(Received for publication, July 17, 1995; and in revised form, September 25, 1995)
From the
FcR chain has previously been shown to interact with the
TCR-CD3 complex, the IgE Fc receptor I (Fc
RI), and the class I and
IIIA IgG receptors (Fc
RI and Fc
RIIIa). Here, we demonstrate
that the Fc receptor
chain associates with Fc
R in
transfected IIA1.6 B lymphocytes. Fc
R could be expressed at the
surface of IIA1.6 B cells by itself, but was devoid of signaling
capacity. Upon co-expression of FcR
chain, a physical interaction
with Fc
R could be demonstrated. This association proved crucial
for the triggering of both proximal (intracellular calcium increase and
tyrosine phosphorylation), as well as distal (IL-2 release), signal
transduction responses. We next tested the hypothesis that a positively
charged arginine residue (Arg
) within the transmembrane
domain of Fc
R promotes association with FcR
chain. We
therefore constructed Fc
R molecules where Arg
was
mutated to either a positively charged histidine, a negatively charged
aspartic acid, or an uncharged leucine. A functional association
between Fc
R and FcR
chain was observed only with a
positively charged residue (Arg
or His
)
present within the Fc
R transmembrane domain. These data show that
transmembrane signal transduction by the Fc
R is mediated via FcR
chain, and that Fc
R requires a positively charged residue
within the transmembrane domain to promote functional association.
IgA is the primary immunoglobulin in bodily secretions and plays
a critical role in protection against the constant environmental
challenges at mucosal sites. Although the protective mechanisms are
incompletely defined, a significant role in IgA-mediated immune defense
has been proposed for IgA Fc receptors. These molecules have been
detected on most populations of phagocytic cells in blood and mucosal
tissues. Engagement of these molecules can trigger phagocytosis,
degranulation, oxidative burst, inflammatory mediator release, and
antibody-dependent cellular cytotoxicity(1, 2) .
FcR on monocytes/macrophages and neutrophils has been defined as a
55-75-kDa glycoprotein(3, 4) , whereas the
eosinophil Fc
R is more heavily glycosylated (70-100
kDa)(5) . Both types of myeloid receptors are recognized by the
CD89 mAb (
)panel (4, 5) and bind both IgA1
and IgA2 via their Fc regions(1) . The cDNA encoding the
myeloid Fc
R has been characterized and was found to encode a
30-kDa peptide, with two extracellular Ig-like domains, a hydrophobic
transmembrane region and a cytoplasmic tail devoid of recognized
signaling motifs(2, 6) . Additionally, we have
recently isolated and characterized the human gene encoding the CD89
molecule. The gene structure indicates Fc
R to represent a more
distantly related member of the immunoglobulin receptor gene family (7) .
To explore the capacity of FcR to trigger
biological functions, we have now generated different transfectants in
the mouse IIA1.6 B cell line. This line, derived from the A20 B cell
lymphoma, lacks the 5`-end of the Fc
RII gene and, consequently, is
Fc receptor-negative(8) . Previous work showed this line to
represent an excellent model for assaying Fc
R-mediated
functioning(9, 10, 11) . Following
transfection, Fc
R was expressed at the surface of IIA1.6 cells by
itself, but lacked signaling capacity. We therefore hypothesized that
Fc
R may associate with a specialized signaling molecule. The FcR
chain was known previously to associate with all three classes of
Fc
R, Fc
RI, and the TCR-CD3
complex(12, 13, 14, 15) . FcR
chain is responsible for coupling these receptors to intracellular
signaling pathways(16) . By co-transfection experiments, we
tested whether FcR
chain could mediate signal transduction via
Fc
R. Our results show that co-expression of Fc
R and FcR
chain in IIA1.6 B cells conferred both proximal and distal signaling
capacity to Fc
R. During the preparation of this manuscript, it was
shown that, in the U937 cell line, Fc
R was associated with FcR
chain and that
chain was phosphorylated on tyrosine
residues following Fc
R cross-linking (17) . The data
presented here indeed confirm that Fc
R associates with FcR
chain in transfected IIA1.6 B cells and, furthermore, suggest that
Fc
R and FcR
chain can associate in normal blood PMN. The
present experiments demonstrate FcR
chain to be critical for
Fc
R-mediated transmembrane signal transduction.
We,
furthermore, explored the molecular basis for FcR/FcR
chain
association. The transmembrane (TM) domain of Fc
R is unusual as it
contains a single positively charged arginine (Arg
)
residue. Since the TM domain of the FcR
chain contains a single
negatively charged aspartic acid residue, we hypothesized that these
oppositely charged residues may promote association. A similar
mechanism involving charged TM amino acids has previously been shown to
be involved in the assembly of the TCR-CD3
complex(18, 19, 20, 21) . Using PCR,
we mutated the Arg
found in the wild type Fc
R (R209)
to either a positively charged histidine (R209H), a negatively charged
aspartic acid (R209D), or an uncharged leucine (R209L). Mutated
Fc
R cDNAs were transfected together with FcR
chain to IIA1.6
cells and assessed for their ability to trigger an increase in
[Ca
]
following
Fc
R cross-linking. Our data show a positively charged residue
within the TM domain of Fc
R to be required for functional
association with FcR
chain.
Figure 1:
Expression of FcR and FcR
chain in transfected IIA1.6 cells. A, surface expression of
Fc
R in IIA1.6 B cells. Cells transfected with Fc
R alone
(Fc
R
) or with both Fc
R and
chain
(Fc
R
/
) were incubated with
CD89 mAb A77 (solid line) or with immunofluorescence buffer
alone (dotted line) followed by FITC-conjugated GAM IgG1 Ab. B, detection of FcR
chain message. Total cellular RNA
was isolated, reverse-transcribed to cDNA, and FcR
chain and
hypoxanthine-guanine phosphoribosyltransferase (HPRT)
transcripts were detected via PCR. C, expression of FcR
chain. Permeabilized cells were incubated with anti-
chain serum
followed by FITC-conjugated anti-rabbit serum and analyzed by flow
cytometry. Fluorescence intensity of untransfected IIA1.6 cells was
identical with that of Fc
R
transfectants (data
not shown).
We found untransfected IIA1.6 cells not to express
endogenous FcR chain (Fig. 1B). This observation
suggests that, in contrast to Fc
RIIIa and
Fc
RI(12, 16) , mere cell surface expression of
Fc
R in IIA1.6 cells is not dependent on the presence of FcR
chain. Previously, it was also shown that Fc
R could be expressed
on the surface of COS cells in the absence of FcR
chain(6) . Similarly, the high affinity IgG receptor Fc
RI
(CD64), which also associates with FcR
chain(13, 14) , can be expressed by itself in COS (13) and 3T3 transfectants(30) .
Figure 2:
Physical association between FcR and
FcR
chain in transfected IIA1.6 B cells and PMN. Surface
radioiodinated cells were lysed in 1% digitonin lysis buffer and
immunoprecipitated with either mAb A77 or anti-FcR
chain
antiserum. Immunoprecipitates were separated by gel electrophoresis
under reducing conditions, followed by autoradiography. A,
radiolabeled cell lysates from IIA1.6 cells (lanes 1 and 4), Fc
R
(lanes 2 and 5), and Fc
R
/
(lanes 3 and 6) transfectants were immunoprecipitated
with either A77 (lanes 1-3) or anti-
chain serum (lanes 4-6). Positions of Fc
R are marked by arrowheads. B, 1% digitonin cell lysates (as
indicated) were immunoprecipitated with either A77 mAb (lanes
1-4) or anti-
chain serum (lane 5).
Precipitates were separated by nonreducing SDS-PAGE, transferred to
nitrocellulose, and probed with anti-
chain serum. C,
control precipitations were also performed using beads coated with an
irrelevant murine IgG1 Ab (mIgG1), as isotype control for A77 (lanes 1-4), or normal rabbit serum (Rbt serum)
as a control for anti-
chain serum (lane 5). D,
mouse and human FcR
chains were precipitated from lysates of
different mouse (lanes 1 and 2) or human (lanes 3 and 4) cells as detailed under ``Materials and
Methods.'' The position of molecular mass standards are marked on
the left.
Western blotting analysis of
immunoprecipitated proteins further supported the presence of a
physical interaction between FcR and FcR
chain in
transfectants and suggest that Fc
R and FcR
chain can also
associate in peripheral blood PMN. Proteins were precipitated from
digitonin-solubilized cells with beads coated with either A77, anti-FcR
chain serum, or control serum (see ``Materials and
Methods''), transferred to nitrocellulose, and probed with
anti-FcR
chain serum. In IIA1.6 cells, a specific band of
20
kDa, corresponding to the expected size of FcR
chain
homodimers(12) , was co-precipitated by anti-Fc
R mAb A77
from Fc
R
/
cells only (Fig. 2B, lane 3). No bands of this size were
precipitated by A77 from either untransfected IIA1.6 or
Fc
R
transfectant cell lysates (lanes 1 and 2). Similarly sized bands were detected in PMN cell
lysates following immunoprecipitation with either A77 (lane 4)
or anti-
chain serum (lane 5). No specific bands of this
size were seen following immunoprecipitation with either an irrelevant
mouse IgG1 antibody (Fig. 2C, lanes 1-4;
A77 control) or with normal rabbit serum (Fig. 2C, lane 5; anti-
chain control). The 28-kDa bands seen in Fig. 2B, lanes 1-5, are considered to be
nonspecific since these bands were also seen in the control
immunoprecipitations (Fig. 2C, lanes
1-5). We, furthermore, observed a slight difference in
mobility of the FcR
chain between transfectants (murine
chain) and PMN (human
chain) (Fig. 2B, lane 3
versus lanes 4 and 5). This different migration profile
was surprising in view of the high homology between murine and human
chains(12) . Therefore, we next precipitated murine
chain from Fc
R
/
transfectants and murine peritoneal macrophages, and human
chain from PMN and U937 cells. Following SDS-PAGE and transfer to
nitrocellulose, the blots were probed with anti-
chain serum.
Results presented in Fig. 2D, clearly showed a slight,
albeit significant, difference in mobility between murine (lanes 1 and 2) and human (lanes 3 and 4) FcR
chains. The observed difference in the mobilities of human and
mouse
chains may be explained by the slightly different amino
acid sequences of these two chains. However, the phosphorylation state
of FcR
chain has also been shown to affect its mobility in
SDS-PAGE(31) ; therefore, differential phosphorylation between
human and murine cells may also explain the observed size difference.
Previously it has been demonstrated that FcR chain homodimers
associate with TCR-CD3, Fc
RI, and all three classes of Fc
R (12, 13, 14, 15) . During the
preparation of this manuscript, it was demonstrated that FcR
chain may also be found in membrane complexes with Fc
R in the U937
cell line(17) . Our results confirm this observation in a
transfectant model system and, furthermore, provide evidence to suggest
that Fc
R may associate with FcR
chain in normal peripheral
blood PMN.
Our results
show FcR chain to be required for generation of both proximal and
distal signaling events via Fc
R. Cross-linking of Fc
R in
Fc
R
/
transfectants leads to
a rapid rise in [Ca
]
that was
maintained for at least 2 min (Fig. 3A). In the absence
of FcR
chain, no [Ca
]
increase was observed. Following Fc
R cross-linking, a rapid
tyrosine phosphorylation of cellular proteins was also observed only in
Fc
R
/
cells (Fig. 3B). Tyrosine phosphorylation was detected within
20 s and reached a maximum between 40 s and 2 min in all experiments.
In all experiments, no detectable signaling responses were initiated
when the cells were incubated with GAM IgG1 mAb alone, indicating that
this antibody does not react with the surface IgG2a expressed by the
IIA1.6 cells (as reported previously; (33) ).
Figure 3:
Signalling events triggered by
cross-linking FcR in transfected IIA1.6 B cells. A,
calcium mobilization triggered by Fc
R in SNARF-1/Fluo-3-loaded
transfectants. Fc
R
(dotted line) or
Fc
R
/
(solid line)
transfectants were incubated with CD89 mAb A77 for 20 min at room
temperature, and Fc
R was subsequently cross-linked with GAM IgG1
F(ab`)
(arrow).
[Ca
]
levels were
analyzed by flow cytometry as described under ``Materials and
Methods.'' Data are representative of five individual experiments. B, tyrosine phosphorylation of cellular proteins upon
cross-linking Fc
R in Fc
R
(left
panel) or Fc
R
/
(right panel) IIA1.6 B cells. Transfectants were incubated
with mAb A77 for 30 min at room temperature and washed twice with RPMI
1640 medium. Cells were then incubated with GAM IgG1 Ab for the
indicated time periods. As control, cells were also incubated either
with A77 alone (A77) as negative control or with GAM IgG (GAM) to cross-link the surface IgG (positive control).
Samples were separated by SDS-PAGE, transferred to nitrocellulose, and
probed with an anti-phosphotyrosine mAb. C, induction of IL-2
production following cross-linking of Fc
R in transfected IIA1.6 B
cells. Fc
R
(open boxes) and
Fc
R
/
(filled
boxes) transfectants were incubated for 24 h in wells coated with
either serum IgA (IgA) or polymeric IgA (pIgA).
Alternatively, transfectants were incubated with CD89 mAb A77 for 30
min at room temperature, washed, and seeded into wells. A cross-linking
GAM IgG1 Ab was then added to the culture supernatant for 24 h (A77+GAM
1). Control cells were incubated with
culture medium alone (medium) or had GAM
Ab added to
cross-link sIgG2a (GAM). Results shown are representative of
data obtained for IL-2 release in three separate
experiments.
Recently, it
has been noted that FcR cross-linking triggers phosphorylation of
FcR
chain in U937 cells(17) , supporting the hypothesis
that FcR
chain is critically involved in the generation of
Fc
R signal transduction responses.
Very little is known
concerning signal transduction pathways associated with FcR.
Interestingly, it has recently been proposed that Fc
R surface
expression may be regulated by
[Ca
]
, since treatment of
neutrophils with known calcium agonists leads to up-regulated Fc
R
expression (34) . This observation, taken together with our
data that Fc
R/
chain cross-linking mediates a rapid rise in
[Ca
]
, may explain the phenomena
of IgA-induced Fc
R expression-up-regulation, a function apparently
unique to Fc
R, at least among other FcRs(2) .
We,
furthermore, examined the capacity of FcR
and
Fc
R
/
transfectants to
trigger the release of IL-2 from IIA1.6 B cells. Secretion of IL-2,
triggered via Fc
R cross-linking, also proved to be dependent on
FcR
chain co-expression (Fig. 3C). IL-2 was
secreted following Fc
R cross-linking by polymeric IgA, serum IgA,
and CD89 mAb A77. The ability of polymeric IgA to trigger a higher
level of IL-2 release than serum IgA may indicate Fc
R to have
higher affinity for this molecular species. IL-2 release represents a
distal signaling event which can be triggered via cross-linking sIgG2a
in IIA1.6 B cells(35) . IIA1.6 sIgG2a is part of the B cell
antigen receptor signaling complex which includes at least four
ITAMs(36) . In contrast, previous data from our laboratory
utilizing a panel of Fc
R IIA1.6 transfectants showed that
cross-linking of Fc
RIIa (CD32; with only one ITAM) was sufficient
to induce a Ca
response and tyrosine kinase
activation but not IL-2 release(10, 32) . Taken
together, these data suggest the number of ITAMs within signaling
complexes to be of importance for determining the type of signals
generated via receptor complexes in IIA1.6 B cells. This hypothesis is
supported by recent work in Jurkat cells, where the number of ITAMs
within the TCR
chain was found to quantitatively affect T cell
responses(37) .
Several reports in the literature suggest
FcR to be capable of synergizing with Fc
R in promoting ADCC,
phagocytosis, and the respiratory
burst(38, 39, 40) . Our results provide a
model for such co-operation in which FcR
chain mediates signal
transduction via both types of receptors. The importance of FcR
chain in triggering phagocytosis via Fc
R has recently been
demonstrated in FcR
chain knock-out mice (41) and
transfection studies(42, 43) . Since our data suggest
that Fc
R functioning also depends on FcR
chain association,
we hypothesize that FcR
chain-deficient mice may display
aberrations in IgA-mediated mucosal immune responses.
Figure 4:
Protein sequence within the region of the
transmembrane domains of FcR, Fc
RI, and FcR
chain.
Predicted transmembrane spanning residues are underlined.
Positively (
) and negatively (
) charged residues which
may mediate association between Fc
R, Fc
RI, and FcR
chain are indicated.
Although, in
general, charged residues are uncommon within the TM domains of
integral membrane proteins, they are not unknown. For example, the
TCR- and TCR-
chains have conserved positively charged
residues within their TM domains while the invariant chains of the CD3
complex contain negatively charged TM residues. Elegant site-directed
mutagenesis experiments have shown these charged residues to be of
critical importance for surface expression of the TCR-CD3
complex(18, 19, 20, 21) .
Therefore, based upon the predicted TM regions of FcR and FcR
chain (Fig. 4), we hypothesized the positively charged
Arg
to be important for association with FcR
chain.
To test this hypothesis, we constructed mutant Fc
R molecules using
overlap extension PCR (see ``Materials and Methods''), in
which the wild type Arg
residue was replaced by either a
positively charged histidine (Fc
R-R209H), a negatively charged
aspartic acid (Fc
R-R209D), or an uncharged leucine
(Fc
R-R209L). Mutant Fc
R cDNAs were transfected together with
FcR
chain to IIA1.6 B cells. Fc
R and FcR
chain mRNA
transcripts were readily detectable by RT-PCR, and the resulting
mutated Fc
R proteins were well expressed at the cell surface as
shown by FACS analysis (Fig. 5, A and B).
Surprisingly, however, FcR
chain protein was only observed in
transfectants co-expressing an Fc
R molecule possessing a
positively charged residue within the TM domain, i.e. wild
type Fc
R and Fc
R-R209H (Fig. 5C). We next
assayed mutant Fc
Rs for their ability to form functional
Fc
R/FcR
chain signaling complexes, by measuring the increase
in [Ca
]
triggered upon Fc
R
cross-linking (see ``Materials and Methods''). Predictably,
only the Fc
R-R209H mutant resulted in intact functional integrity
of the Fc
R/FcR
chain complex comparable to the wild type
Fc
R (Fig. 5D). These data demonstrate that a
positively charged residue within the Fc
R TM domain promotes
functional association with FcR
chain. Our studies suggest
furthermore that, in IIA1.6 cells, FcR
chain molecules unable to
associate with Fc
R are degraded. This occurs possibly via a
mechanism recognizing the charged aspartic acid residue within the TM
domain (Fig. 4). The presence of charged residues within the TM
domain of some proteins can result in their retention and degradation
in the endoplasmic reticulum(44) .
Figure 5:
A, cells transfected with either
FcR-R209D, -R209H, or -R209L and FcR
chain were incubated
with CD89 mAb My43 (curve 2) or with immunofluorescence buffer
alone (curve 1) followed by FITC-conjugated GAM IgM Ab. B, detection of Fc
R and FcR
chain mRNA transcripts
by RT-PCR as described under ``Materials and Methods.'' C, detection of FcR
chain protein by Western analysis.
Digitonin cell lysates (as indicated) were immunoprecipitated (IP
Ab) with either anti-
chain serum (upper panel) or
normal rabbit serum (Rbt serum) as control (lower
panel). Precipitates were separated by nonreducing SDS-PAGE,
transferred to nitrocellulose, and probed with anti-
chain serum (WB Ab). The position of molecular mass standards are marked
on the left. D, calcium mobilization triggered by
mutant Fc
R/FcR
chain in SNARF-1/Fluo-3-loaded transfectants.
Transfectants were incubated with CD89 mAb My43 for 20 min at room
temperature, and Fc
Rs were subsequently cross-linked with GAM IgM.
[Ca
]
levels were
analyzed by flow cytometry as described under ``Materials and
Methods.'' The lines represent
Fc
R-R209H/
(
),
Fc
R-R209D/
(
), and
Fc
R-R209L/
(
).
Fc
R
/
(
) and
Fc
R
(
) are also shown for comparison. Data
are representative of three individual
experiments.
A similar charge-based
mechanism may also be operational for FcRI-FcR
chain
association, since a positively charged histidine residue is located
directly preceding the predicted TM domain(45) . Charged
residues located at or near the extracellular/TM boundary may also be
able promote association between protein subunits as suggested for the
and
subunits of the major histocompatibility complex class
II molecule(46) . The fact that surface expression of both
Fc
R and Fc
RI appears independent of FcR
chain (in
contrast to Fc
RI and Fc
RIIIA) may, indeed, argue for a
different type of FcR
chain association between these two
receptors and either Fc
RI or Fc
RIIIa.
In conclusion, our
data demonstrate that FcR is capable of associating with the FcR
chain in a transfectant model system and also provides evidence
that these two proteins can associate in peripheral blood PMN. We,
further, show Fc
R signal transduction responses to be critically
dependent upon co-expression of FcR
chain, and that a positively
charged residue within the TM domain of Fc
R is involved in the
functional association between these two molecules. Implications of
these observations in terms of cooperation (and competition) between
immunoglobulin receptors in cellular activation processes remain to be
elucidated.