(Received for publication, September 25, 1995)
From the
Fc receptors for the Fc part of IgG are the mediators for
antibody effector functions. Fc
RIII and Fc
RII are low
affinity receptors that, through the interaction with immune complexes,
initiate a variety of immunological responses, such as phagocytosis,
antibody-dependent cellular cytotoxicity, and release of inflammatory
mediators. We set out to define the IgG binding site on human
Fc
RIII. We assumed that potential
-turns in Ig-like domains
are the most probable determinants for ligand binding, and chimeric
Fc
RIIIB/Fc
RI receptors as well as single residue mutants were
constructed in these regions of Fc
RIIIB. Substitution of four
amino acids in the membrane-proximal domain (Gln
,
Arg
, Lys
, Val
) resulted in
decreased binding of human IgG1. Lys
and Val
were found also to be crucial for the interaction with the
IgG-binding inhibitory monoclonal antibody 3G8. In a putative
three-dimensional model constructed in this study, these residues map
on the CC` loop (Gln
), on F
-sheet
(Arg
), and on the FG loop (Lys
,
Val
). Our data are consistent with the study about human
Fc
RII (Hulett, M. D., Witort, E., Brinkworth, R. I., McKenzie, I.
F. C., and Hogarth, P. M.(1994) J. Biol. Chem. 269,
15287-15293), suggesting that common structural determinants, i.e. FG loop or the GFC surface of the membrane-proximal
domain, can be involved in interactions with IgG by both low affinity
receptor classes Fc
RII and Fc
RIII.
Fc receptors constitute a group of membrane proteins that
interact with IgG Fc regions. The three classes of human Fc
receptors (Fc
RI, Fc
RII, Fc
RIII) belong to the Ig gene
superfamily and are widely expressed in hematopoietic cell lineages (2, 3, 4, 5) . Fc
RI (CD64)
binds IgG with high affinity, whereas Fc
RII (CD32) and Fc
RIII
(CD16) are low affinity receptors, interacting predominantly with
immune complexes of the IgG3 and IgG1
subclasses(6, 7) .
Human class III receptors are
represented by two isoforms that differ in their membrane
anchors(8) , expression patterns, and affinities to IgG. The
transmembrane FcRIIIA (9) receptor is expressed on NK
cells, macrophages, on subsets of monocytes, and T cells in association
with dimers of the
-chain of Fc
RI (10, 11, 12) and/or the
-chain of T cell
receptor(13) . Expression of the
glycosylphosphatidylinositol-anchored Fc
RIIIB isoform is
restricted to
neutrophils(8, 14, 15, 16) .
Tissue-specific expression of the two isoforms can be regulated at the
transcriptional level(17, 18) . Fc
RIIIA binds
IgG1 and IgG3 complexes with higher affinity (K
3
10
M
)
than the B isoform (K
10
M
) and is able to interact with
monomeric IgG(19, 20, 21) . Fc
RIIIB is
represented by two allelic forms, NA1 and NA2, which can be detected
with certain specific CD16 monoclonal
antibodies(22, 23) . NA1 and NA2 (24) differ
in their glycosylation patterns (25) and in their ability to
trigger phagocytosis by neutrophils(26, 27) .
The
Ig-binding extracellular regions of Fc receptors contain two
(FcRII, Fc
RIII, Fc
RI) or three (Fc
RI) Ig-like
disulfide-bonded domains (2, 3, 4, 5) composed of seven
antiparallel
-sheets(28) . Loops between the
-sheets
are likely to be involved in interactions with the ligands.
The
membrane-proximal domain is crucial for IgG binding by most of the Fc
receptors studied, i.e. human
FcRII(1, 29, 30) , human
Fc
RI(30, 31, 32) , mouse
Fc
RII(33, 34) , and rat Fc
RI(31) .
The membrane-distal domain of Fc
RIIIB, when fused to domains
3-5 of ICAM-1, did not react with IgG(35) . Thus, to
identify the IgG binding sites of Fc
RIII, we focused on the
second, membrane-proximal domain of Fc
RIIIB. We predicted
potential
-turn regions of the second Ig-like domain with the aid
of the PC Gene program, and within these regions, amino acid residues
were exchanged with the equivalent ones in the
-chain of the human
high affinity receptor for IgE, Fc
RI (36) . The resulting
chimeric Fc
RIII/Fc
RI receptors should show diminished IgG
binding affinity, since the extracellular part of Fc
RI reveals
significant amino acid identity (41%) with Fc
RIIIB, but Fc
RI
does not interact with IgG(30) .
Chimeric
FcRIII/Fc
RI receptors revealed several regions on
Fc
RIIIB (amino acids 125-127, 152-158, 160-163),
substitution of which resulted in decreased interaction with IgG.
Following single residue mutagenesis and molecular modeling of the
receptor indicated that amino acids critical for ligand binding are
apparently located on the loops connecting C and C`
-sheets
(Gln
) as well as F and G
-sheets (Lys
,
Val
) and on the F
-sheet (Arg
) all on
the second extracellular domain of Fc
RIIIB. These residues may
constitute one discontinuous binding area on the GFC
-sheet
surface for the ligand, IgG1 or IgG3 complexes. This was further
supported by the finding that the epitope for the IgG-binding
inhibitory monoclonal antibody (mAb) (
)3G8 (37) was
localized on the same FG loop of the membrane-proximal domain.
Monoclonal antibodies to CD16 (3G8(37) , CLB-Fcgran1(38) , DJ130c, MEM-154, LNK16, and B88-9) were clustered and characterized in the Fifth International Workshop of Leukocyte Differentiation Antigens (22) . W6/32 (39) reacts with a monomorphic determinant of human HLA-A, B, and was used as a positive control.
Human myeloma cell line SKO-007 (41) secreting IgE was a gift from Dr. R. Lamers (Max Planck Institute for Immunobiology, Freiburg, Germany). The cells were cultured in Iscove's modified Eagle`s medium (Life Technologies, Inc., Eggenstein, Germany) supplemented with 1% fetal calf serum (PAA, Linz, Austria), 2 mML-glutamine, 100 units/ml penicillin, 100 µg/ml streptomycin (Biochrom KG, Berlin, Germany). IgE was purified from culture supernatants on a NHS-activated HiTrap column (Pharmacia) coupled with polyclonal antibody to hIgE (The Binding Site, Heidelberg, Germany).
Chimeric FcRIIIA/B receptor was constructed by cloning the
536-bp SphI-HincII fragment coding for the
extracellular part of the Fc
RIIIA gene into the Fc
RIIIB gene.
Figure 1:
Alignment of the membrane-proximal
domains of hFcRIIIB, hFc
RI
, and hFc
RIIA. Locations
of the regions of highest
-turn probability in Fc
RIIIB
predicted by the Chou and Fasman method are indicated as gray bars over the sequence. Chimeric Fc
RIIIB/Fc
RI receptors were
designed based on this analysis. The amino acid residues exchanged
between Fc
RIIIB and Fc
RI in the chimeras are boxed.
Residues involved in ligand binding in Fc
RIIA are boxed on that sequence.
The wild-type and chimeric
receptors were transiently expressed in 293 cells, and the structural
integrity of the receptors was assessed with a panel of six CD16 mAbs
(3G8, DJ130c, Gran1, MEM-154, LNK16, and B88-9). No significant
differences between the interaction with wild-type FcRIIIB and
five of the eight chimeras were observed with the mAbs (Table 1),
indicating that the mutations had caused no major alterations in the
structure of these chimeric receptors. In contrast, the chimera
160-163 was not recognized by the ligand-binding inhibitory mAb
3G8, and the binding with the mAbs Gran1, MEM-154, and B88-9 was
affected (about 30-60% of wild-type receptor). mAb DJ130c reacted
at the wild-type level with all the chimeras (Table 1).
Recognition of two chimeric receptors, 113-118 and 134-138,
was significantly decreased by most of the CD16 mAbs (Table 1).
Thus, we assumed extensive structural alterations in the
membrane-proximal domains of these receptors.
The epitopes for the
mAbs used have not been extensively studied yet. DJ130c is considered
to bind the membrane-distal domain of FcRIII (46) . This
explains the reactivity of the antibody with all the chimeras. The
other five mAbs used are directed against the membrane-proximal
domain(46) . (
)3G8 is known to interfere with the
ligand binding of the receptor(37, 46) . Thus, the
complete loss of binding with 3G8 indicates a major alteration within
the IgG binding site on the chimera 160-163.
In binding assays
of transfected 293 cells with hIgG1 complexes, a K of 53.1 nM was calculated for the wild-type Fc
RIIIB (Table 1). The affinity of the receptor to covalently linked
highly purified hIgG1 dimers was about 10 times lower (K
= 4.6
10
M, Table 1), presumably due to the lower valency of the dimeric
ligand.
A FcRIIIA/B chimera that maintained the extracellular
domains of Fc
RIIIA in the glycosylphosphatidylinositol-anchored
molecule revealed similar to wild-type Fc
RIIIB affinities to IgG1
complexes (K
= 50.7 nM, Table 1and Fig. 2) as well as to dimers (K
= 2.7
10
M, Table 1). Hence, we suppose that the minor differences in the
amino acid sequences of the extracellular domains of these two isoforms
do not account for the higher IgG binding capacity of
Fc
RIIIA(20) .
Figure 2:
Binding of the chimeric receptors to hIgG1
complexes. Binding of serial dilutions of heat-aggregated hIgG1 to 293
cells transfected with cDNAs of wt FcRIIIB (
, dashed
line), chimeras Fc
RIIIA/B (
, dashed line),
97-99 (
), 125-127 (
), 129-131
(&cjs3560;), 147-148 (*), 152-158 (
), 160-163
(
), and of mock cells (&cjs3649;, dashed line). The panel represents results of a typical experiment. Fraction of
bound IgG1 was calculated according to the formula f = FL - FL
/FL
- FL
, where FL =
anti-IgG fluorescence of the given receptor at the provided
concentration of IgG complexes, FL
=
anti-IgG fluorescence in the absence of IgG, and FL
= anti-IgG fluorescence of the wt
Fc
RIIIB at saturation. Binding assays were performed in
triplicates.
293 cells bearing the chimeras
97-99, 129-131, and 147-148 showed binding affinities
similar to wt FcRIIIB, (Fig. 2). In contrast, chimeras
125-127 and 152-158 bound hIgG1 complexes as well as dimers
at significantly lower levels (K
values for the
chimeras are shown in Table 1). Chimera 160-163 had almost
completely lost the capacity to bind IgG (Fig. 2). Since this
chimera was still recognized by most of the CD16 mAbs except 3G8, we
speculate that we have rather replaced residues functional in ligand
and 3G8 binding than destroyed the overall structure of the second
domain.
Binding affinities of the receptors to chemically
cross-linked IgG dimers were lower than to- heat-aggregated complexes (Table 1). However, the chimeric receptors reacted with hIgG1
dimers and large complexes in the same manner, i.e. the
chimeras 127-127, 152-158, and 160-163 revealed
decreased binding capacities to both the ligands, whereas the other
three chimeras (97-99, 129-131, and 147-148) showed
binding affinities comparable to the wild-type FcRIIIB.
Chimeras 113-118 and 134-138 did not bind IgG as well as most of the CD16 mAbs mapped to react with the membrane-proximal domain. As discussed below, the loss of IgG binding was considered to result from the destroyed structure of the domain, and these chimeras were excluded from further mutational analysis.
None of the
transfectants expressing chimeric receptors were able to interact with
hIgE (data not shown). Evidently, the FcRI-derived amino acid
residues in the chimeras are not directly involved in IgE binding or
are not sufficient for the binding detectable in our assays.
Figure 3:
Molecular model of the membrane-proximal
domain of FcRIIIB. Ribbon diagram presentation of a
``MOLSCRIPT'' (58) and ``RASTER
three-dimensional'' (59) drawing showing the predicted
-strands G, F, C in the front (light) and A, B, E in the
back (dark). The regions that were replaced with the
equivalent ones from the Fc
RI
in the chimeric receptors are
indicated. The cysteines that form the disulfide bond are shown on F
and B
-sheets.
Amino acids substituted in the chimeras that
did not have any effect on ligand binding (97-99, 129-131,
and 147-148) are positioned apart from the binding surface on
this model. Residues 97-99 stretch between the two extracellular
domains, and 147-148 are located on the E -sheet. Both the
regions are located on the ABE surface of the membrane-proximal domain,
thus on the opposite side of the potential binding area (Fig. 3). Residues 129-131 are placed on the CC` loop,
opposite to the 125-127 region. Replacement of these amino acids
seems not to affect the function of the neighboring putative binding
residues 125-127, since the substitution did not influence the
interaction with IgG (Table 1, Fig. 2).
The overall
structure of two of the chimeric receptors (113-118 and
134-138) was considered to be disrupted according to the
monoclonal antibody data. Location of the amino acids 134-138 on
the C` -sheet that is presumably stabilizing the two
-sheet
surfaces would explain the disruption of the structure of the domain
when these residues were substituted. According to the model, the
region 113-118 within the BC loop is also connecting the two
-sheet surfaces and, therefore, likely to be stabilizing the
conformation of the domain. In addition, this region can be involved in
generating the binding site due to the close proximity to the FG loop,
the potential binding structure for IgG.
The GFCC" surface of the
membrane-proximal domain of hFcRII has been reported to be crucial
for IgG binding(1) . The key residues on hFc
RIIA (Fig. 1) are shown to locate on the FG loop of the domain on a
molecular model of this receptor(1) . The FG loop is also
involved in ligand binding in mouse
Fc
RII(32, 33) . These data support our hypothesis
that the FG loop of the membrane-proximal domain is the main binding
determinant in Fc
RIII, as was demonstrated by the loss of IgG
binding capacity after substituting the residues 160-163 on the
putative FG loop.
Arg/His influences the interaction
of hFc
RIIA with hIgG2(47, 48) . The low
responsive Fc
RIIA isoform harbors histidine in this position and
interacts with human IgG2 but not with mouse IgG1, whereas arginine in
the position 131 in Fc
RIIA abolishes the binding to hIgG2.
Arg/His
is located on the C`E loop on the Fc
RIIA
model(1) . In hFc
RIII, His
is corresponding
to the His/Arg
in hFc
RIIA (Fig. 1) and is
putatively positioned on the C`
-sheet. Since hFc
RIII does
not bind IgG2 and, according to our model, the C`
-sheet remains
conformationally distant from the GFC surface, we suggest the residues
on this
-sheet are not directly involved in hIgG1 binding.
Within the region 125-127 (LQN-YKD),
replacement of Leu with Tyr and of Gln
with
Lys resulted in lower IgG binding capacities (K
= 182 and 116 nM for IgG complexes, respectively, Table 1, Fig. 4A), comparable to that of the
responsive chimera (K
= 150 nM).
In contrast, changing Asn
to Asp did not alter the
binding affinity of the mutant receptor (Fig. 4A, Table 1). Interaction with IgG1 dimers followed the same pattern, i.e. mutants Leu
and Gln
harbored
decreased ligand binding capacities as compared to the wild-type
receptor. Analyzing the receptor model, we supposed that only
Glu
and not Leu
is directly involved in
binding. We assume that replacing the small polar side chain of leucine
with that of aromatic tyrosine in the mutant Leu
has led
to immense structural changes in the CC` loop and, thus, to interrupted
ligand binding.
Figure 4:
Binding of the single residue mutant
receptors to hIgG1 complexes. Representative IgG1 binding experiments
of the mutant receptors are grouped according to the chimeras they
originate from. Panel A, 293 cells were transfected with cDNAs
of wt FcRIIIB (
, dashed line), chimera
125-127 (
), mutant receptors Leu
(
),
Gln
(
), Asn
(
), and vector DNA
(&cjs3649;, dashed line). Panel B, transfectants of
wt Fc
RIIIB (
), chimera 152-158 (
), Phe
(
), Arg
(
), and Leu
(
). Panel C, transfectants of wt Fc
RIIIB
(
), chimera 160-163 (
), Ser
(
),
Lys
(
), Asn
(
), Val
(&cjs3560;), and Ser
(
). Experiments were
performed in triplicates, and fractions bound were calculated as
described in Fig. 2.
A IgG binding site of FcRIIIB has been
described by Hibbs et al.(35) who identified a
continuous binding region on the CC` loop of the second domain,
Gln
-Tyr
, by alanine-scanning mutagenesis.
In contrast to these data, our mutational analysis revealed no linearly
continuous binding sites. In our experiments, replacement of the
sequence Lys
-Asp-Arg
with Glu-Ala-Leu from
Fc
RI did not influence IgG binding. Only conversions of the polar
Leu
and Gln
(Fig. 6) to aromatic Tyr
or positively charged Lys, respectively, disrupted ligand-receptor
interaction, whereas changing the charge of the adjacent side chain
(NH
group of Asn
to negatively charged
O
of Asp) did not affect the interaction at all. We
suppose that substitution of every amino acid to alanine by Hibbs et al.(35) affected the structure of the CC` loop
and, hence, the binding capacity, even when the residues neighboring to
functional ones were changed. Gly
was also recognized to
be involved in IgG binding in that study. Glycine with only one
hydrogen atom as the side chain can adopt a wider range of main chain
conformations than other residues (28) and should play an
important role in maintaining the structure of the CC` loop. This might
as well explain the absence of ligand binding by mutated
Gly
, also demonstrated in that study(35) .
Figure 6:
Space-filling presentation of the ligand
binding domain of FcRIIIB. Amino acid residues putatively involved
in IgG binding are colored. The N (Leu
) and C termini
(Ile
) of the domain are indicated by cyan. The
computer model was generated based on the previously described
structure of a IgG Fab fragment (44) .
We
constructed three mutants (Phe, Arg
, and
Leu
) in the second region, 152-158. Analyzing the
molecular model, Ser
was found to be placed apart from
the other residues and excluded from further studies. Replacement of
the positively charged Arg
(Fig. 4B, Table 1) with polar Thr resulted in the decrease of receptor
function, while exchanging the neighboring residues Phe
with Tyr and Leu
with Lys did not have significant
effects on IgG binding. Substitution of only the Arg
with
Thr disrupted receptor-ligand interaction to a considerably higher
extent than did the replacement of the longer region, 152-158 (Table 1, Fig. 4B). We assume that the single
residue replacement destroyed also the possible
-sheet structure
and ablated IgG binding capacity of the receptor. In contrast, when the
whole F
-sheet was exchanged in the chimera 152-158, IgG
binding was decreased, apparently, due to the missing of the residue of
direct interaction with the ligand.
Based on the chimera
160-163, the amino acid Ser was converted to Gln,
Lys
to Leu, Asn
to Asp, Val
to Tyr, and Ser
to Glu. Two of the substitutions,
Lys
to Leu and Val
to Tyr, resulted in
decreased ligand binding capacities of the respective receptors. The
mutation of Val
almost abolished the ability to bind IgG (Fig. 4C, Table 1). Both the mutants reacted
weakly with the mAbs 3G8 and B88-9, indicating that Lys
and Val
are located also within the epitopes for
these antibodies. The three other mutant receptors, Ser
,
Asn
, and Ser
, resembled the wild-type
receptor (Fig. 4C, Table 1), although in every
instance a substantial change in charge and/or configuration of the
side chain was generated. We conclude that Lys
and
Val
, which are located according to the molecular model
on the FG loop, are crucial for IgG binding, either being involved in
direct interactions with the ligand or determining the conformation of
the FG loop.
Figure 5:
Binding of transfectants with mAb 3G8. 293
cells transfected with the cDNAs of wt FcRIIIB (
, dashed
line), mutant receptors Lys
(
), Val
(
), Gln
(
), Arg
(
), and vector DNA (&cjs3649;, dashed line) were
incubated with serial dilutions of 3G8 (0.1-20 µg/ml). Bound
mAb was detected with fluorescein isothiocyanate-labeled anti-mouse
antibody by fluorescence-activated cell sorter analysis. Fractions of
3G8 bound were calculated as described in the legend for Fig. 2.
On IgG1 and IgG3, the natural ligands for these
receptors, the lower hinge region (amino acids 233-237) has been
identified to constitute a binding pocket for all Fc
receptors(49, 50, 51, 52) . The
lower hinge regions are different in IgG2 and IgG4, which explains also
the failure of these subclasses to interact with Fc
receptors.
Based on these data, we suppose that the same structural elements on
the low affinity Fc
receptors, e.g. FG loops, are
interacting with the same binding site, the lower hinge region on the
IgG molecules. Although the amino acid residues found to be involved in
ligand binding in Fc
RIIIB are not conserved among Fc receptors (Fig. 1), we think that the FG loops of the membrane-proximal
domains of different Fc
receptors constitute similar binding
structures for IgG, whereas different single residues are involved in
direct interactions.
The extensive amino acid sequence homology
between FcRI and Fc
receptors suggests that a similar folding
pattern might be adopted by these receptors, and the FG loops could be
even more widely used as an interaction site with immunoglobulins.
A
relatively higher affinity to IgG has been reported for the
FcRIIIA receptor(19, 20) . The minor differences
in the extracellular polypeptide sequences of the Fc
RIII A and B
were shown not to be responsible for the improved binding capacity of
the A isoform, since the chimera Fc
RIIIA/B harboring the
extracellular part of the A isoform in the B gene bound hIgG1 complexes
and dimers similarly to the Fc
RIIIB. An additional binding site
for Fc
RIIIA has been proposed in the CH3 domain of
IgG(53, 54, 55) . Binding curves obtained in
our experiments do not refer to the existence of a second binding site
on Fc
RIIIB. However, the possibility remains to be studied that on
NK cells, which express Fc
RIIIA in association with the
-chain of the Fc
RI, the receptor exposes different
interaction characteristics with IgG and thus endures higher affinity
to the ligand.
Understanding the molecular basis of the interactions
between Fc receptors and immunoglobulins is of great importance,
since the use of antibodies as therapeutical agents is increasing. The
low affinity Fc
receptors are also believed to play an important
role in inducing antibody-mediated
inflammation(56, 57) . Several chronic inflammatory
diseases like rheumatoid arthritis and leukocytoclastic vasculitis are
linked to constant presence of antigen-antibody complexes and
continuous activation of effector cells expressing Fc
receptors.
Identification of the binding sites on the receptors may provide new
possibilities for treatment of these diseases by blocking Fc
receptor-IgG interaction.