
220-kDa (SP-R210S) and long 230 –250-kDa protein species reflecting
the expression of short and long variants of MyoXVIIIA mRNA species
in lung, bone marrow, and the immunopoietic organs spleen and lymph
(Fig. 4A). Smaller species closer to 150 kDa were also detected. Previously we showed that SP-R210 isolated from U937 is highly labile to
proteolysis (44). More recently, Hamilton and co-workers (66) identified a 110-kDa form of MyoXVIIIA in U937 cells. Most interestingly, the
MyoXVIIIAct antibodies did not detect MyoXVIIIA protein in heart,
liver or muscle extracts (Fig. 4A) suggesting low MyoXVIIIA protein
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FIGURE 2. Primary structure analysis of MyoXVIIIA. A, N-terminal 485 amino acids of the longest MyoXVIIIA contain a KE motif, a PDZ domain, clusters of phosphorylation (PO3) sites
for casein kinases, protein kinases A and B, and a proline-rich SH3-binding site. An N-terminal KE-rich sequence is present in MysPDZ. Additional phosphorylation sites were also
detected in the motor domains and the unique C terminus. The myosin heavy chain motor domain is found between amino acids 485 and 1186. The neck region between amino acids
1186 and 1246 contains an IQ motif and a C-mannosylation site. The dimeric coiled-coil domain spans amino acids 1246 and 1938, and the N-terminal sequence from 1938 to
2035/2054 is unique to MyoXVIIIA. The square box in the C terminus indicates an alternatively spliced coiled-coil insertion. The vertical shaded box in the motor domain outlines a
putative transmembrane helix (TM). The short isoform of MyoXVIIIA lacks the N-terminal 485 amino acids. The long and short isoforms are designated as MyoXVIIIA␣/MysPDZ␣/SPR210L and the short as MyoXVIIIA␤/MysPDZ␤/SP-R210S, respectively; their boundaries indicated by solid arrowheads. Antibodies were made against recombinant neck MyoXVIIIAn
and C-terminal MyoXVIIIAct domains; the boundary of each domain is indicated by opposite arrows. B, the amino acid sequence of the MyoXVIIIA␤/SP-R210S isoform (see Fig. 3B) is
shown in boldface letters. The underlined peptides in lowercase letters were identified by MS/MS (TABLE TWO). The boxed sequence is a putative transmembrane helix. The putative
signal peptidase cleavage site MyoXVIIIA␤/SP-R210S is underlined at position 48 of the boldface sequence.
levels despite the strong mRNA expression of MyoXVIIIA (Fig. 3A) or
additional C-terminal splicing of the MyoXVIIIA RNA in these tissues.
Kidney has low but detectable expression of MyoXVIIIA and SP-R210
(Fig. 4, A and B). Western analysis using antibodies to the originally
described rat SP-R210 (44) and commercial antibodies to MyoIIA indicate overlapping tissue distribution of MyoXVIIIA (Fig. 4A), SP-R210
(Fig. 4B), and MyoIIA (Fig. 4C), all being highly expressed in immunopoietic organs. However, the proteins detected by both MyoIIA and
SP-R210 antibodies in the lung were significantly more intense compared with the MyoXVIIIAct antibodies, suggesting that these antibodies detect only a fraction of MyoXVIIIA isoforms having the cognate
C-terminal domain. In this regard, during the course of this work, we
identified two additional splice variants of the C-terminal domain differing by the insertion of a 15-amino acid coiled-coil domain (gray box,
Fig. 2A), the isoform with the longer C-terminal domain being highly
expressed in lung (60). However, the overlapping tissue distribution
may also be related to cross-reactivity of SP-R210 antibodies with both
MyoXVIIIA and MyoIIA. To establish the specificity of these antibodies, we generated stable COS-1 cells transfected with either the MyoXVIIIA␤/SP-R210S (67) isoform or MyoIIA. COS-1 cells do not express
MyoIIA (68). Fig. 5 demonstrates that control COS cells express an
endogenous 250-kDa protein cross-reacting only with MyoXVIIIAct
antibodies, suggesting that this endogenous MyoXVIIIA does not display the epitopes recognized by SP-R210 antibodies (top and bottom
34452 JOURNAL OF BIOLOGICAL CHEMISTRY
panels of Fig. 5, A1 and A2). Both SP-R210 and MyoXVIIIAct antibodies
readily recognize MyoXVIIIA␤/SP-R210S (top and bottom panels of Fig.
5, A1 and A2) but not MyoIIA, whereas antibodies to MyoIIA are specific to MyoIIA-expressing cells only (middle panels of Fig. 5, A1 and
A2). Together, these results indicate that SP-R210 antibodies recognize
the MyoXVIIIA␤ isoform. Given the identification of MyoIIA shown on
TABLE TWO, we also wanted to determine whether MyoXVIIIA and
MyoIIA coisolate as a complex in the absence of SP-A. Immunoprecipitation experiments using mAM cells and MyoXVIIIAct antibodies were
carried out, and immunoprecipitated proteins were probed with SPR210, MyoXVIIIAct, and MyoIIA antibodies. The results on Fig. 5B
demonstrate that both short and long MyoXVIIIA/SP-R210 variants
were readily precipitated by MyoXVIIIAct antibodies that were recognized by both SP-R210 and MyoXVIIIAct antibodies, but only a single
MyoIIA protein was recognized by the MyoIIA antibodies. The presence of a single species recognized by anti-MyoIIA indicates that
MyoXVIIIA and MyoIIA coprecipitate as a complex that is not because
of cross-reactivity of the SP-R210 antibody with MyoIIA.
Expression of MyoXVIIIA␤/SP-R210S Confers SP-A Binding—We
next hypothesized that if indeed MyoXVIIIA␤/SP-R210S confers SP-A
binding, it is likely to be localized on the surface of COS-1 cells. The flow
cytometry results of Fig. 6, A and B, demonstrate that MyoXVIIIA␤/SPR210S is readily localized to the cell-surface of stably transfected COS-1
cells by both MyoXVIIIAct (Fig. 6A) and original SP-R210 antibodies
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SP-A Receptor SP-R210
FIGURE 3. Expression of MyoXVIIIA mRNA variants. A, Northern hybridization using a human
300-bp 32P-labeled C-terminal cDNA was carried
out as described under “Experimental Procedures.” Each lane was loaded with 10 ␮g of total
RNA. The C-terminal probe hybridized to tissueand cell-specific mRNAs ranging between 6.0 and
9.0 kb. S, spleen; Lu, lung; BM, bone marrow; M,
muscle; Lv, liver; K, kidney; H, heart. B, comparison
of 5⬘-UTR DNA sequence of mouse MysPDZ␤ and
human SP-R210S. Two arrows indicate the start
codons of MysPDZ␤ and SP-R210S, respectively.
(Fig. 6B). A low level of the endogenous MyoXVIIIA is also present on
the surface of control COS-1 cells. In contrast, stable transfection of
MyoIIA (Fig. 6C) did not render this protein to the cell surface. This
finding indicates that the targeting of MyoXVIIIA␤/SP-R210S to the cell
surface is specific to this protein and not likely an artifact of exogenous
protein overexpression. Moreover, Fig. 7 demonstrates that expression
of MyoXVIIIA␤/SP-R210S induced saturable 125I-SP-A binding, conferring over 2.5-fold increase in SP-A binding from 500,000 ⫾ 70,000
sites/cell in control cells to 1,200,000 ⫾ 120,000 sites/cell in MyoXVIIIA␤/SP-R210S-COS-1 cells. The dissociation constants are similar
with a Kd of 15.8 ⫾ 3.8 nM in control and 13.1 ⫾ 2.5 nM in MyoXVIIIA␤/
SP-R210S-expressing COS-1 cells, suggesting that endogenous MyoXVIIIA also confers some SP-A binding.
OCTOBER 14, 2005 • VOLUME 280 • NUMBER 41
Mapping of Inhibitory Antibodies—In order to generate inhibitory
antibodies to block SP-A binding, we produced recombinant neck (MyoXVIIIAn) and C-terminal (MyoXVIIIAct) domains (Fig. 2A) with His tags at
their C terminus (60). The 47-kDa MyoXVIIIAn and 16-kDa MyoXVIIIAct
domains are shown on the colloidal blue-stained gel on Fig. 8A, lanes 1 and
2, respectively. The MyoXVIIIAn domain is heterogeneous with smaller
fragments that are also His-tagged at the C terminus (60). The Western blot
analysis on Fig. 8A, lanes 3 and 4, indicates that the SP-R210 antibodies
described earlier (17, 44–46) recognize MyoXVIIIAn but not MyoXVIIIAct. Because the SP-R210 antibodies recognized mainly the full-length
recombinant protein, these results indicate that some SP-R210 epitopes are
located within a 7–10-kDa region of MyoXVIIIA between the IQ motif and
part of the coiled-coil domain. To determine whether the MyoXVIIIAn
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FIGURE 4. Tissue expression of MyoXVIIIA, SP-R210, and MyoIIA. Western analysis
was carried out using anti-MyoXVIIIAct (A), anti-SP-R210 (B), or anti-MyoIIA (C) antibodies. Proteins were separated on 8 (A) or 7% SDS-polyacrylamide gels (B and C). Each lane
was loaded with 20 ␮g of protein except lung that was loaded with 40 ␮g of protein. S,
spleen; Lu, lung; BM, bone marrow; M, muscle; Lv, liver; K, kidney; H, heart; LN, lymph
node.
FIGURE 6. Cell-surface localization of MyoXVIIIA␤/SP-R210S. Control-, MyoXVIIIA␤/
SP-R210S-, and MyoIIA-COS-1 cells were obtained as described under “Experimental Procedures.” Flow cytometric analysis on intact cells using either MyoXVIIIAct (A) or original
SP-R210 (B) (44) antibodies demonstrated cell-surface expression of MyoXVIIIA␤/SPR210S. In contrast, MyoIIA antibodies did not detect MyoIIA on the surface of MyoIIACOS-1 cells (C). The open histograms show staining with nonimmune polyclonal rabbit
IgG (A–C). Gray histograms show staining with rabbit anti-MyoXVIIIAct (A), SP-R210 (B), or
anti-MyoIIA (C) antibodies.
FIGURE 5. Identification of SP-R210 as MyoXVIIIA and coimmunoprecipitation with
MyoIIA. A, control, MyoXVIIIA␤/SP-R210S (A1), and MyoIIA-expressing COS-1 (A2) cells
were obtained as described under “Experimental Procedures” and were probed with
MyoXVIIIAct, MyoIIA, or SP-R210 antibodies on Western blots. Control cells were transfected with plasmids having either MyoXVIIIA␤/SP-R210S or MyoIIA cDNA cloned in the
antisense orientation. Each lane was loaded with Laemmli lysates from 70,000 cells. B,
cell lysates from mAM cells were incubated with MyoXVIIIAct antibodies, and immunoprecipitated (i.p.) proteins were analyzed on Western blots (w.b.) using SP-R210, MyoXVIIIAct, or MyoIIA antibodies as indicated. Proteins were separated on 7% SDS-polyacrylamide gels; the gels were allowed to run off until the 37-kDa marker reached the bottom
of the gel to allow separation of long and short isoforms of MyoXVIIIA.
domain mediates SP-A binding, we generated new polyclonal antibodies
against MyoXVIIIAn (Fig. 8A, lane 5). Next, we evaluated the effect of
MyoXVIIIAn antibodies on SP-A binding. Fig. 8B shows that the MyoX-
34454 JOURNAL OF BIOLOGICAL CHEMISTRY
FIGURE 7. Expression of MyoXVIIIA␤/SP-R210S in COS-1 cells confers SP-A binding.
Binding of SP-A to control and MyoXVIIIA␤/SP-R210S-expressing cells was determined
using 125I-labeled SP-A. The saturation isotherm and Scatchard analysis of binding data
(inset) show 2.5-fold increase in SP-A binding to MyoXVIIIA␤/SP-R210S-expressing cells.
VIIIAn antibodies reduced SP-A binding 70% compared with control level
of SP-A binding in the presence of preimmune IgG. Antibodies to the
C-terminal MyoXVIIIAct domain also did not block SP-A binding (not
shown). Furthermore, the anti-MyoXVIIIAn antibodies blocked SP-A
binding in a concentration-dependent manner to a maximum 60% of con-
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SP-A Receptor SP-R210
FIGURE 8. The neck domain of MyoXVIIIA/SP-R210 mediates SP-A binding to macrophages. A, purified recombinant MyoXVIIIAn (lane 1) and MyoXVIIIAct (lane 2) proteins
are shown on a colloidal blue-stained 10% SDS-PAGE. Western blot analysis indicated
that SP-R210 antibodies (44) recognized only the MyoXVIIIAn domain (lane 3) but not
MyoXVIIIAct (lane 4). Specific antibodies against MyoXVIIIAn were generated in rabbits
(lane 5). Each lane was loaded with 100 ng of protein. For Western blotting antibodies
were used at 0.1 ␮g/ml. Antibodies were pre-adsorbed against E. coli extracts. B, the
effect of MyoXVIIIAn antibodies on the binding of biotinylated SP-A (bSP-A) was determined by flow cytometry. Bound bSP-A was measured using PE-conjugated streptavidin. Anti-MyoXVIIIAn antibodies (right panel) decreased the mean fluorescence intensity
of bound SP-A by 70% compared with preimmune IgG (left panel). Antibodies were used
at 50 ␮g/ml. C, concentration-dependent inhibition of SP-A binding by MyoXVIIIAn antibodies. Data are means ⫾ S.E., n ⫽ 6.
trol (Fig. 8C). Higher concentrations of MyoXVIIIAn antibodies did not
lead to complete inhibition, suggesting that either mAM cells express additional SP-A-binding sites or that the coverage of SP-A-binding epitopes by
the MyoXVIIIAn antibodies is incomplete. Together, these results indicate
that the SP-A receptor SP-R210 is identical to cell-surface isoforms of
MyoXVIIIA.
DISCUSSION
Previous studies utilizing inhibitory antibodies ascribed functional
roles for SP-R210 on macrophage phagocytosis and activation (17, 44,
45), T lymphocyte proliferation (46), and lipid secretion in alveolar type
II epithelial cells (44), suggesting an important physiological role of
SP-R210 in regulating SP-A functions in the lung. Here we have determined by several lines of evidence, including mass spectrometry, cell-
OCTOBER 14, 2005 • VOLUME 280 • NUMBER 41
surface localization by flow cytometry on live cells, heterologous expression, and development of blocking antibodies, that SP-R210 is a cellsurface form of MyoXVIIIA acting as a high affinity SP-A receptor.
Previous studies identified two major isoforms of this novel myosin in
stromal and hematopoietic cells and tissues (61, 62). Here we also demonstrate multiple mRNA variants being expressed in a tissue- and cellspecific manner, but, in addition, we demonstrate the distinct and dominant distribution of MyoXVIIIA␤/SP-R210S in immune cells and
organs implying that MyoXVIIIA isoforms have important roles in
immune modulation. The present work is the first to assign a functional
role for the MyoXVIIIA myosin class as cell-surface proteins. In parallel
with the finding that MyoXVIIIA and MyoIIA coimmunoprecipitate,
our findings also support a model where SP-A functions are mediated
through exofacial MyoXVIIIA and submembrane MyoIIA.
The MyoXVIIIA gene encodes diverse mRNA and protein species,
the latter having the ability to assume different subcellular localizations.
Mori et al. (69) and Isogawa et al. (70) generated MysPDZ fusion proteins having fluorescent protein at the N or C terminus of mouse and
human MyoXVIIIA, respectively, to visualize their subcellular localization. These studies showed that MyoXVIIIA localized to the actin
cytoskeleton by a mechanism that required the KE motif at the N terminus (69, 70) but without the need for ATP hydrolysis unlike other
myosins (70). Mori et al. (69) also determined that the PDZ domain is
sufficient for the localization of MysPDZ␣ to the inner surface of the
plasma membrane. In addition, MysPDZ␣ was found to colocalize with
ER/Golgi membranes, suggesting a potential role in membrane traffic.
In contrast, MysPDZ␤, lacking both KE and PDZ sequences, had a diffuse cytoplasmic localization. The exofacial targeting of MyoXVIIIA
shown in the present work is based on cell-surface localization with
specific antibodies, and functional expression analyses demonstrating
the presence of heterologous and endogenous protein on the cell surface. Based on these results, it is possible that some of the MyoXVIII〈
proteins fused to N- or C-terminal fluorescent proteins are also
expressed on the cell surface (69, 70). The mechanisms that mediate the
exit of MyoXVIIIA isoforms to the cell surface as shown in the present
report are not presently known. Computational analysis of the primary
structure of MyoXVIIIA␤/SP-R210S (Fig. 2A) suggests a signal peptidase site preceded by a short hydrophobic stretch having the potential
to serve as an N-terminal signal sequence. A putative transmembrane
helix is present within the motor domain (Fig. 2A). Most interestingly,
there are several proximal methionine residues that may potentially be
utilized as start codons at the N terminus of both long and short variants
of MyoXVIIIA to express multiple MyoXVIIIA isoforms with different
subcellular localizations. For instance, utilization of alternative initiation codons in the plectin mRNA has been shown to generate plectin
variants with different subcellular sites (71). In addition, the MysPDZ␤
and MyoXVIIIA␤/SP-R210S isoforms of MyoXVIIIA have different
5⬘-UTR sequences (Fig. 3B), indicating that alternative splicing of small
exons may contribute further to the diversity and function of MyoXVIIIA isoforms. On this note, there are alternative small exon variants at
the C terminus of MyoXVIIIA (Fig. 2A) (60), and several mRNA species
from different tissues having internal deletions or insertions of small
exons have already been published in NCBI (GenBankTM accession
numbers AY692137, AY692138, and AY692139, NM_078471,
BC039612, and NM_203318). The generation of functional diversity by
alternative splicing of small exons occurs frequently in the myosin family genes (72). In addition, it is also possible that internal motifs targeting
MyoXVIIIA to ER/Golgi membranes may facilitate its secretion to the
cell surface. For instance, internal targeting motifs are thought to be
responsible for ER transport of the intermediate filament vimentin to
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the cell surface of a subset of microvascular endothelial cells and activated macrophages (73, 74). Computational analysis of the MyoXVIIIA
primary structure also indicates a C-mannosylation motif (75), convertase-type dibasic motifs (76), and more ubiquitous casein kinase and
protein kinase phosphorylation sites, each having a potential role in
post-translational processing, cellular signaling, and localization. Posttranslational cleavage of MyoXVIIIA is thought to be responsible for the
generation of a 110-kDa isoform in U937 cells (66). It has also been
shown that PDZ domains play a role in polarized protein secretion (77).
In the present case, it is notable that both long and short isoforms of
MyoXVIIIA can be expressed in the same cell, possibly having a role in
targeting of MyoXVIIIA isoforms to the cell surface.
Based on our findings we postulate that surface MyoXVIIIA/SP-R210
isoforms link a variety of SP-A functions via intracellular MyoIIA. Previously, it was demonstrated that the binding of SP-A to macrophages
signals the reorganization of the actin cytoskeleton (78). Downstream, a
number of critical cell functions have been ascribed to MyoIIA in live
cells such as receptor capping (79), cell shape (80), cytokinesis (81), and
vesicle transport (68). In macrophages, multiple myosin motor proteins
are involved in phagosome formation (82, 83), although the role of
MyoIIA in phagocytosis is less clear. In addition, MyoIIA associates with
the uropod during T cell motility, tethering the formation of immune
synapses during antigen recognition (12, 84). In the same context,
MyoIIA interacts directly with the chemokine receptor CXCR4 in T
lymphocytes (85). The results of TABLE TWO suggest the presence of
an additional cellular MyoII isoform coisolating with MyoXVIIIA/SPR210. Adelstein and co-workers (86) have already identified a third nonmuscle myosin II family member, MyoIIC. Mechanistically, the aforementioned intracellular functions of MyoIIA are related to a variety of
functional activities of SP-A on cell motility and migration (25, 26), lipid
secretion, lymphocyte proliferation (46), and phagocytosis (13, 17, 21,
22, 82).
The mediation of SP-A function by MyoXVIIIA/SP-R210 could
occur directly by transversing the cell membrane or indirectly by binding to additional surface molecules. The topology of long and short
MyoXVIIIA/SP-R210 isoforms on the cell membrane requires additional study. In recent studies a theme has emerged regarding the ability
of SP-A to stimulate a variety of surface molecules indirectly. For example, it has been demonstrated that SP-A augments the activity of the
phagocytic scavenger and mannose receptors indirectly (13, 16, 19),
although the acting SP-A-binding site stimulating these receptors is not
known. Two more examples linking SP-A function to more than one
surface molecule indirectly are CD14 (42) and calreticulin (23). Thus,
binding of SP-A to CD14 could be responsible for the indirect activation
or inhibition of TLR4 and the binding of SP-A to calreticulin bridges
SP-A-mediated apoptotic cell clearance to CD91. It becomes compelling to determine the role of MyoXVIIIA/SP-R210 as a primary SP-Abinding site on linking the internalization of SP-A-opsonized pathogens
to macrophage phagocytic and signaling receptors.
It was postulated above that extracellular MyoXVIIIA/SP-R210 links
to MyoIIA inside the cell. However, Wright and co-workers (87) also
demonstrated that SP-A interacts with MyoIIA, and their results indicated a role for SP-A in the clearance of cellular myosin released from
dead cells. The identification of MyoIIA in the present report is consistent with this finding, but our immunoprecipitation results also indicate
that MyoXVIIIA and MyoIIA are intimately linked at the plasma membrane, having exofacial and intracellular locations, respectively. However, there is evidence that MyoIIA can be accessible to cell-surface
iodination of intact cells (88). Conditions that may expose MyoIIA to
the cell surface may occur during membrane repair as a consequence of
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injury to the cell membrane. On this note, it was recently demonstrated
that MyoIIA facilitates exocytosis-dependent cell membrane sealing
(68), but this process in live cells is too rapid to allow detection of
intracellular MyoIIA by bulky proteins such as antibodies and SP-A.
The cells would have to sustain significant damage before SP-A could
gain access to intracellular MyoIIA. On the other hand, MyoIIA is subject to specific degradation by caspases in apoptotic cells (89) and consequently may have a role in SP-A-mediated clearance of apoptotic cells
as well (22). Thus, it is conceivable that externalization of MyoIIA peptides on the cell surface marks apoptotic cells for recognition by SP-A
and clearance by bridging MyoIIA-tagged apoptotic cells to MyoXVIIIA/SP-R210-expressing macrophages.
In summary, we have identified SP-R210 as cell-surface MyoXVIIIA.
Additional studies are required to dissect the functional consequences
of SP-A binding to MyoXVIIIA expressed on macrophages and other
immune cells. The distinct expression of MyoXVIIIA/SP-R210 suggests
that this receptor has more functions in hematopoietic and immune
cells. Understanding the expression patterns of MyoXVIIIA isoforms is
critical for the generation of appropriate animal models. The mechanism of interaction between MyoXVIIIA/SP-R210, SP-A, and other collectins requires additional investigation.
REFERENCES
1. McCormack, F. X., and Whitsett, J. A. (2002) J. Clin. Investig. 109, 707–712
2. van de Wetering, J. K., van Golde, L. M., and Batenburg, J. J. (2004) Eur. J. Biochem.
271, 1229 –1249
3. Sato, M., Sano, H., Iwaki, D., Kudo, K., Konishi, M., Takahashi, H., Takahashi, T.,
Imaizumi, H., Asai, Y., and Kuroki, Y. (2003) J. Immunol. 171, 417– 425
4. Guillot, L., Balloy, V., McCormack, F. X., Golenbock, D. T., Chignard, M., and SiTahar, M. (2002) J. Immunol. 168, 5989 –5992
5. Hohlfeld, J. M., Erpenbeck, V. J., and Krug, N. (2002) Pathobiology 70, 287–292
6. Murakami, S., Iwaki, D., Mitsuzawa, H., Sano, H., Takahashi, H., Voelker, D. R.,
Akino, T., and Kuroki, Y. (2002) J. Biol. Chem. 277, 6830 – 6837
7. Hickman-Davis, J. M., Fang, F. C., Nathan, C., Shepherd, V. L., Voelker, D. R., and
Wright, J. R. (2001) Am. J. Physiol. 281, L517–L523
8. Borron, P., McIntosh, J. C., Korfhagen, T. R., Whitsett, J. A., Taylor, J., and Wright,
J. R. (2000) Am. J. Physiol. 278, L840 – 847
9. Stamme, C., Walsh, E., and Wright, J. R. (2000) Am. J. Respir. Cell Mol. Biol. 23,
772–779
10. Song, M., and Phelps, D. S. (2000) Infect. Immun. 68, 6611– 6617
11. Pasula, R., Wright, J. R., Kachel, D. L., and Martin, W. J., II (1999) J. Clin. Investig. 103,
483– 490
12. Alcorn, J. F., and Wright, J. R. (2004) Am. J. Physiol. 286, L129 –L136
13. Kuronuma, K., Sano, H., Kato, K., Kudo, K., Hyakushima, N., Yokota, S. I., Takahashi,
H., Fujii, N., Suzuki, H., Kodama, T., Abe, S., and Kuroki, Y. (2004) J. Biol. Chem. 279,
21421–21430
14. Oberley, R. E., Ault, K. A., Neff, T. L., Khubchandani, K. R., Crouch, E. C., and Snyder,
J. M. (2004) Am. J. Physiol. 287, L296 –L306
15. van Iwaarden, F., Welmers, B., Verhoef, J., Haagsman, H. P., and van Golde, L. M.
(1990) Am. J. Respir. Cell Mol. Biol. 2, 91–98
16. Gaynor, C. D., McCormack, F. X., Voelker, D. R., McGowan, S. E., and Schlesinger,
L. S. (1995) J. Immunol. 155, 5343–5351
17. Weikert, L. F., Edwards, K., Chroneos, Z. C., Hager, C., Hoffman, L., and Shepherd,
V. L. (1997) Am. J. Physiol. 272, L989 –L995
18. Madan, T., Eggleton, P., Kishore, U., Strong, P., Aggrawal, S. S., Sarma, P. U., and Reid,
K. B. (1997) Infect. Immun. 65, 3171–3179
19. Beharka, A. A., Gaynor, C. D., Kang, B. K., Voelker, D. R., McCormack, F. X., and
Schlesinger, L. S. (2002) J. Immunol. 169, 3565–3573
20. Lopez, J. P., Clark, E., and Shepherd, V. L. (2003) J. Leukocyte Biol. 74, 523–530
21. Tino, M. J., and Wright, J. R. (1996) Am. J. Physiol. 270, L677–L688
22. Schagat, T. L., Wofford, J. A., and Wright, J. R. (2001) J. Immunol. 166, 2727–2733
23. Vandivier, R. W., Ogden, C. A., Fadok, V. A., Hoffmann, P. R., Brown, K. K., Botto, M.,
Walport, M. J., Fisher, J. H., Henson, P. M., and Greene, K. E. (2002) J. Immunol. 169,
3978 –3986
24. Kramer, B. W., Jobe, A. H., Bachurski, C. J., and Ikegami, M. (2001) Am. J. Respir. Crit.
Care Med. 163, 158 –165
25. Schagat, T. L., Wofford, J. A., Greene, K. E., and Wright, J. R. (2003) Am. J. Physiol.
284, L140 –L147
26. Wright, J. R., and Youmans, D. C. (1993) Am. J. Physiol. 264, L338 –L344
27. Borron, P., Veldhuizen, R. A., Lewis, J. F., Possmayer, F., Caveney, A., Inchley, K.,
VOLUME 280 • NUMBER 41 • OCTOBER 14, 2005
SP-A Receptor SP-R210
McFadden, R. G., and Fraher, L. J. (1996) Am. J. Respir. Cell Mol. Biol. 15, 115–121
28. Wang, J. Y., Shieh, C. C., You, P. F., Lei, H. Y., and Reid, K. B. (1998) Am. J. Respir. Crit.
Care Med. 158, 510 –518
29. Borron, P. J., Mostaghel, E. A., Doyle, C., Walsh, E. S., McHeyzer-Williams, M. G., and
Wright, J. R. (2002) J. Immunol. 169, 5844 –5850
30. Brinker, K. G., Garner, H., and Wright, J. R. (2003) Am. J. Physiol. 284, L232–L241
31. Linke, M. J., Harris, C. E., Korfhagen, T. R., McCormack, F. X., Ashbaugh, A. D.,
Steele, P., Whitsett, J. A., and Walzer, P. D. (2001) J. Infect. Dis. 183, 943–952
32. LeVine, A. M., Whitsett, J. A., Gwozdz, J. A., Richardson, T. R., Fisher, J. H., Burhans,
M. S., and Korfhagen, T. R. (2000) J. Immunol. 165, 3934 –3940
33. LeVine, A. M., Kurak, K. E., Bruno, M. D., Stark, J. M., Whitsett, J. A., and Korfhagen,
T. R. (1998) Am. J. Respir. Cell Mol. Biol. 19, 700 –708
34. LeVine, A. M., Kurak, K. E., Wright, J. R., Watford, W. T., Bruno, M. D., Ross, G. F.,
Whitsett, J. A., and Korfhagen, T. R. (1999) Am. J. Respir. Cell Mol. Biol. 20, 279 –286
35. Hickman-Davis, J. M., Gibbs-Erwin, J., Lindsey, J. R., and Matalon, S. (2004) Am. J.
Respir. Cell Mol. Biol. 30, 319 –325
36. Haddad, I. Y., Milla, C., Yang, S., Panoskaltsis-Mortari, A., Hawgood, S., Lacey, D. L.,
and Blazar, B. R. (2003) Am. J. Physiol. 285, L602–L610
37. Hawgood, S., Ochs, M., Jung, A., Akiyama, J., Allen, L., Brown, C., Edmondson, J.,
Levitt, S., Carlson, E., Gillespie, A. M., Villar, A., Epstein, C. J., and Poulain, F. R. (2002)
Am. J. Physiol. 283, L1002–L1010
38. Yang, S., Milla, C., Panoskaltsis-Mortari, A., Hawgood, S., Blazar, B. R., and Haddad,
I. Y. (2002) Am. J. Respir. Cell Mol. Biol. 27, 297–305
39. Li, G., Siddiqui, J., Hendry, M., Akiyama, J., Edmondson, J., Brown, C., Allen, L., Levitt,
S., Poulain, F., and Hawgood, S. (2002) Am. J. Respir. Cell Mol. Biol. 26, 277–282
40. Chabot, S., Koumanov, K., Lambeau, G., Gelb, M. H., Balloy, V., Chignard, M., Whitsett, J. A., and Touqui, L. (2003) J. Immunol. 171, 995–1000
41. LeVine, A. M., Hartshorn, K., Elliott, J., Whitsett, J., and Korfhagen, T. (2002) Am. J.
Physiol. 282, L563–L572
42. Sano, H., Chiba, H., Iwaki, D., Sohma, H., Voelker, D. R., and Kuroki, Y. (2000) J. Biol.
Chem. 275, 22442–22451
43. Gardai, S. J., Xiao, Y. Q., Dickinson, M., Nick, J. A., Voelker, D. R., Greene, K. E., and
Henson, P. M. (2003) Cell 115, 13–23
44. Chroneos, Z. C., Abdolrasulnia, R., Whitsett, J. A., Rice, W. R., and Shepherd, V. L.
(1996) J. Biol. Chem. 271, 16375–16383
45. Weikert, L. F., Lopez, J. P., Abdolrasulnia, R., Chroneos, Z. C., and Shepherd, V. L.
(2000) Am. J. Physiol. 279, L216 –L223
46. Borron, P., McCormack, F. X., Elhalwagi, B. M., Chroneos, Z. C., Lewis, J. F., Zhu, S.,
Wright, J. R., Shepherd, V. L., Possmayer, F., Inchley, K., and Fraher, L. J. (1998) Am. J.
Physiol. 275, L679 –L686
47. Brown, S. D., Twells, R. C., Hey, P. J., Cox, R. D., Levy, E. R., Soderman, A. R., Metzker,
M. L., Caskey, C. T., Todd, J. A., and Hess, J. F. (1998) Biochem. Biophys. Res. Commun.
248, 879 – 888
48. Hey, P. J., Twells, R. C., Phillips, M. S., Yusuke, N., Brown, S. D., Kawaguchi, Y., Cox,
R., Guochun, X., Dugan, V., Hammond, H., Metzker, M. L., Todd, J. A., and Hess, J. F.
(1998) Gene (Amst.) 216, 103–111
49. Haagsman, H. P., Sargeant, T., Hauschka, P. V., Benson, B. J., and Hawgood, S. (1990)
Biochemistry 29, 8894 – 8900
50. Zhu, Q., Zelinka, P., White, T., and Tanzer, M. L. (1997) Biochem. Biophys. Res.
Commun. 232, 354 –358
51. Shibata, Y., Berclaz, P. Y., Chroneos, Z. C., Yoshida, M., Whitsett, J. A., and Trapnell,
B. C. (2001) Immunity 15, 557–567
52. Jensen, O. N., Wilm, M., Shevchenko, A., and Mann, M. (1999) in 2-D Proteome
Analysis Protocols (Andrew, J. L., ed) Vol. 112, pp. 487–512, Humana Press Inc.,
Totowa, NJ
53. Wilkins, M. R., and Williams, K. L. (1997) J. Theor. Biol. 186, 7–15
54. Sanchez, J. C., Hochstrasser, D., and Rabilloud, T. (1999) Methods Mol. Biol. 112,
221–225
55. Shabanowitz, J., Settlage, R. E., Marto, J. A., Christian, R. E., White, F. M., Russo, P. S.,
Martin, S. E., and Hunt, D. F. (2000) in Mass Spectrometry in Biology and Medicine
(Burlingame, A.L., Carr, S.A., and Baldwin, M.A., eds) pp.163–177, Humana Press,
Totowa, NJ
OCTOBER 14, 2005 • VOLUME 280 • NUMBER 41
56. Yates, J. R., III, Eng, J. K., and McCormack, A. L. (1995) Anal. Chem. 67, 3202–3210
57. Bateman, A., Coin, L., Durbin, R., Finn, R. D., Hollich, V., Griffiths-Jones, S., Khanna,
A., Marshall, M., Moxon, S., Sonnhammer, E. L., Studholme, D. J., Yeats, C., and Eddy,
S. R. (2004) Nucleic Acids Res. 32, D138 –D141
58. Hofmann, K., and Stoffel, W. (1993) Biol. Chem. Hoppe-Seyler 374, 166
59. Puntervoll, P., Linding, R., Gemund, C., Chabanis-Davidson, S., Mattingsdal, M.,
Cameron, S., Martin, D. M., Ausiello, G., Brannetti, B., Costantini, A., Ferre, F., Maselli, V., Via, A., Cesareni, G., Diella, F., Superti-Furga, G., Wyrwicz, L., Ramu, C.,
McGuigan, C., Gudavalli, R., Letunic, I., Bork, P., Rychlewski, L., Kuster, B., HelmerCitterich, M., Hunter, W. N., Aasland, R., and Gibson, T. J. (2003) Nucleic Acids Res.
31, 3625–3630
60. Szeliga, J., Jordan, J., Yang, C.-H., Sever-Chroneos, Z., and Chroneos, Z. C. (2005)
Anal. Biochem., in press
61. Furusawa, T., Ikawa, S., Yanai, N., and Obinata, M. (2000) Biochem. Biophys. Res.
Commun. 270, 67–75
62. Mori, K., Furusawa, T., Okubo, T., Inoue, T., Ikawa, S., Yanai, N., Mori, K. J., and
Obinata, M. (2003) J. Biochem. (Tokyo) 133, 405– 413
63. Berg, J. S., Powell, B. C., and Cheney, R. E. (2001) Mol. Biol. Cell 12, 780 –794
64. Di Cunto, F., Calautti, E., Hsiao, J., Ong, L., Topley, G., Turco, E., and Dotto, G. P.
(1998) J. Biol. Chem. 273, 29706 –29711
65. Ghosh, S., Hevi, S., and Chuck, S. L. (2004) Blood 103, 2369 –2376
66. Cross, M., Csar, X. F., Wilson, N. J., Manes, G., Addona, T. A., Marks, D. C., Whitty,
G. A., Ashman, K., and Hamilton, J. A. (2004) Biochem. J. 380, 243–253
67. Nagase, T., Seki, N., Ishikawa, K., Ohira, M., Kawarabayasi, Y., Ohara, O., Tanaka, A.,
Kotani, H., Miyajima, N., and Nomura, N. (1996) DNA Res. 3, 321–329, 341–354
68. Togo, T., and Steinhardt, R. A. (2004) Mol. Biol. Cell 15, 688 – 695
69. Mori, K., Matsuda, K., Furusawa, T., Kawata, M., Inoue, T., and Obinata, M. (2005)
Biochem. Biophys. Res. Commun. 326, 491– 498
70. Isogawa, Y., Kon, T., Inoue, T., Ohkura, R., Yamakawa, H., Ohara, O., and Sutoh, K.
(2005) Biochemistry 44, 6190 – 6196
71. Rezniczek, G. A., Abrahamsberg, C., Fuchs, P., Spazierer, D., and Wiche, G. (2003)
Hum. Mol. Genet. 12, 3181–3194
72. Kim, K. Y., Kovacs, M., Kawamoto, S., Sellers, J. R., and Adelstein, R. S. (2005) J. Biol.
Chem. 280, 22769 –22775
73. Xu, B., deWaal, R. M., Mor-Vaknin, N., Hibbard, C., Markovitz, D. M., and Kahn,
M. L. (2004) Mol. Cell. Biol. 24, 9198 –9206
74. Mor-Vaknin, N., Punturieri, A., Sitwala, K., and Markovitz, D. M. (2003) Nat. Cell
Biol. 5, 59 – 63
75. Furmanek, A., and Hofsteenge, J. (2000) Acta Biochim. Pol. 47, 781–789
76. Nakayama, K. (1997) Biochem. J. 327, 625– 635
77. Ikemoto, M., Arai, H., Feng, D., Tanaka, K., Aoki, J., Dohmae, N., Takio, K., Adachi,
H., Tsujimoto, M., and Inoue, K. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 6538 – 6543
78. Tino, M. J., and Wright, J. R. (1999) Am. J. Physiol. 276, L164 –L174
79. Pasternak, C., Spudich, J. A., and Elson, E. L. (1989) Nature 341, 549 –551
80. Xu, X. S., Lee, E., Chen, T., Kuczmarski, E., Chisholm, R. L., and Knecht, D. A. (2001)
Dev. Biol. 232, 255–264
81. Knecht, D. A., and Loomis, W. F. (1987) Science 236, 1081–1086
82. Swanson, J. A., Johnson, M. T., Beningo, K., Post, P., Mooseker, M., and Araki, N.
(1999) J. Cell Sci. 112, 307–316
83. Cox, D., Berg, J. S., Cammer, M., Chinegwundoh, J. O., Dale, B. M., Cheney, R. E., and
Greenberg, S. (2002) Nat. Cell Biol. 4, 469 – 477
84. Wulfing, C., and Davis, M. M. (1998) Science 282, 2266 –2269
85. Rey, M., Vicente-Manzanares, M., Viedma, F., Yanez-Mo, M., Urzainqui, A., Barreiro,
O., Vazquez, J., and Sanchez-Madrid, F. (2002) J. Immunol. 169, 5410 –5414
86. Golomb, E., Ma, X., Jana, S. S., Preston, Y. A., Kawamoto, S., Shoham, N. G., Goldin,
E., Conti, M. A., Sellers, J. R., and Adelstein, R. S. (2004) J. Biol. Chem. 279, 2800 –2808
87. Michelis, D., Kounnas, M. Z., Argraves, W. S., Sanford, E. D., Borchelt, J. D., and
Wright, J. R. (1994) Am. J. Respir. Cell Mol. Biol. 11, 692–700
88. Olden, K., Willingham, M., and Pastan, I. (1976) Cell 8, 383–390
89. Kato, M., Fukuda, H., Nonaka, T., and Imajoh-Ohmi, S. (2005) J. Biochem. (Tokyo)
137, 157–166
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