(Received for publication, May 8, 1995; and in revised form, June 15, 1995)
From the
The low affinity receptor for IgG, FcRII (CD32), has a wide
distribution on hematopoietic cells where it is responsible for a
diverse range of cellular responses crucial for immune regulation and
resistance to infection. Fc
RII is a member of the immunoglobulin
superfamily, containing an extracellular region of two Ig-like domains.
The IgG binding site of human Fc
RII has been localized to an
8-amino acid segment of the second extracellular domain,
Asn
-Ser
. In this study, evidence is
presented to suggest that domain 1 and two additional regions of domain
2 also contribute to the binding of IgG by Fc
RII. Chimeric
receptors generated by exchanging the extracellular domains and
segments of domain 2 between Fc
RII and the structurally related
Fc
RI
chain were used to demonstrate that substitution of
domain 1 in its entirety or the domain 2 regions encompassing residues
Ser
-Val
and Ser
-Thr
resulted in a loss of the ability of these receptors to bind
hIgG1 in dimeric form. Site-directed mutagenesis performed on
individual residues within and flanking the
Ser
-Val
and Ser
-Thr
domain 2 segments indicated that substitution of
Lys
, Pro
, Leu
,
Val
, Phe
, and His
profoundly
decreased the binding of hIgG1, whereas substitution of Asp
and Pro
increased binding. These findings suggest
that not only is domain 1 contributing to the affinity of IgG binding
by Fc
RII but, importantly, that the domain 2 regions
Ser
-Val
and Phe
-Thr
also play key roles in the binding of hIgG1. The location of
these binding regions on a molecular model of the entire extracellular
region of Fc
RII indicates that they comprise loops that are
juxtaposed in domain 2 at the interface with domain 1, with the
putative crucial binding residues forming a hydrophobic pocket
surrounded by a wall of predominantly aromatic and basic residues.
Cell surface receptors for the Fc portion of IgG (FcR) are
expressed on most hematopoietic cells, and through the binding of IgG
they play a key role in homeostasis of the immune system and host
protection against infection. Three structurally related but
functionally distinct classes of Fc
R have been defined: Fc
RI,
Fc
RII, and Fc
RIII(1, 2, 3) .
Fc
RII is a low affinity receptor for IgG that binds only IgG
immune complexes and is expressed on a diverse range of cells such as
monocytes, macrophages, neutrophils, eosinophils, platelets, and B
cells(1, 2, 3) . Fc
RII is involved in a
number of immune responses including antibody-dependent cell-mediated
cytotoxicity, clearance of immune complexes, release of inflammatory
mediators, and regulation of antibody
production(1, 2, 3, 4, 5, 6) .
The extracellular region of FcRII comprises two Ig-like
disulfide-bonded extracellular domains that are related to the Ig
superfamily proteins and are most closely related to the C2 set of Ig
domains(7, 8, 9, 10, 11, 12) .
The two Ig-like domain extracellular region of Fc
RII is
structurally conserved in all of the Ig superfamily leukocyte FcRs
(including Fc
RI, Fc
RIII, Fc
RI, and Fc
RI) and
presumably represents an Ig-interactive
motif(13, 14, 15, 16, 17) .
The elucidation of the molecular basis of FcR-Ig interactions is
fundamental for understanding the mechanisms by which these receptors
mediate biological functions such as activation of inflammatory cells,
induction of cytokine release, and destruction of pathogens. In
previous studies we utilized chimeric Fc receptors to identify the IgG
binding region of human Fc
RII(18, 19) . Chimeric
Fc
RII/Fc
RI
chain receptors were used to demonstrate
that the second extracellular domain of Fc
RII was responsible for
the binding of IgG, with a single direct binding region located between
residues Asn
and Ser
. Site-directed
mutagenesis of the Asn
-Ser
region
identified 5 residues as playing crucial roles in the binding of human
and mouse IgG1 by Fc
RII: Ile
, Gly
,
Leu
, Phe
, and
Ser
(20) .
However, despite the direct
demonstration of only a single region involved in the binding of IgG,
there is compelling evidence to suggest that other regions of
FcRII contribute to binding. A genetic polymorphism of human
Fc
RIIa, the so called ``responder/non-responder''
system, results in an amino acid substitution in domain 2 at residue
131 (Arg
His), which has been shown to influence the binding of
mouse IgG1 and human IgG2(21, 22, 23) .
Similarly, in the mouse a genetic polymorphism of Fc
RII,
identified as differences at residues 116 and 161, defines the epitope
of the anti-Ly17.2 mAb (
)that blocks the binding of IgG to
this receptor(24, 25) . Our previous molecular
modeling studies of Fc
RII domain 2 (wherein the
Asn
-Ser
binding region was located to an
exposed loop region; the F/G loop) suggest that these functionally
important amino acid changes are situated in the B/C and C`/E loops
(containing residues 116 and 131, respectively), which are juxtaposed
to the F/G loop (contains residue 161) at or near the interface with
domain 1(20) . Furthermore, the studies using chimeric
Fc
RII/Fc
RI receptors have identified three regions in the
structurally homologous receptor, Fc
RI, capable of directly
binding IgE: residues 87-128, 130-135, and 154-161,
which encompass the B/C, C`/E, and F/G loops
respectively(1, 18, 19) . Taken together,
these findings suggest that the B/C and C`/E loops of Fc
RII may in
addition to the F/G loop also play a role in the binding of IgG by
Fc
RII. Also of interest is that while the role of domain 2 of
Fc
RII in Ig binding has been clearly defined, a role for domain 1
of Fc
RII has not been determined. However, domain 1 of Fc
RI,
although demonstrated to not have a direct role in IgE binding, has
been shown to play an important role in high affinity binding (18, 26) possibly by maintaining the structural
integrity of the receptor or by providing additional contact sites.
Since Fc
RII is structurally related to Fc
RI, domain 1 of
Fc
RII may also play a similar role.
The possibility that domain
1 and the B/C or C`/E loop regions of domain 2 also contribute to the
binding of IgG1 by FcRII is addressed herein, using both chimeric
receptor and site-directed mutagenesis strategies.
The chimeric FcRII/Fc
RI
chain
receptors were generated as follows. For chimera
109-116
, oligonucleotide pairs NR1 + CHM10 and
CHM09 + EG5 were used to produce two fragments, which were spliced
together using oligonucleotides NR1 and EG5. For chimera
130-135
, oligonucleotide pairs NR1 + PM12 and PM11
+ EG5 followed by NR1 and EG5. The sequences of the
oligonucleotides used and their positions of hybridization with the
Fc
RIIa
cDNA are as follows: NR1,
5`-TACGAATTCCTATGGAGACCCAAATGTCTC-3` (nucleotide positions
10-30); EG5, 5`-TTTGTCGACCACATGGCATAACG-3`(967-981);
CHMO9, 5`-CACATCCCAGTTCCTCCAACCGTGGCACCTCAGCATG-3` (419-437 with
nucleotides 442-462 of Fc
RI
chain); CHM10,
5`-AGGAACTGGGATGTGTACAAGGTCACATTCTTCCAG-3` (462-487 with
446-462 of Fc
RI
chain), PM11,
5`-GTGGTTCTCATACCAGAATTTCTGGGGATTTTCC-3` (473-490 with
492-506 of Fc
RI
chain); PM12,
5`-CTGGTATGAGAACCACACCTTCTCCATCCCAC-3` (516-531 with
491-506 of Fc
RI
chain).
Sequences derived from
FcRI
chain are underlined, Fc
RII is not underlined, and
nonhomologous sequences including restriction enzyme sites used in
cloning of the PCR products are in boldface type. Nucleotide positions
refer to the previously published Fc
RIIa and Fc
RI
chain
cDNA sequences(8, 16) .
The FcRII alanine
point mutant cDNAs were generated using the following oligonucleotide
combinations: Lys
-Ala, GBCO3 + EG5 and GBCO4 +
NR1; Pro
-Ala, GBCO1 + EG5 and GBCO2 + NR1;
Leu
-Ala, GBCO5 + EG5 and GBCO6 + NR1;
Val
-Ala, GBCO7 + EG5 and GBCO8 + NR1;
Phe
-Ala, GCEO1 + EG5 and GCEO2 + NR1;
Ser
-Ala, GCEO3 + EG5 and GCEO4 + NR1;
Arg
-Ala GCEO5 + EG5 and GCEO6 + NR1;
Leu
-Ala, GCEO7 + EG5 and GCEO8 + NR1;
Asp
-Ala, GCEO9 + EG5 and GCE10 + NR1;
Pro
-Ala, GCE11 + EG5 and GCE12 + NR1.
Oligonucleotides NR1 and EG5 were used to splice together the two
component fragments of each mutant to produce the point-substituted
cDNAs. The sequences of the oligonucleotides used and their positions
of hybridization with the Fc
RIIa
cDNA are as follows:
GBCO1, 5`-GAAGGACAAGGCTCTGGTCAAG-3` (nucleotide positions
443-464); GBCO2, 5`-CTTGACCAGAGCCTTGTCCTTC-3`(443-464);
GBCO3, 5`-CTGGAAGGACGCTCCTCTGGTC-3`(440- 461); GBCO4,
5`-GACCAGAGGAGCGTCCTTCCAG-3`(440-461); GBCO5,
5`-GGACAAGCCTGCTGTCAAGGTC-3`(446-467); GBCO6,
5`-GACCTTGACAGCAGGCTTGTCC-3`(446-467); GBCO7,
5`-GACAAGCCTCTGGCTAAGGTCAC-3`(447-469); GBCO8,
5`-GTGACCTTAGCCAGAGGCTTGTC-3`(447-469); GCEO1,
5`-CCCAGAAAGCTTCCCGTTTGG-3`(490-511); GCEO2,
5`-CCAAACGGGAAGCTTTCTGGG-3`(490-511); GCEO3,
5`-CAGAAATTCGCTCGTTTGGATC-3`(492-514); GCEO4,
5`-GATCCAAACGAGCGAATTTCTG-3`(492-514); GCEO5,
5`-GAAATTCTCCGCTTTGGATCCC-3`(494-516); GCEO6,
5`-GGGATCCAAAGCGGAGAATTTC-3`(494-516); GCEO7,
5`-ATTCTCCCGTGCTGATCCCACC-3`(497-519); GCEO8,
5`-GGTGGGATCAGCACGGGAGAAT-3`(497-519); GCEO9,
5`-CTCCCGTTTGGCTCCCACCTTC-3`(500-522); GCE10,
5`-GAAGGTGGGAGCCAAACGGGAG-3`(500-522); GCE11,
5`-CCGTTTGGATGCTACCTTCTCC-3`(503-525); GCE12,
5`-GGAGAAGGTAGCATCCAAACGG-3`(503-525). The alanine codon GCT or
its complement AGC are underlined. Oligonucleotides NR1 and EG5 are
described above.
Chimeric and mutant receptor cDNA expression
constructs were produced by subcloning the cDNAs into the eukaryotic
expression vector pKC3(29) . Each cDNA was engineered in the
PCRs to have an EcoRI site at its 5` end (the 5`-flanking
oligonucleotide primer NR1 containing an EcoRI recognition
site) and a SalI site at the 3` end (the 3`-flanking
oligonucleotide primer EG5, containing a SalI recognition
site), which enabled the cDNAs to be cloned into the EcoRI and SalI sites of pKC3. The nucleotide sequence integrities of the
chimeric cDNAs were determined by dideoxynucleotide chain termination
sequencing (30) using Sequenase (U.S. Biochemical
Corp.) as described(31) .
The substitution of the FcRII
domain 1 with that of the Fc
RI
chain produced a receptor
(designated D1
D2
), which as expected retained the capacity to
bind the multivalent IgG-EA complexes, as did the wild-type Fc
RII (Fig. 1a). However, in contrast to the wild-type
receptor the D1
D2
chimeric FcR did not bind dimeric-hIgG1 at
any concentration (Fig. 2). This suggests that domain 1 is
necessary for optimal Ig binding as demonstrated by the binding of
highly substituted but not small dimeric complexes.
Figure 1:
IgG complex binding of chimeric Fc
receptors. COS-7 cell monolayers were transfected with the following
chimeric cDNA constructs: D1D2
(a),
109-116
(b),
130-135
(c), or an expressible form of the Fc
RI
chain (d). The binding of IgG immune complexes was assessed directly
on the monolayers by EA rosetting using mouse IgG1-sensitized
erythrocytes. The transfections were performed using a transient
expression system, resulting typically in 30-50% of cells
expressing the chimeric FcR. IgG binding of the chimeric FcR is evident
by COS-7 cells binding IgG-sensitized eythrocytes, i.e. forming ``rosettes,'' which appear as cell outlines
covered in eythrocytes. Cells not expressing FcR or expressing FcR
incapable of binding IgG do not bind the sensitized
erythrocytes.
Figure 2:
Human IgG1 dimer binding of chimeric Fc
receptors. Radiolabeled dimeric human IgG1 was titrated on COS-7 cells
transfected with wild-type FcRII (
), an expressible form of
the Fc
RI
chain (
), or the following chimeric
receptor cDNAs: D1
D2
(
),
109-116
(
),
130-135
(
). All of the chimeras were
expressed on the cell surface as determined by EA rosetting, outlined
in the Fig. 1legend.
The previous
analysis of genetic polymorphisms of FcRII (21, 22, 23, 24, 25) in
conjunction with our molecular modeling studies described
above(20) , suggest that the region around residue 114 (human
equivalent of polymorphic residue 116 in mouse Fc
RII) in the
predicted B/C loop may be important in Ig binding. To investigate this
possibility a chimeric FcR (
109-116
) was constructed
wherein the B/C loop of Fc
RII (residues Ser
,
Trp
, Lys
, Asp
,
Lys
, Pro
, Leu
,
Val
) was replaced with the homologous region of the
Fc
RI
chain (Gly
, Trp
,
Arg
, Asn
, Trp
,
Asp
, Val
, Tyr
). After
transfection into COS-7 cells, this receptor was clearly able to bind
Ig in the form of multivalent immune complexes, i.e. erythrocytes highly sensitized with IgG (IgG-EA) (Fig. 1b). By contrast, this receptor was unable to
bind dimeric hIgG1 at any concentration, implying that the B/C loop is
essential for optimal Ig binding (Fig. 2). Similarly, the region
surrounding residue 131 responsible for the responder/nonresponder
phenotype of Fc
RIIa, i.e. the C`/E loop
(Ser
, Arg/His
, Leu
,
Asp
, Pro
, Thr
) was replaced
with the equivalent Fc
RI
chain sequence (Trp
,
Tyr
, Glu
, Asn
,
His
, Asn
), generating a chimeric receptor
(
130-135
) that upon transfection into COS-7 cells was
able to bind IgG-EA (Fig. 1c) but not dimeric IgG1 (Fig. 2). As expected COS-7 cells transfected with an
expressible form of the Fc
RI
chain (18) did not bind
hIgG1 dimers or IgG-EA (Fig. 1d and 2). Thus the
ability of the chimeric Fc
RII containing B/C or C`/E domain 2
substitutions to bind the highly sensitized EA complexes but not
dimeric hIgG1 suggests that these receptors bind IgG less avidly than
wild-type Fc
RII and clearly indicates that the B/C and C`/E
regions also make a contribution to the binding of IgG by Fc
RII.
Figure 3:
Human IgG1 dimer binding by FcRIIa
alanine point mutants. Radiolabeled dimeric human IgG1 was titrated on
COS-7 cells transfected with wild-type Fc
RII or Fc
RII
containing alanine point mutations. A, B/C loop mutants:
wild-type Fc
RII (
), Lys
Ala (
),
Pro
Ala (
), Leu
Ala
(
), Val
Ala (
). B, C`/E loop
mutants: wild-type Fc
RII (
), Phe
Ala
(+), Ser
Ala (
), Arg/His
Ala (
), Leu
Ala (
),
Asp
Ala (⊞), Pro
Ala
(
). A comparison of the levels of human IgG1 dimer binding to
Fc
RII mutants relative to wild-type Fc
RIIa is shown. C, B/C loop mutants; D, C`/E loop mutants. The
binding of wild-type Fc
RIIa was taken as 100% and mock-transfected
cells as 0% binding. Results are expressed as ±S.E. To control
for variable receptor expression between the mutant Fc
RII COS-7
cell transfectants, levels of expression were determined using a
radiolabeled monoclonal anti-Fc
RII antibody 8.2, and dimer binding
was normalized to that seen for wild-type Fc
RII. Typical levels of
8.2 binding in cpm ±S.E.: WT Fc
RII, 95,279; Lys
Ala, 71,660; Pro
Ala, 61,636;
Leu
Ala, 44,696; Val
Ala,
110,722; Phe
Ala, 74,707; Ser
Ala, 139,802; Arg/His
Ala, 140,475; Leu
Ala, 121,096; Asp
Ala, 100,149;
Pro
Ala, 172,047.
The replacement of the B/C loop residues
(Lys, Pro
, Leu
,
Val
) in turn with Ala in each case resulted in diminished
hIgG1 dimer binding (Fig. 3). The most dramatic effect was seen
on substitution of Lys
or Leu
, which
exhibited only 15.9 ± 3.4% (mean ± S.D.) and 20.6
± 4.0% binding compared with wild-type Fc
RII. The
replacement of Pro
or Val
with Ala had a
lesser effect, these receptors displaying 53.5 ± 13.5% and 73.5
± 7.9% wild-type binding respectively. It is interesting to note
that the individual replacement of these amino acids did not result in
the complete abolition of dimer binding seen in chimera
109-116
. These results suggest that each of these
residues in the B/C loop contribute to the binding of IgG by Fc
RII
either as direct contact residues or indirectly by maintaining the
correct conformation of the binding site. The same approach was used to
analyze the role of individual amino acids within the C`/E loop
(Phe
, Ser
, Arg/His
,
Leu
, Asp
, Pro
). In contrast
to that observed for residues of the B/C loop, mutation of individual
residues of the C`/E loop resulted in both loss and enhancement of IgG
binding. Substitution of Phe
and Arg/His
dramatically decreased hIgG1 dimer binding by approximately 90
and 80%, respectively, to 8.2 ± 4.4 and 21.9 ± 3.9
compared with that seen for wild-type Fc
RII (Fig. 3).
Interestingly, replacement of residues Asp
and
Pro
increased binding to 113.5 ± 8.8% and 133.5
± 3.2% of the wild-type receptor. The substitution of
Ser
or Leu
had no significant effect on the
binding of hIgG1 dimers, since these mutants exhibited binding
comparable with that seen for wild-type Fc
RII (Fig. 3).
These findings suggest that Phe
and Arg/His
may play an important role in the binding of hIgG1, and the
observation that the substitution of Asp
and Pro
increase binding also suggests an important role for these
residues, which appears distinct from Phe
and
Arg/His
. Again, a distinction between a possible direct
binding role or contribution to structural integrity of the receptor
cannot be made; however, these findings clearly identify both the B/C
and C`/E loops as playing a role in the binding of IgG by Fc
RII.
Site-directed mutagenesis was also performed on 3 residues of the
C`/C loop, a region predicted to be distant from the putative binding
region, i.e. the B/C, C`/E, and F/G loop regions. The
substitution of residues Asn, Gly
, and
Lys
had no effect on the binding of hIgG1 dimer, since
each of these mutants exhibited similar binding to the wild-type
receptor (data not shown).
Figure 4:
Molecular modeling of the extracellular
region of human FcRII (domains 1 and 2) and location of residues
putatively involved in the interaction with hIgG1. A,
Fc
RII domain 1-domain 2 model structure. Domain 1 is shown in green and domain 2 in darkblue. The three
regions of domain 2 putatively involved in IgG binding (B/C, C`/E, and
F/G loops) are highlighted in paleblue. The side
chains of amino acids implicated in hIgG1 binding as described under
``Results'' are indicated. Those side chains that when
substituted result in decreased or increased binding are shown in paleyellow or red, respectively. The brightyellow regions represent the A/B and G strands
of domain 1, predicted to be in close proximity to the domain 2 active
binding region. B, location of residues putatively involved in
the interaction of Fc
RII with hIgG1. Domain 2 and the domain 1
interface region of the Fc
RII domain 1-domain 2 model is shown to
highlight the putative binding region. Residues implicated in IgG1
binding are indicated as described above. The computer model of
Fc
RII domain 1domain 2 was generated by molecular modeling based
on the structure of the related CD4 domains 1 and 2 as described under
``Materials and Methods.''
The studies described herein provide evidence to suggest that
the interaction of IgG with human FcRII involves multiple regions
juxtaposed in the receptor. Previously, we have described the
localization of a single region of Fc
RII capable of directly
binding IgG situated in the second extracellular domain between
residues Asn
and Ser
(20) . Of the
entire extracellular region, only the 154-161 segment was
demonstrated to directly bind IgG, since placement of only this region
in the corresponding region of the human Fc
RI
chain, imparted IgG binding function to the IgE receptor Fc
RI.
Moreover, replacement of this region in Fc
RII with that of
Fc
RI
resulted in the total loss of IgG binding
including large complexes, implying that residues
Asn
-Ser
comprise the key IgG1 interactive
site of Fc
RII. However, the generation of further chimeric
Fc
RII/Fc
RI
receptors as described herein indicates that
two additional regions of Fc
RII domain 2 also influence the
binding of IgG by Fc
RII. The replacement of the regions
encompassing Ser
-Val
(B/C loop) and
Ser
- Thr
(C`/E loop) of Fc
RII with the
equivalent regions of the Fc
RI
chain, produced receptors
that, despite containing the putative binding site
(Asn
-Ser
) and retaining the ability to bind
large complexes (IgG-EA), lost the capacity to bind small complexes
(dimeric hIgG1). Indeed, site-directed mutagenesis performed on
residues of the B/C and C`/E regions identified a number of amino acids
that appear to play crucial roles in hIgG1 binding by Fc
RII. The
replacement of Lys
, Pro
, Leu
,
and Val
of the B/C loop and Phe
and
Arg/His
of the C`/E loop with alanine all resulted in
diminished hIgG1 binding. Furthermore, the substitution of Asp
and Pro
of the C`/E loop increased hIgG1 binding.
Therefore, these findings provide strong evidence to suggest that the
B/C and C`/E loops of Fc
RII, in addition to the F/G loop, also
contribute to the binding of IgG.
A number of other studies have
provided evidence to support the proposed IgG binding roles of the B/C
and C`/E loop regions of FcRII. Studies of genetic polymorphisms
of mouse and human Fc
RII have implicated residues 114, 131, and
159 in the binding of IgG by human Fc
RII. These residues are
located in the B/C (residue 114), C`/E (131), and F/G (159) loops,
respectively. The Ly-17 polymorphism of mouse Fc
RII has been
described at the molecular level as two allelic variants (Ly17.1 and Ly17.2) that differ only at residues 116 and 161 (the
equivalent of residues 114 and 159 in the human). Monoclonal antibodies
specific for Ly17.2 inhibit the binding of IgG to the
receptor, implying that residues 116 and/or 161 (and therefore their
human equivalents) are involved in binding themselves or closely
situated to residues crucial in the interaction of Fc
RII with
IgG(24, 25) . Furthermore, the high responder/low
responder polymorphism of hFc
RIIa results in an amino acid
substitution at residue 131, which has been shown to influence the
binding of mIgG1 and hIgG2(21, 22, 23) . The
findings described herein also indicate that the nature of the residue
at 131 plays a role in the binding of hIgG1, since replacement with
alanine results in almost complete loss in binding of this isotype to
Fc
RII. Thus, although the F/G loop of Fc
RII is clearly a
major region involved in the direct interaction with IgG, as
demonstrated by the fact that only this region has been definitively
shown to directly bind IgG(20) , residue 131 also appears to
play a binding role. However, the question of whether residue 131 is
directly participating in IgG binding or providing a secondary or
indirect influence remains to be answered.
The mutagenesis data
clearly implicate a number of distinct regions within FcRII in the
interaction with IgG complexes as described above. The spatial
relationship of these regions, i.e. residues 109-116
(B/C loop), 129-135 (C`/E loop), and 154-161 (F/G loop) is
postulated in our model of Fc
RII (Fig. 4). This model
suggests that these regions are juxtaposed to each other in domain 2 at
the interface with domain 1 and form a hydrophobic pocket surrounded by
a wall of additional residues. The data supporting this model include
the following. 1) Mutagenesis of the hydrophobic residues
Ile
, Gly
, Pro
, Leu
almost completely abolishes binding of dimeric hIgG1 complexes.
2) Substitution of residues that may contribute the wall (Lys
in the B/C loop, Phe
and Arg
in the
C`/E loop, and Leu
and Phe
in the F/G loop)
also modify binding of immune complexes. 3) It may also be expected
that such a wall would be accessible to anti-FcR antibodies. Indeed
several anti-Fc
RII monoclonal antibodies detect epitopes in the
B/C, C`/E, and F/G loops. For example, the epitope detected by the
anti-human Fc
RII antibody 41H16 (39) is dependent on
residue 131 of the C`/E loop, and the Ly-17 epitope of mouse Fc
RII
is dependent on residues that equate to residues 114 and 159 in human
Fc
RII (25) that are located in the B/C and F/G loops,
respectively. 4) The studies described herein demonstrate that domain 1
of Fc
RII, although it does not appear to play a direct role in IgG
binding, does play an important role in the affinity of IgG binding by
Fc
RII. This is suggested since replacement of domain 1 of
Fc
RII with domain 1 of Fc
RI reduced the capacity of
Fc
RII to bind IgG, as shown by the failure of this receptor to
bind dimeric hIgG1. These data imply that the IgG binding role of
domain 1 is likely to be an influence on receptor conformation,
stabilizing the structure of domain 2 to enable efficient IgG binding
by Fc
RII. Again this proposal is consistent with the molecular
modeling, which suggests the localization of the IgG binding site of
Fc
RII to loop regions in domain 2 at the interface with domain 1.
The binding site would therefore be in close proximity to domain 1 and
as such predicted to be influenced in conformation, presumably by the
loop and strand regions at the ``bottom'' of domain 1. These
regions include the G strand and the A/B and E/F loops, which may
therefore interact with the ``active'' binding region of
domain 2.
Further support for the involvement of the B/C and C`/E
loops of FcRII domain 2 in the binding of IgG has been provided in
the cloning and subsequent Ig binding studies of rat
Fc
RIII(40) , which is structurally and functionally
homologous to Fc
RII. Two rat Fc
RIII isoforms, IIIA and IIIH, which have extensive amino acid differences in
their second extracellular domains, have been shown to bind rat and
mouse IgG subclasses differently. Both isoforms bind rtIgG1, rtIgG2a,
and mIgG1; however, they differ in that only the IIIH form
binds rtIgG2b and mIgG2b. Significantly, the amino acid differences
between rat Fc
RIIIA and IIIH isoforms are
situated predominantly in the predicted B/C and C`/E loops of domain 2.
However, it should be noted that the F/G loop regions of rat
Fc
RIIIA and IIIH are almost totally conserved,
which together with the observation that both forms bind rtIgG1,
rtIgG2a, and mIgG1, is consistent with the proposal that the F/G loop
region is the major IgG interactive region and that the B/C and C`/E
loop regions provide supporting binding roles. In addition, a recent
mutagenesis study of human Fc
RIII has also implicated residues in
the B/C and C`/E loops of this receptor in the binding of
IgG(41) . It is also interesting to note that in this study the
C/C` region of Fc
RIII was suggested to play a major role in IgG
binding, which is in marked contrast to our findings with Fc
RII.
Indeed, the substitution of 3 residues in the C/C` loop of Fc
RII
with alanine, namely Asn
, Gly
, and
Lys
, did not have any effect on the binding of dimeric
hIgG1. Therefore, these findings somewhat surprisingly suggest that
Fc
RII and Fc
RIII, which exhibit substantial amino acid
sequence conservation and similar IgG binding affinities and
specificities, may interact differently with IgG.
It is interesting
to note that a number of parallels are apparent in the molecular basis
of the interaction of FcRII with IgG and that of Fc
RI with
IgE. The Ig binding roles of the two extracellular domains of Fc
RI
are similar to Fc
RII, with domain 2 responsible for the direct
binding of IgE and domain 1 playing a supporting structural role (18, 26, 42) . Furthermore, as described for
Fc
RII, we and others have also identified multiple IgE binding
regions in domain 2 of Fc
RI. Using chimeric Fc
RII/Fc
RI
receptors we have demonstrated that domain 2 of Fc
RI contains at
least three regions, each capable of directly binding IgE, since the
introduction of the Fc
RI regions encompassed by residues
Trp
-Lys
,
Tyr
-Asp
, and Lys
-Glu
into the corresponding regions of Fc
RII was found to impart
IgE binding to Fc
RII(1, 18, 20) . A
similar study using chimeric Fc
RIII/Fc
RI receptors has
implicated 4 regions of Fc
RI domain 2 in IgE binding since the
regions Ser
-Phe
,
Arg
-Glu
,
Asp
-Ser
, and Lys
-Ile
of Fc
RI when replaced with the corresponding regions of
Fc
RIII resulted in the loss or reduction of IgE
binding(42) . Taken together, these data suggest that at least
four regions of Fc
RI domain 2 contribute to the binding of IgE,
Ser
-Phe
,
Arg
-Glu
,
Tyr
-Ser
, and
Lys
-Glu
. Three of these regions correspond
to the three regions identified herein as important in the binding of
IgG by Fc
RII, Arg
-Glu
,
Tyr
-Ser
, and
Lys
-Glu
, which encompass the B/C, C`/E, and
F/G loops, respectively. In addition, studies with anti-Fc
RI
chain mAb have indicated that the region encompassed by residues
100-115 contains an epitope detected by mAb 15A5, which can
completely block the binding of IgE to Fc
RI(43) . Thus,
these findings implicate the B/C, C`/E, and F/G loops juxtaposed in
domain 2 at the domain 1 interface as the crucial IgE-interactive
region of Fc
RI. Clearly, the findings described herein for
Fc
RII together with those discussed for Fc
RI provide evidence
to suggest that the Ig-interactive regions of Fc
RII and Fc
RI
are conserved between the two receptors, with the domain 1-domain 2
interface forming the Ig binding site.
In conclusion, the results
presented herein demonstrate that multiple regions of hFcRII are
involved in the binding of IgG, with three putative loop regions
juxtaposed in the second extracellular domain at the domain 1 interface
comprising the IgG binding site. The proposition that the functionally
distinct receptor Fc
RI also interacts with IgE in a structurally
similar fashion, in conjunction with the conserved nature of the
extracellular regions of the Ig superfamily FcR, strongly suggests that
this region will also comprise the key Ig-interactive site of all
members of this family.