The Lung Biology Center, Department of Medicine, University of California, San Francisco, California 94143
Adhesion of blood leukocytes to the endothelium involves multiple steps including initial attachment (tethering), rolling, and firm arrest. Presentation
of adhesion molecules on leukocyte microvilli can substantially enhance tethering. Localization of L-selectin
to microvilli and of CD44 to the planar cell body have been shown to depend upon their transmembrane and
cytoplasmic domains. We investigated the role of leukocyte integrin transmembrane and cytoplasmic domains in initiating adhesion under flow and in microvillous localization. Integrins 4
7,
L
2, and
M
2 were heterologously expressed in K562 cells.
4
7 initiated
adhesion under flow and localized to microvilli,
whereas
2 integrins did not initiate adhesion and localized to the cell body. Chimeric integrins were produced by replacing the
4
7 cytoplasmic and/or transmembrane domains with the homologous domains of
L
2 or
M
2. Unexpectedly, these chimeras efficiently mediated adhesion to the
4
7 ligand mucosal
addressin cell adhesion molecule-1 under flow and localized to microvilli. Therefore, differences between the transmembrane and cytoplasmic domains of
4 and
2 integrins do not account for differences in ability to
support attachment under flow or in membrane localization. Integrins
4
1,
5
1,
6A
1,
v
3, and
E
7
also localized to microvilli. Transmembrane proteins
known or suspected to associate with extracellular domains of microvillous integrins, including tetraspans
and CD47, were concentrated on microvilli as well.
These findings suggest that interactions between the
extracellular domains of integrins and associated proteins could direct the assembly of multimolecular complexes on leukocyte microvilli.
LEUKOCYTE recruitment to tissues from blood involves a
series of adhesive interactions between leukocytes
and the vascular endothelium (Springer, 1994 Presentation of certain leukocyte adhesion molecules on
microvilli substantially enhances the ability of these molecules to support tethering and rolling on endothelial ligands.
The importance of receptor distribution was highlighted
by studies of the adhesion molecules L-selectin and CD44
(von Andrian et al., 1995 Available evidence about the distribution of integrins
on the leukocyte surface is also consistent with a role for
microvillous presentation in initial adhesion under flow.
Integrins We sought to examine the role of leukocyte integrin
transmembrane and cytoplasmic domains in microvillous
localization and in initial adhesion under flow. Here we report that replacement of Cell Lines
K562 human erythroleukemia cells (CCL 243; American Type Culture
Collection, Rockville, MD) were maintained in growth medium: RPMI
1640 supplemented with 10% FBS, penicillin (50 IU/ml), streptomycin (50 µg/ml), and glutamine (2 mM). Stably transfected K562 lines expressing
human integrin cDNAs
The pCDM8-integrin
Transfections and Expression
K562 cells in log phase growth were washed twice in electroporation
buffer (HBSS, 20 mM Hepes, 6 mM dextrose). 8 × 106 cells were resuspended in 0.2 ml buffer and transferred to 2 mm electroporation cuvettes
(BTX, San Diego, CA). Stable transfections were performed using the
pCDM8- Antibodies
Fib 504 (anti-integrin Immunoelectron Microscopy
Transfectants were immunolabeled in suspension. Cells were prefixed for
20 min in 0.2% paraformaldehyde/PBS at 4°C. After washing twice with
PBS containing 10% goat serum, cells were incubated with primary antibody diluted in PBS containing 10% goat serum. After a 30-min incubation at 4°C and a wash with PBS, gold-conjugated secondary antibody in
PBS was added. Cells were incubated for 30 min at 4°C, washed twice with
PBS, and fixed in 0.1 M sodium cacodylate buffer, pH 7.4, with 3% glutaraldehyde. Before embedding, cells were rinsed in 0.1 M cacodylate buffer
and postfixed in 1% osmium tetroxide/0.1 M cacodylate buffer. After rinsing, cells were dehydrated through a graded series of acetone washes, and infiltrated and embedded in Spurr's epoxy resin (Ted Pella, Inc., Redding,
CA). Sections (70-nm thick) were stained with uranyl acetate and lead citrate, and examined with a CM120 Phillips electron microscope (Philips
Electron Optics, Inc., Mahwah, NJ). 50-100 cells were examined and representative cells were photographed. Each experiment was repeated at
least twice. Colloidal gold distribution on immunolabeled cells was determined by analysis of electron micrographs (×19,500-×40,000). Gold particles associated with cell body or microvilli were counted from 3-11 micrographs that represented different individual cells.
Static Adhesion Assay
Static adhesion assays were performed as previously described (Tidswell
et al., 1997 Adhesion under Flow
Capillary tubes (100-µl capacity; Drummond, Broomall, PA) were coated
at 4°C overnight with 20 µl of solution containing MAdCAM-IgG (0.2 µg/
ml) or ICAM-1-C Generation of Transfectants Expressing Chimeric and
Mutant To examine the role of cytoplasmic and transmembrane
domains of
Adhesion of Transfectants to MAdCAM-1 under
Static Conditions
We analyzed the ability of integrin-transfected K562 cells
to adhere to MAdCAM-1, an
Adhesion of Transfectants to MAdCAM-1 under Flow
We next examined the ability of the wild-type and chimeric integrins to initiate adhesion under flow (Fig. 4).
K562-
Membrane Localization of Wild-type and
Chimeric Previous reports demonstrated that integrin
Table I.
Integrin Distribution on Transfected K562 Cells
; Butcher
and Picker, 1996
). In some cases, initial binding of leukocyte adhesion molecules to their endothelial ligands can
lead to the transient arrest, or tethering, of the leukocyte
followed by leukocyte rolling. Rolling cells can then be
"activated" via incompletely understood mechanisms, which lead to an increase in the activity of certain adhesion molecules and the arrest of the leukocyte on the lumenal surface of the endothelium. In other cases, leukocytes may arrest immediately, without rolling. After arrest,
leukocytes can extravasate into the underlying tissue. Different leukocyte adhesion molecules are used for different
steps in this process (von Andrian et al., 1991
). L-selectin
and the E- and P-selectin ligands are expressed on some
leukocytes and mediate initial adhesion (tethering and rolling), but do not support firm arrest. In contrast, the
leukocyte
2 integrins
L
2 (LFA-1, CD11a/CD18) and
M
2 (Mac-1, CD11b/CD18) mediate firm arrest but not
initial adhesion. Another integrin subfamily, the
4 integrins
4
1 (VLA-4, CD49d/CD29) and
4
7 (LPAM-1),
can support both initial adhesion and firm arrest (Sriramarao et al., 1994
; Alon et al., 1995
; Berlin et al., 1995
).
). L-selectin is located primarily
on microvilli, whereas CD44 is concentrated on the planar
cell body. L-selectin-CD44 chimeras were used to examine the role of cytoplasmic and transmembrane domains in
receptor localization and the ability to roll on ligands. A
chimera comprising the L-selectin extracellular domain
fused to the CD44 transmembrane and cytoplasmic domains (L/CD44) localized to the cell body, and a CD44 extracellular, L-selectin transmembrane and cytoplasmic domain chimera (CD44/L) localized to microvilli. Although
replacement of the transmembrane and cytoplasmic domains of L-selectin or CD44 did not alter their ability to
adhere under static (no flow) conditions, it affected adhesion under flow. L-selectin (on microvilli) supported initial
attachment better than the L/CD44 chimera (cell body),
whereas CD44/L (microvilli) supported initial attachment better than CD44 (cell body). These results indicate that
the transmembrane and/or cytoplasmic domains account
for the differences in localization of L-selectin and CD44,
and strongly suggest that microvillous localization is important for optimal initial adhesion under flow.
L
2 and
M
2 are concentrated on the planar
cell body and do not support initial adhesion, whereas
4
1 and
4
7 localize primarily to microvilli and do support tethering and rolling (Erlandsen et al., 1993
; Berlin et
al., 1995
). The mechanism underlying the differential topography of these integrins on nonadherent leukocytes is
not known. However, studies of other integrins have established a central role for the cytoplasmic domains of integrin
subunits in localization of integrins on membranes
of adherent cells (LaFlamme et al., 1992
, 1994
; Briesewitz
et al., 1993
; Sastry and Horwitz, 1993
; Ylanne et al., 1993
;
Pasqualini and Hemler, 1994
). Interactions with several
cytoskeletal proteins such as talin and
-actinin (demonstrated in vitro) are suggestive of links to microfilament fibers that may regulate protein localization. In addition,
several transmembrane proteins, such as tetraspan proteins (including CD9, CD53, CD63, CD81, and CD82)
(Slupsky et al., 1989
; Rubinstein et al., 1994
; Berditchevski
et al., 1995
, 1996
, 1997
), CD32 (Fc
RIIA) (Worth et al.,
1996
), and CD47 (integrin-associated protein) (Lindberg et al., 1993
), have been shown to associate with integrins.
These interactions are known or suspected to involve the
extracellular domains of these proteins, and their role (if
any) in integrin localization is unknown.
4
7 transmembrane and cytoplasmic domains with the homologous domains of
2 integrins does not alter membrane localization or initiation of
adhesion under flow. This unexpected result suggests that
differences in localization of leukocyte integrins to microvilli are determined by the extracellular domain.
Materials and Methods
4 (K562-
4
1) and
4
7 (K562-
4
7) were described
previously (Tidswell et al., 1997
). Additional K562 integrin transfectants
were provided by other investigators: K562-
L
2 and K562-
M
2 (I. Graham, Washington University, St. Louis, MO) (Graham et al., 1994
);
K562-
6A
1 (A. Sonnenberg, The Netherlands Cancer Institute, Amsterdam, The Netherlands) (Hogervorst et al., 1993
); K562-
v
3 (S. Blystone,
Washington University) (Blystone et al., 1994
).
4 cDNA plasmid (Kamata et al., 1995
) was a gift
from Y. Takada (Scripps Research Institute, La Jolla, CA). The cloning of
the
7 cDNA has been previously described (Erle et al., 1991
). Integrin
2
cDNA (Hickstein et al., 1988
) was provided by D. Hickstein (University
of Washington, Seattle, WA). Integrin
L (Larson et al., 1989
) and
M
(Corbi et al., 1988
) cDNAs were provided by T. Springer (Center for
Blood Research, Boston, MA).
E cDNA (Shaw et al., 1994
) was a gift
from G. Russell and M. Brenner (Harvard Medical School, Boston, MA).
Chimeric integrin subunits were constructed using splice overlap extension PCR (Horton et al., 1989
). The amino acid splice sites for each construct are shown in Fig. 1. Chimeric
subunits were subcloned into
pCDM8 (Invitrogen, San Diego, CA) and chimeric
subunits were subcloned into pCEP4 (Invitrogen). The integrity of the constructs was confirmed by DNA sequencing.
Fig. 1.
Schematic representation of wild-type and chimeric
4
7 integrins. The amino acid sequence at the splice site is shown
with conserved regions in bold, and the
L
2 or
M
2 sequences
underlined.
[View Larger Version of this Image (27K GIF file)]
4 and pCEP-
7 constructs (25 µg each) plus 5 µg pBK-neo
(Stratagene, La Jolla, CA) at 900 µFarad, 200 V, 13
(Electro Cell Manipulator 600; BTX). Samples were left at room temperature for 10 min
before and after electroporation. Cells were then placed in 20 ml growth
medium and maintained at 37°C in a 5% CO2 incubator. After 48 h, transfectants were selected using growth medium containing 500 µg/ml each of
Hygromycin B (Calbiochem-Novabiochem Corp., La Jolla, CA) and G418
(GIBCO BRL, Gaithersburg, MD). Stably transfected clones were obtained by limiting dilution and analyzed by flow cytometry as previously described (Tidswell et al., 1997
). Transfectants were maintained in selection medium.
7) was a gift of E.C. Butcher (Stanford University,
Palo Alto, CA) (Andrew et al., 1994
). HP1/2 was used to detect the integrin
4 subunit (Pulido et al., 1991
). 7E4 (anti-
2) and GoH3 (anti-
6)
were purchased from Immunotech (Westbrook, ME). L230 (anti-
v) and
B11G2 (anti-
5) were provided by D. Sheppard and C. Damsky (University
of California, San Francisco, CA). Antibodies against the integrin-associated proteins CD53 (clone HI29; PharMingen, San Diego, CA), CD63
(MAB1787; Chemicon International, Inc., Temecula, CA), and CD32
(clone IV.3; Medarex, East Annandale, NJ) were obtained from commercial sources. The anti-CD47 antibody B6H12 was a gift from E. Brown
(Washington University). Hybridoma supernatants or purified IgG preparations were used for flow cytometry analysis and immunoelectron microscopy. Gold-conjugated secondary antibodies (6- or 12-nm particles)
were purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA).
). Briefly, 21-well glass slides (Structure Probe, West Chester,
PA) were coated with mucosal addressin cell adhesion molecule-1 (MAdCAM-1)1-IgG fusion protein (4 ng/well) or intercellular adhesion molecule-1 (ICAM-1)-C
fusion protein (0.23 µg/well) overnight at 4°C.
MAdCAM-1-IgG (Tidswell et al., 1997
) was a gift of M. Briskin (Leukosite Inc., Boston, MA). ICAM-1-C
(Piali et al., 1995
) was a gift of B. Imhof (Centre Medicale Universitaire, Geneva, Switzerland). After blocking with 4% BSA for 2 h, 4 × 104 cells were resuspended in 15 µl of 10 mM
Hepes, 150 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, with or without 1 mM
MnCl2, and added to the wells. Cells were allowed to adhere for 90 min at
room temperature. After washing, cells were fixed with 3% glutaraldehyde and stained with 0.5% crystal violet. Adherent cells were counted using a microscope. Assays were performed in triplicate.
(0.7 mg/ml) and blocked with 4% BSA for 2 h at 37°C.
K562 transfectants were washed with HBSS containing 1 mM EDTA, and
resuspended at 5 × 105 cells/ml in HBSS/10 mM Hepes containing 1 mM
each of Ca2+ and Mg2+. To measure adhesion under flow, cells were perfused
through the coated capillary tube using a syringe pump (74900 series; Cole-Parmer Instrument Co., Vernon Hills, IL) at a flow rate of 0.67 ml/
min. Calculated shear stress was 1.0 dyne/cm2 according to Pousille's law
of dynamic shear (Berlin et al., 1995
). Results were captured using a TMS
microscope (Nikon, Garden City, NJ), CCD video camera (Sony, Park
Ridge, NJ), and time lapse SVMS videocassette recorder (Panasonic, Secaucus, NJ). After 2 min of flow, five randomly chosen fields from the
coated region were analyzed for 5 s each. All adherent cells (rolling or arrested) were counted. Cells did not adhere to areas coated with 4% BSA alone (control).
Results
4
7 Constructs
4
7 in adhesion and membrane localization,
we expressed chimeric integrin heterodimers (Fig. 1).
Each construct included the extracellular domains of
4
and
7. In one construct, designated
4
7(
L
2c), most
of the cytoplasmic domains of
4 and
7 were replaced
with homologous regions of
L and
2. In a second construct,
4
7(
M
2tc), all of the transmembrane and cytoplasmic domains of
4 and
7 were replaced with homologous domains of
M and
2. The integrin
and
subunit
cDNAs were cotransfected into K562 human erythroleukemia cells, which do not normally express
4,
L,
M,
2,
or
7. The levels of protein expression on the transfectants
K562-
4
7, K562-
4
7(
L
2c), and K562-
4
7(
M
2tc)
were determined to be similar by flow cytometry (Fig. 2,
A-C). Two truncated cDNAs,
4
(truncated after amino acids GFFKR) and
7
(truncated after amino acids
VLAYR), were also produced. The
4
was expressed in
combination with
7 on transfected K562 cells (K562-
4
7),
although at levels somewhat below those seen with other
constructs (data not shown). We were unable to detect expression of
7
on cells cotransfected with
4, despite a
previous report that the homologous truncation mutant of
mouse
7 was expressed on transfected cells (Crowe et al., 1994
). We were able to document heterologous expression
of other wild-type integrins, including
L
2,
M
2,
6A
1,
v
3, and
E
7, on appropriate K562 transfectants by
flow cytometry (Fig. 2, D-H).
Fig. 2.
Cell surface expression of wild-type and chimeric integrins. K562-4
7 (A), K562-
4
7 (
L
2c) (B), and K562-
4
7
(
M
2tc) (C) cells were stained with the anti-
7 antibody, Fib
504, as shown. Each of these three transfectants was also recognized by other antibodies specific for the
4 subunit or the
4
7
heterodimer, but were not recognized by anti-
E antibodies (not
shown). K562-
L
2 were recognized by antibodies to
2 (D) and
L (not shown). K562-
M
2 cells were recognized by antibodies
to
2 (E) and
M (not shown). K562-
E
7 cells stained with antibodies to
7 (F) and
E, but not with anti-
4 antibodies (not
shown). K562-
6A
1 cells were stained with the anti-
6 antibody
GoH3 (G). There was low level expression of
v on nontransfected K562 cells, and higher expression on K562-
v
3 cells as
determined using the anti-
v antibody L230 (H). Fluorescence
intensity is shown on a log scale (one log per division). Dotted
and solid histograms represent staining with nontransfected and
transfected K562 cells, respectively.
[View Larger Version of this Image (36K GIF file)]
4
7 ligand, and ICAM-1,
an
L
2 ligand, under static (no flow) conditions (Fig. 3).
After transfection with
4 cDNA alone, K562 cells express
4
1 but not
4
7 (Tidswell et al., 1997
). These cells
failed to adhere to MAdCAM-1. In contrast, cells transfected with both
4 and
7 (K562-
4
7) adhered efficiently to MAdCAM-1. As previously reported, adhesion
was increased in the presence of Mn2+. The chimeric transfectants, K562-
4
7(
L
2c) and K562-
4
7(
M
2tc), also
adhered to MAdCAM-1, and the extent of adhesion was
very similar for chimeric and wild-type
4
7 transfectants.
As expected, K562-
4
7 cells did not adhere to ICAM-1,
whereas K562-
L
2 cells did.
Fig. 3.
Static adhesion of
transfectants to immobilized
ligands. Adhesion of various
integrin transfectants to
MAdCAM-1 (top) and ICAM-1
(bottom) was measured in
the presence and absence of
Mn2+. Bars indicate SEM.
nd, not determined.
[View Larger Version of this Image (20K GIF file)]
4
7 cells adhered to MAdCAM-1 under flow. This
adhesion was dependent upon both
4
7 and MAdCAM-1
because K562 cells transfected with
4 alone (K562-
4
1)
did not adhere to MAdCAM-1, and K562-
4
7 cells did
not adhere to capillary tubes coated with other ligands,
such as ICAM-1. The chimeric integrins,
4
7(
L
2c) and
4
7 (
M
2tc), both supported adhesion to MAdCAM-1
under flow. Wild-type
4
7 and the chimeric integrins
were very similar in their ability to initiate adhesion under
flow. We also examined the resistance to detachment from
MAdCAM-1 at increasing shear stress conditions (up to
10 dynes/cm2). We did not find differences between wild-type and chimeric transfectants in this assay (data not
shown). At a shear stress of 1 dyne/cm2, most cells remained adherent during the time interval of cell counts.
Transfectants expressing a truncated
4 subunit (K562-
4
7) also adhered to MAdCAM-1 under flow, although
at a somewhat lower rate (perhaps related to lower levels
of expression of this construct, data not shown). As expected from previous reports (von Andrian et al., 1991
),
K562-
L
2 cells were unable to initiate adhesion to their
ligand, ICAM-1, under flow (Fig. 4).
Fig. 4.
Adhesion to ligands
under flow. Adhesion of various integrin transfectants to
MAdCAM-1 (top) and ICAM-1
(bottom) measured at a wall
shear stress of 1 dyne/cm2 in
the absence of Mn2+, as described in Materials and
Methods. Bars indicate SEM.
[View Larger Version of this Image (20K GIF file)]
4
7,
L
2, and
M
2 Integrins on
Transfected K562 Cells
4
7 localizes
primarily to microvilli of mouse TK-1 lymphoma cells,
whereas integrins
L
2 and
M
2 localize primarily to
the cell body of TK-1 cells and human neutrophils respectively (Erlandsen et al., 1993
; Berlin et al., 1995
). We began by using immunoelectron microscopy to determine
whether these integrins would localize similarly in transfected K562 cells. K562 cells had numerous microvillous
projections.
4
7 was found primarily on microvilli (Fig. 5 A).
In contrast,
L
2 and
M
2 integrins were located primarily on the cell body (Fig. 5, B-D). Both
4
7(
L
2c) and
4
7(
M
2tc) localized to microvillous projections (Fig.
5, E and F). A quantitative analysis of the distributions of
these integrins is shown in Table I. The
4
7 construct
was also concentrated on microvilli (not shown). These results indicate that the transmembrane and cytoplasmic domains are not responsible for the differential localization of
4
7 versus
L
2 and
M
2.
Fig. 5.
Localization of
4
7,
L
2,
M
2, and chimeric integrins by immunoelectron microscopy. K562
transfectants were stained
for protein expression using
12-nm gold particles (arrows)
as described in Materials and
Methods. Wild-type
4
7
(identified using the anti-
7
antibody, Fib 504) was localized predominantly to microvilli of K562-
4
7 tranfectants (A). K562-
L
2 (B
and C) and K562-
M
2 (D)
were stained with antibodies
to
L (B) or
2 (C and D),
demonstrating that
L
2 and
M
2 were expressed mostly
on the cell body. K562 cells
transfected with the chimeric
integrins
4
7(
L
2c) (E) and
4
7(
M
2tc) (F) were
stained with Fib 504. These
chimeric integrins were found
predominantly on microvilli.
Photomicrographs are representative of integrin distribution on the 50-100 cells examined in each sample. Bar,
0.5 µm.
[View Larger Version of this Image (155K GIF file)]
Presentation of Other Leukocyte Integrins on Microvilli
In addition to 4
7,
L
2, and
M
2, leukocytes express
other integrins that play roles in adhesion to endothelial
cells, to other cells, and to extracellular matrix proteins.
One of these,
4
1, can initiate adhesion to vascular cell
adhesion molecule-1 (VCAM-1) under flow and has been
reported to be expressed on lymphocyte microvilli (Alon
et al., 1995
; Berlin et al., 1995
). We confirmed that
4
1
was also expressed preferentially on microvilli of K562-
4
1 transfectants (Table I). We found that the T cell integrin
E
7 (Cepek et al., 1994
), which can mediate adhesion
to epithelium but has no established role in endothelial adhesion, was also localized to microvilli of K562-
E
7 cells
(Fig. 6 A, and Table I). Integrin
6A
1, a laminin receptor
which is expressed on monocytes and some lymphocytes,
was concentrated on microvilli of K562 transfectants (Fig.
6 B, and Table I). The vitronectin receptor, integrin
v
3,
is also a receptor for the endothelial cell ligand platelet/endothelial cell adhesion molecule-1 (Piali et al., 1995
) and
is expressed on monocytes and other cells. We found that
v
3 localized predominantly to microvilli (Fig. 6 C, and
Table I). The fibronectin receptor, integrin
5
1, is constitutively expressed on K562 cells and was also expressed predominantly on microvilli (not shown).
Localization of Transmembrane Proteins Known to Associate with Integrins
Integrins have been shown to associate with a variety of
intracellular and transmembrane proteins. Some of these
interactions occur within the cell and are mediated by integrin cytoplasmic domains, whereas others are known or
suspected to be extracellular. Since our results suggested
that extracellular (and not transmembrane or cytoplasmic)
domains determine integrin localization, we performed immunoelectron microscopy to localize several cell surface
proteins known or suspected to interact with integrin extracellular domains. Several members of the tetraspan
family of transmembrane proteins have been shown to associate with 4
1,
4
7,
6
1, and some other integrins
(Berditchevski et al., 1996
; Mannion et al., 1996
). These
associations are likely to involve the extracellular domains of tetraspans and integrins (see Discussion). Several tetraspans are constitutively expressed on K562 cells. CD53
(82 ± 5% on microvilli, Fig. 7 A), CD63 (92 ± 4% on microvilli, Fig. 7 B), and CD81 and CD82 (data not shown)
are all localized predominantly to microvilli. Another transmembrane protein, CD47 (integrin-associated protein), has been shown to associate with
v
3 via its extracellular
domain. CD47, like
v
3, was distributed mostly on microvilli (Fig. 7 C). CD32 is a transmembrane protein that
has been reported to associate with
M
2 (which is on the
cell body; Fig. 5 C) and possibly with the tetraspan CD82
(found on microvilli, see above) (Lebel-Binay et al., 1995
).
CD32 was expressed on both microvilli (62 ± 15%) and
the cell body (38 ± 15%) (Fig. 7 D).
In this study, we examined the role of leukocyte integrin
cytoplasmic and transmembrane domains in adhesion under flow and microvillous localization. We began by confirming that previously described differences in leukocyte
integrin adhesive activity and membrane localization were
also seen in K562 cell integrin transfectants. As expected,
4
7 was able to initiate adhesion under flow and localized
to microvilli, whereas
L
2 and
M
2 mediated adhesion
only under static conditions and localized to the planar cell
body. Two chimeras,
4
7(
L
2c) and
4
7(
M
2tc), were expressed to examine the roles of the extracellular,
transmembrane, and cytoplasmic domains in adhesion and
membrane localization. Both chimeras were as efficient in
initiating adhesion under flow as the wild-type integrin
4
7. The chimeras were found predominantly on microvilli, indicating that the extracellular domain (and not the transmembrane or cytoplasmic domains) determined
membrane localization of
4
7. We have so far been unable to determine whether
2 integrin localization to the
cell body is also independent of the transmembrane and
cytoplasmic domains. We attempted to address this issue
by expressing a chimeric integrin composed of the extracellular domain of
M
2 and the transmembrane and cytoplasmic domains of
4
7, but have not yet been successful in these experiments. In addition to
4
7, several other
integrins (
4
1,
5
1,
6A
1,
v
3, and
E
7) were localized to microvilli. Transmembrane proteins known or
suspected to interact with integrin extracellular domains,
including tetraspan family members and CD47, were also
found to be concentrated on microvilli.
The ability of leukocyte adhesion molecules to support
initial adhesion under flow is influenced by several factors
including affinity, avidity, and accessibility to endothelial
ligands. Previous studies of nonintegrin adhesion molecules indicate that cytoplasmic domains can have dramatic
effects upon initiating adhesion under flow without altering static adhesion. Experiments involving the use of L-selectin-CD44 chimeras suggest that cytoplasmic domains may
influence adhesion under flow by targeting these receptors to the microvillous or cell body (see Introduction). However, it is clear that the cytoplasmic domain of L-selectin
also has effects on initiation of adhesion that are independent of receptor positioning. A truncation mutant of L-selectin lacking the 11 COOH-terminal amino acid residues of
the cytoplasmic domain was localized to microvilli and retained the ability to bind ligand, but was unable to support
rolling on endothelium (Kansas et al., 1993; Pavalko et al.,
1995
). This mutant lost the ability to associate with the cytoskeletal proteins
-actinin and vinculin, suggesting that
interactions between adhesion molecule cytoplasmic domains and cytoskeletal proteins can be important in regulation of initial adhesive interactions under flow. We
found that replacement of the cytoplasmic and/or transmembrane domains of
4
7 (which does support adhesion
under flow) with the homologous domains of
L
2 or
M
2 (which do not) did not affect adhesion to the
4
7
ligand MAdCAM-1 under either static or flow conditions.
This indicates that the cytoplasmic domains cannot account for the differences in ability of these integrins to
support adhesion under flow. We also found that truncation of the
4 subunit after the conserved GFFKR motif
had little if any effect on initiation of
4
7-mediated adhesion to MAdCAM-1. Others have previously shown that
the same truncation of
4 did not affect the ability of
4
1
to initiate adhesion to its ligand, VCAM-1 (Kassner et al.,
1995
). The
4 truncation was reported to decrease the
cell's resistance to detachment from VCAM-1 in the face
of increasing shear force, suggesting that the
subunit cytoplasmic domain plays a role in strengthening adhesion.
We were unable to detect any difference in resistance to
detachment between wild-type and chimeric
4
7 integrins, suggesting that
4
7,
L
2, and
M
2 integrin cytoplasmic domains are similar in their ability to mediate
adhesion strengthening.
Many of the adhesion molecules that initiate adhesion
to endothelium under flow are concentrated on leukocyte
microvilli.These include L-selectin (Picker et al., 1991; Erlandsen et al., 1993
; Pavalko et al., 1995
), P-selectin glycoprotein ligand-1 (Moore et al., 1995
; Bruehl et al., 1997
),
and the integrins
4
7 and
4
1 (Berlin et al., 1995
; and
this report). Other adhesion molecules, including CD44
and the integrins
L
2 and
M
2, are found predominantly on the cell body (Erlandsen et al., 1993
; Berlin et
al., 1995
; von Andrian et al., 1995
; and this report). Little
information is available about the mechanisms that lead to
the selective display of certain adhesion molecules on microvilli. It seems likely that interactions between the cytoplasmic domains of adhesion molecules and specific cytoskeletal elements can play an important role. In support
of this concept, the localization of L-selectin-CD44 chimeras was shown to be determined by the cytoplasmic and/or transmembrane domains, and not by the extracellular domains (see Introduction). Concentration of integrins in
other structures, such as focal adhesions and hemidesmosomes, is known to depend upon the
subunit cytoplasmic
domain. We were surprised to find that our analysis of integrin chimeras did not demonstrate a role for the cytoplasmic or transmembrane domains of either the
or
subunit in determining membrane localization. Replacement of the
4
7 cytoplasmic and/or transmembrane domains with homologous domains of
L
2 or
M
2 did not
interfere with microvillous localization. Put another way,
replacement of the extracellular domain of the
L
2 or
M
2 integrins with the extracellular domain of
4
7 resulted in a shift from cell body to microvillous localization.
These results indicate an important role for the extracellular domain in directing localization of integrins to the microvillous versus the cell body.
Integrin extracellular domains can interact with other cell
surface proteins. For example, the transmembrane protein
CD47 interacts with v
3 and this interaction depends
upon the extracellular domain of CD47 (Lindberg et al.,
1996
). Several members of the tetraspan family of transmembrane proteins, including CD53, CD63, CD81, and
CD82, have been shown to coprecipitate with some integrins, including
4
1,
6
1, and
4
7, but not with
L
2
or some other integrins (Berditchevski et al., 1996
; Mannion et al., 1996
). These interactions are likely to involve
the integrin extracellular domain, since mutations of the
4 subunit extracellular domain substantially reduce association whereas alterations of the
subunit cytoplasmic
domain have no effect. We found that CD47, CD53, CD63,
CD81, and CD82, all known to associate with integrins that we localized to microvilli, were themselves concentrated on microvilli. Our data are consistent with the hypothesis
that interactions with tetraspans and CD47 help target certain integrins to microvilli. The widespread expression of
tetraspans and CD47 on leukocytes and other cells makes
this hypothesis difficult to test directly. At least one integrin that we localized to microvilli,
5
1, apparently does
not associate with tetraspans or CD47, suggesting that
other interactions also are important (Berditchevski et al., 1996
). This hypothesis assumes that
4
7 and other microvillous integrins are actively concentrated on microvilli,
but
L
2 and
M
2 are not. An alternative explanation of
our results is that microvillous expression is the "default
pathway" for integrins, and that the extracellular domains
of
L
2 and
M
2 prevent these integrins from being displayed on microvilli. This could be mediated by interactions between
2 integrins and associated cell surface proteins. Although
M
2 has been shown to associate with
CD32, the pattern of expression of CD32 (on both cell
body and microvilli) suggests that this interaction is not responsible for the concentration of
M
2 on the cell body.
We found that many integrins and integrin-associated
proteins were preferentially expressed on microvilli. Some
of these integrins, including 4
7 and
4
1, play important roles in mediating leukocyte adhesion under flow.
Other microvillous integrins, such as
5
1 and
E
7, mediate adhesion to extracellular matrix proteins or epithelial cells, but have no known role in initiating leukocyte- endothelial interactions. This suggests that the assembly of
multimolecular complexes containing integrins and integrin-associated proteins on microvilli may have other important roles in adhesion and signaling.
Received for publication 28 May 1997 and in revised form 24 July 1997.
Address all correspondence to M. Abi Abitorabi, University of California, San Francisco, Box 0854, San Francisco, CA 94143-0854. Tel.: (415) 206-6649. Fax: (415) 206-4123.We thank K.L. McDonald and P. Sicurello (Robert D. Ogg Electron Microscope Laboratory, University of California, Berkeley, CA) and G. Antipa and G. Lum (San Francisco State University, San Francisco, CA) for their expert advice and courtesy in allowing us to use their electron microscope facilities. We thank S. Wu for optimizing the static adhesion assay protocol and are grateful to R. Pytela and D. Sheppard for critically reviewing this manuscript.
This study was supported by National Institutes of Health grants HL50024 (to D.J. Erle) and HL03230 (to M. Tidswell). M. Abi Abitorabi was supported by National Institutes of Health training grant HL07155 and National Research Service Award 1F32HL09364.
ICAM-1, intercellular adhesion molecule-1; MAdCAM-1, mucosal addressin cell adhesion molecule-1; VCAM-1, vascular cell adhesion molecule-1.
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