The

two binding-site binding JANOS

models

GERGELY

AND

of Immunology,

GABRIELLA EOtvOs Lordnd

SARMAY University,

Abstract Fc receptors (FcR) are immunoglobulinbinding molecules that enable antibodies to perform several biological functions by forming a link between specific antigen recognition and effector cells. FcRs are involved in regulating antibody production as well. Most FcRs belong to the immunoglobulin superfamily, and show structural homology with each other and their

ligands.

Recent

data

on the structure

of IgG

binding FcRs obtained from monoclonal antibodies and gene cloning studies, as well as on ligand binding capacity and fine specificity of the receptor binding site (or sites), are reviewed. The binding capacity and fine specificity of receptor binding sites, as well as the structure and conformation of the immunoglobulin ligands, play important roles in triggering FcR-mediated signals. In induction of signals, the interaction of the FcR with the CH2 domain of the IgGFc is decisive. The high-affinity FcyRI possess one active binding site specific for contact residues that is located at the Nproximal end of the CH2 domain and is able to mediate both

binding

Fc’yRIII

has two active binding

sites: the CH3 domain-

and

specific

site,

only

which

CH2 domain-specific binding and signaling.

signal mediates

transfer.

The binding;

low-affinity and

the

site, which is responsible for Similarly, the low-affinity Fc’yRII

on resting B cells has one site for CH2 and another for CH3 binding. The expression, release, and fine specificity of Fc-yRII on B cells correlates with the cell cycle. -GERGELY, J.; SARMAY, G. The two binding-site models of human IgG binding Fc’y receptors. FASEBJ 4: 3275-3283; 1990. Key site

IgG

Fc-y receptors

Department

with

of human

Words: Fcy receptors extracellular domain - binding antibody dependent - cytotoxicity signal transduction

Fc RECEPTORS (FcR)1 REPRESENT binding structures that specifically

A

family

of antibody

recognize and bind homologous immunoglobulins (Ig) via their Fc portion (1). A wide range of cells express FcR; most belong to the immune system (including monocytes, macrophages, granulocytes, K cells, B cells, and some T cells), but FcRs are present in membranes of other cell types as well. These receptors enable antibodies to perform many biological functions by forming a link between

God, 2131

Hungary

specific antigen recognition (provided by the antibody) and effector cells. Besides triggering cytotoxicity and inducing secretion of mediators and endocytosis of opsonized particles, FcRs play an important role on different levels in the regulation of antibody production (reviewed in refs 2-4). The functional variety of FcRs results partly from their structural diversity; however,

the biological functions of FcRs are also determined by their specific structure and tissue distribution. DNA cloning and sequencing studies have revealed that FcRs (with one exception: the low-affinity Fc#{128}RII) belong to and form a subgroup of the Ig superfamily (5). In this review we concentrate on human receptors for IgG and summarize recent data on the structure, ligand sites. HUMAN

binding

capacity,

and

fine

specificity

of binding

Fc-yR

Three classes of human IgG binding FcRs have been defined by Anderson and Looney (6) by using monoclonal antibodies (MAb) recognizing FcR epitopes. The three types of receptors are expressed on various and overlapping populations of cells (Table 1). The high-affinity Fc’yRI (CD64) is a 72-kDa glycoprotein (7) that is expressed mainly on mononuclear phagocytes. The rank order of affinity of Fc-yRI for IgG isotypes is IgGI = IgG3 > IgG4, although it does not interact with IgG2. The receptor is trypsin-resistant;

the core protein (after removal of N-linked carbohydrates) has an Mr of 72,000 (8). Fc-yRII (CD32) is a protein of 40,000 Mr (9) that is widely distributed in a variety of cells such as monocytes, platelets, neutrophils, and B cells. FcyRII has a lower affinity (10); hence it requires multiple Fc interaction (aggregated IgG), binds IgG1 and IgG3 equally well, but binds the other subclasses less readily. The receptor is structurally polymorphic, and monocytes from different donors bind murine IgGi either strongly or weakly (11). The low-affinity Fc’yRIII (CD16) ex-

‘Abbreviations: ADCC, antibody-dependent cellular cytotoxicity; FcR, Fc receptor; IBF, immunoglobulin binding factor; Ig, immunoglobulin; MAb, monoclonal antibody; PIG, phosphatidylinositol glycan; TM, transmembrane; SP, signal peptide; EC, extracellular domain; TM, transmembrane domain; CP, cytoplasmic domain; K, killer; NK, natural killer.

0892-6638190/0004-3275/$01 .50. © FASEB 3275 www.fasebj.org by Kaohsiung Medical University Library (163.15.154.53) on September 05, 2018. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNumber

TABLE

1. Human

FcR FcyRI

Fcy receptors

Mr

72,000

Characteristics High

affinity

Affinity

Expression Mononuclear

IgGi does

phagocytes

CD64 FcyRII

40,000

Low

affinity

CD32 Fc1RIII CDI6

50,000-70,000

Low

affinity

Monocytes,

platelets,

IgGI

neutrophils,

B cells, K562

IgG2

Neutrophils, mononuclear

NK, K cells, phagocytes

IgG1

pressed primarily on neutrophils, natural killer (NK) and killer (K) cells, and to some extent on monocytes and macrophages (12), has an Mr of 50,000-70,000. Fc-yRIII expressed on neutrophils is different from that present on tissue macrophages (13). The receptor shows preferential binding of IgG1 and IgG3. All Fc-yRs except the phosphatidylinositol glycanlinked form of Fc-yRIII (Fc-yRIII-PIG) are integral membrane proteins with a glycosylated extracellular part, a single transmembrane segment, and a COOHterminal cytoplasmic tail. The NH2-terminal extracellular portion contains two domains that show homology with the CH2 set of Ig domains (5). Murine FcyRIIs are encoded by two genes (a and /3) with 95% homology in the region corresponding to the extracellular domains. Alternative splicing of the /3 gene results in two transcripts (/3k and 132), which differ in a 138 bp-long deletion within the region coding for the cytoplasmic domain. Recent reports from several groups (14-22) have indicated that as in the mouse, the human Fc’yRII comprises a family of at least three highly related isoforms, designated Fc’yRIIA, FcyRIIBI, and Fc’1RIIB2. Different reactivities of certain monoclonal antibodies had previously suggested the possible diversity of Fc7RII. It was found that MAbIV.3 recognized molecules on monocytes but not on B cells (10), whereas MAb KuFc79 reacted with molecules on monocytes and B cells, but not on platelets (17). Although several laboratories published data recently on the structure of human Fc7RII deduced on the basis of gene cloning and sequencing, the pattern of expression of mRNA corresponding to the various types of human cDNA is not entirely clear. The structural heterogeneity of the human Fc’yRII has been elucidated by Brooks et al. (18) through the isolation, characterization, and expression of cDNA clones derived from myeloid and lymphoid RNA. The predicted amino acid sequences were consistent with integral membrane glycoproteins with single membranespanning domains. The extracellular domains displayed sequence homology to other Fc-yR. They also found that a minimum of three genes (Fc’yRIIa, lIa’, and lib) encode the transcripts that demonstrate highly related extracellular and membrane-spanning domains. IIa/IIa’ differed in the intracytoplasmic domain from IIb. According to Brooks et al. (18), alternative splicing of the IIb gene generates further heterogeneity of the predicted protein.

for human =

not

isotypes

IgG3>IgG4, bind IgG2

IgG3, less readily and IgG4

Binding of murine IgG IgG2a

References

7, 2

and IgG3

IgGI

2, 11

IgG3

33-35

Seki (16), in good agreement with other groups (19-21), described two distinct but similar cDNA clones encoding isoforms of the FcyRII differentially expressed either by human B cells or activated T cells and monocytes. The nucleotide sequence of the cDNA clone from B cells (/3Fc-yRII) contains an open reading frame that encodes a protein with an extracellular domain of 179 amino acids. It has four evenly spaced Cys residues, which suggests the existence of two domains that may contain three potential N-linked glycosylation sites. The presumed transmembrane domain consists of 26 residues, and is followed by four positively charged amino acids that begin a cytoplasmic domain of 44 amino acids. The deduced amino acid sequence of the protein encoded by clones from peripheral T cells and monocytes (aFc-yRII) shares an extracellular domain of 179 amino acids with two presumptive sites for N-linked glycosylation and a transmembrane domain of 26 residues, whereas the cytoplasmic domain consists of 76 amino acids. The predicted relative mass of both molecules is consistent with the observed Mr of 40,000. The protein encoded by /3Fc’yRII has an inferred Mr of 27,000 with three glycosylated sites, whereas the one encoded by aFc-1RII (Mr 31,000) has two N-linked glycosylated sites (16). Comparison of the deduced amino acid sequences of the human /3Fc-yRII, aFc-yRII, and mouse

/32Fc’yRII

gene

products

revealed

that

the

human

receptors have almost identical extracellular and transmembrane domains, which are similar to those of the mouse /32FcyRII product. On the other hand, the human clones code for very different signal peptides and cytoplasmic domains. Recently (22) a cDNA encoding the human /3FcyRII was isolated from a placental cDNA library. It was suggested that the receptor is synthesized with a 42-aminoacid leader sequence; the mature protein consists of 249 amino acids. The leader sequence and the cytoplasmic domain were found to be different from the CD32 antigen, but have shown, in accordance with Seki’s findings (16), great homology to the mouse fl2Fc’yR. Using CD32 and /3Fc-1RII-specific cDNA fragments allowed Engelhardt et al. (22) to determine the distribution and induction of each of these receptors on the mRNA level, whereas others (19-21) did not differentiate between CD32 and /3Fc-yRII transcripts because they used the complete cDNA as a hybridization probe (22). By cloning and sequencing of cDNAs encoding murine Fc-1RII (15), it was shown that the ectodomains

3276 Vol. 4 December 1990 The FASEB journal GERGELY AND SARMAY www.fasebj.org by Kaohsiung Medical University Library (163.15.154.53) on September 05, 2018. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNumber

f Fc-yRIIA,

Fc’yRIIBl,

and

Fc-yRIIB2

are

closely

elated; however, their cytoplasmic domains are disinct. Fc-1RIIB1 and Fc’yRIIB2 are identical except for n in-frame insertion in the cytoplasmic tail of Fc’yRIIBl increasing its length from 47 to 94 amino acids). The structural comparison between human and murine Fc-1RII led to the conclusion (16) that these receptors derive from a common ancestral gene (Fig. 1). Two forms of the low-affinity human Fc-yRIII were described. The cDNA clone for Fc7RIII isolated from a human leukocyte library was found to encode a 46-kDa phosphatidylinositol glycan-linked membrane protein (Fc-yRIII-PIG) (23-27). The receptor has an extracellular domain (consisting of two Ig-like loops) that is most homologous to human Fc-yRII and mouse Fc’yRIIa, yet contains six sites for N-linked glycosylation. Recently a transmembrane form of Fc-yRIII was found in NK cells (Fc-yRIII-TM). The difference between cDNAs corresponding to Fc-yRIII-PIG and FcyRIII-TM was characterized by single nucleotide substitutions. Because one of these converted the termination codon UGA (PIG) to CGA, the predicted cytoplasmic domain

is extended

by 21 amino

acids (28).

Kinet (4) proposed that the processed Fc-yR can be classified into three groups. Group I contains the human Fc-yRI; group II includes several forms of human FcyRII and mouse Fc’yRIIb, whereas human Fc’yRIII-TM and mouse Fc-yRIIa belong to group III. Within a group, the receptors show considerable homology in their extracellular portions, but no more than between groups. Within a group the transmembrane segments show strong homology (50-76%), but between groups the sequence homology is low (0-33%). The cytoplasmic portions show low homology within a group (16-27%) and practically none between groups. The IgG binding FcRs belong to the same superfamily of molecules as their ligands, i.e., their extracellular parts show considerable sequence homology with Ig domains. As the contact residues on the Ig molecules and the binding site (or sites) on the FcR are likely exposed on the surface of the corresponding molecules,

the receptor-ligand interactions may be similar to the interdomain interactions of Ig molecules (29). The homology in the extracellular domains containing the binding site (or sites) on the one hand, and the striking differences in the structure of transmembrane parts, and especially in the cytoplasmic tails, on the other, explain why the interaction of identical/similar ligands with FcRs results in activation of different signaling mechanisms. The importance of type-specific variations among cytoplasmic domains in distinct functions mediated by FcR can be illustrated by the two isoforms of FcyRII (3, 30). FcyRIIB2 mediates uptake and delivery to lysosomes of IgG complexes via internalization in coated pits, whereas FcyRIIB1 is incapable of mediating ligand uptake and degradation, i.e., the 47-amino-acid insert in the Fc-yRIIB1 cytoplasmic portion disrupts a domain necessary for accumulation in coated pits. Despite the resembling extracellular domains, the various isoforms of human FcyRII might differ in fine specificity and binding affinity. Moreover, the ligand binding and signal transfer might be influenced by the actual conformation of the receptor. Finally, the density of the ligand and the actual conformation of the Ig molecules are also important in the interaction of FcR and their ligands. In the remainder of this review we will concentrate on the biological importance of the fine specificity of Fc-1R binding site (or sites) and on that of the structure of its ligand (Ig). BINDING

SPECIFICITY

OF

FcyR

Although the extracellular domains of Fc7R show remarkable homology, the various isoforms exhibit slightly distinctive IgG subclass recognition specificity. Earlier studies pointed to the preferential role of IgGi and IgG3 subclasses both in binding and triggering Fc-yR-mediated functions (31). However, it seems that because of characteristic sequential and/or conformational differences of the various IgG subclasses (reviewed in ref 32) and the number and fine specificity of the receptor binding site (or sites), considerable differences can be found in their interaction and in activation of signaling mechanisms. From this point of view the identification of interacting groups on the ligand

and the characterization of the receptor binding sites) are of great biological importance.

Ii -

cp

I CITMI CP

Figure mily.

1. Schematic Regions equal

model for the evolution to the ancestral gene

of the Fc.yRII gene fa(top) in nucleotide se-

quence are hatched. TM: transmembrane

SP: signal peptide; EC: extracellular domain; CP: cytoplasmic domain.

16, with

from

permission

the author.

domain; From ref

site (or

As already mentioned, the high-affinity Fc’yRI binds not only complexed (antigen plus antibody) but also monomeric IgG; moreover, the IgGi and IgG3 isotypes interact with a tenfold higher association constant than IgG4, whereas IgG2 binding was not demonstrable (2). Fc-1RII binds monomeric IgG with lower affinity than ligands in complexed form (antigen-IgG complex or

aggregated

IgG),

and the affinity

of IgGi

and

IgG3

binding is higher than that of IgG2 and IgG4, whereas Fc’yRIII recognizes only IgGI and IgG3. The interaction of rhesus D-positive red cells sensitized with anti-D

antibodies were

suitable

belonging tools

either

to IgGi

in studying

both

or IgG3 subclasses the FcR-ligand

in-

HUMAN lgG-BINDING Fc-y RECEPTORS 3277 www.fasebj.org by Kaohsiung Medical University Library (163.15.154.53) on September 05, 2018. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNumber

teraction

and

the

subsequent

triggering

of

effector

typic

mechanisms, such as phagocytosis and ADCC. When studying the rosette formation of human monocytes with

anti-D-sensitized

erythrocytes,

we obtained

higher

percentages of EA-rosettes with IgG3-sensitized cells than with the IgG1-sensitized ones (35), which is in good agreement with other findings (33, 34). IgG3 proved to be more efficient in sensitizing erythrocytes for phagocytosis as well. It was also shown that FcyRIIbearing B cells formed rosettes mainly with IgG3sensitized red blood cells, whereas K cells interacted equally well with both subclasses (35’ Although human monocytes express the three types of Fc’yR, the mediation of antibody-dependent cellular cytotoxicity (ADCC) is the function mainly of FcyRI and Fc-1RII (36). The same type of target cell lysis is mediated by FcyRIII when K cells are the effector cells. Comparing the ADCC activity of monocytes and K cells (Fig. 2), it was shown (35) that IgGland IgG3-sensitized erythrocytes were equally efficient in inducing the monocyte-mediated ADCC. Although both isotypes bind to the same receptor (Fc-1RIII), IgGi was more active than IgG3 in K cell-mediated lysis. It was remarkable, however, that the increase in the density of the target cell-sensitizing IgG3 molecules resulted in effective killing induction. The differences in binding and in effector function activating capacity can be explained by dissimilarities in the structure of the ligands and by those in structure and fine specificity of the receptor binding site (or sites). THE

BINDING

SITE

OF

FcyRI

Although earlier studies attributed cytophilic activity to the CH3 domain of IgG (37, 38), Woof et al. (39) showed that CH3 does not express the contact residues forming the human Fc’yRI interaction site, but is involved in a composite CH2/CH3 interaction site. By using MAb specific for isotypic, allotypic, or isoallo-

U937

10

epitopes,

the

topographical

distribution

of group

recognized by the human FcyRI was determine (40-43). The results obtained by using MAb as well a those found in comparative studies of the interaction o

human,

mouse,

and rabbit

IgG isotypes

with humar

Fc-yRI, and investigations with chymeric antibodies an aglycoforms of IgG1 and IgG3, allowed localization o the Fc-yRI binding region on human IgG. Two regions of sequence that may form a continuous protein surface on the CH2 domain seem to be important in the interaction with Fc-yRI expressed on monocytes (Fig. 3). These regions are close to the hinge (Leu 234-Ser 239) and near the COOH-terminal end of the domain (Gly 316-Lys 338). Their average position is close to the two 13-strands and joining bend formed by the residues Gly 316-Lys 338 (44). It is important that although IgGi and IgG3 have identical sequences (GluLeu-Leu-Gly) at residues 233-236, IgG2 is different (Pro-Val-Ala and a deletion at residue 236). This explains the lack of binding of IgG2 to FcyRI. On the other hand, because of the extended rigid structure of the hinge region of IgG3 (32), the accessibility of its interacting site (and consequently the binding affinity of complexed IgG3 to Fc’yRI on monocytes) relative to IgGl is better. It was shown that the two Ig-like extracellular domains of Fc1RI form one active binding site that interacts with a site on IgGl or IgG3 located at the NH2-proximal end of their CH2 domain. The highaffinity binding of these isotypes may depend on the rigid ligand binding site interaction supported by the steric effect of the non-Ig-like third extracellular domain of the receptor. THE

TWO

BINDING

SITES

OF

Fc-yRIII

It is well known that FcRs expressed on K cells use antibodies to recognize their target cells, and that FcRs mediate the killing signals resulting in target cell lysis. Monocytes appear to use mainly FcyRI for ADCC,

CELLS

K CELLS

100

1000

Amount

of

antibody

lull

Figure

2. Antibody-dependent lysis of human red blood cells sensitized with IgGi (-) and IgG3 (---) anti-D antibodies mediated by prestimulated U937 cells and K cells at different levels of sensitization. 5tCr-labeled erythrocytes were sensitized with different amounts of IgGi or IgG3 anti-D antibodies having a similar capacity to bind to red blood cells. Units represent microliters of sera added to the erythrocytes. The sensitized red blood cells were washed and added to K cell-enriched effector cell suspension or PMA prestimulated U937 cells, respectively. The specific 5tCr release was measured in the supernatants of samples after overnight incubation at 37#{176}C.

The FASEB journal GERGELY AND SARMAY 3278 Vol. 4 December 1990 www.fasebj.org by Kaohsiung Medical University Library (163.15.154.53) on September 05, 2018. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNumber

and His 268 as well, whereas the interacting groups within the CH3 domain have been localized in the region of Ser 408-Arg 416 (Fig. 4). In an associative recognition system (47) where the firm binding of the

2711.

268

interacting effector and target cells was mediated dition to IgG by C3b-bridges, a higher enhanced of killing was observed. In such systems, by using or CH3 domain-specific MAb to inhibit ADCC

and

in experiments

deleted 3) lytic

239

applying

paraproteins signals are

(48), mediated

CH2

or CH3

in adrate CH2 (46),

domain-

it was demonstrated only by the interaction

that of

the CH2 domain of the sensitizing antibody with the corresponding binding site of FcR, and 4) the interaction between the CH3 domain and CH3-specific binding site is responsible only for increasing the binding affinity.

The characteristic IgG3 in the induction

differences

can be explained

by the structural

dissimilarities

these

of

region.

difference residues teracting

CH3

and

IgGI lysis

(35)

and conformational

isotypes,

their differing interaction receptor. IgG1 and IgG3 hinge

between

of Fc-yRIII-mediated and

consequently

by

with

the binding sites of the differ in the length of their

Furthermore,

there

is a characteristic

in the hydrophilicity of the CH2 domain that includes the potential contact residues inwith the CH2 domain-specific binding site of

Fc-yRIII (35). The unique primary amino acid sequences of the IgG1 and IgG3 domains, i.e., Lys/Gin 274 and Asn/Lys 276, may result in differences of tertiary structure of these isotypes. Therefore, the similarity in lysis triggering capacity of these two types can be explained by the differing accessibility the CH2 and CH3 domains. Presumably, both

CH2 Figure 3. Suggested molecular location of Fc-yRI (I FcyRIII ( _) binding regions on IgG Fc.

]) and

and CH3

domains

of IgG1 interact

with

the disisoof the

the cor-

responding binding sites of FcyRIII, whereas because of the differences in amino acid residues at critical positions (contact residues), only the CH3 domain of the

IgG3

molecule

is accessible

binding.

Howcellsowing

The Fc-1RIII

triggering site on the CH2 domain. All these observations emphasize the importance of the actual conformation and structure of the ligand in inducing functionally significant signals mediated by the receptor.

lytic

ADCC either

fine specificity was studied

activity

of K cells

was investigated with

complement

and signal-inducing in experiments

CH3

(45,

after

domain-specific

component

(which

46).

capacity monitoring

The

treating soluble

binds

of the

inhibition

of

the target

cells

Fc-yR

the CH2

and

Lys 274-Arg

formation.

might These

events

the disadvantages be overcome lead

to the

of IgG3

in

by changes

in con-

accessibility

of the

THE

TWO

BINDING

SITES

OF

Fc’yRII

or

in some experiments the effector cells were treated with synthetic peptides comprising linear sequences of either CH2 or CH3 domains of IgGi. It was shown that 1) Fc-1RIII mediating the lytic process of K cells possess two active binding sites, and efficient lysis depends on the simultaneous interaction of these with the CH2 and CH3 domains. 2) The groups that react with the CH2 domain-specific binding site are situated in the region

of residues

interactions,

induction

Clq

domain)

or with MAb recognizing epitopes either on CH2 CH3 domains, respectively. On the other hand,

lysis

density of molecules,

the target probably

ever, above a sensitizing IgG3

to inter-Fc

critical antibody

for effective

whereas on K cells, target cell lysis is mediated by Fc-yRIII. The application of differently sensitized target cells allowed us to study separately the lytic activity of monocytes and K cells mediated by Fc-1RI and Fc-yRIII, respectively.

301, and may involve Gly 237

Previously

we showed

that

a subset

of FcR

expressing

human peripheral lymphocytes releases their FcR when incubated at 37#{176}C in serum-free medium; furthermore, functionally active, soluble FcR could be isolated from the supernatants of the cells (49, 50). The released receptors were found to be monomeric, and interacted with the CH3 domain of IgG. Another population of FcR cells did not release their receptors under similar conditions (Table 2): Fc’yRIII expressed on K/NK (45) cells and Fc’yRII expressed on resting B cells (51) were

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A

B

Z of A000 Inhibition

25

CH2

50

____

274

nM

100

281

289

293

298

301

I

I

4Y511’ 1-*

CH3

ZB8

CH2

FlOf (.) G7c (is) A55 (+)

CH3

xSa8 (X) 0F3

IgGi

JL512(D)

1K PREQQYDSTYR

KFDWYVDG

4

CH1 Y48 FY91 Y77

418

48

(U)

(c’)

SKLTVNKSR

407

(T) (Y)

.--

-

--

-pY98 Y49

Figure 4. Effect of synthetic peptides and monoclonal antibodies on ADCC. Effector or target cells were pretreated with different amounts of synthetic peptides comprising sequences of CH2 (Y51, Y48, Y91, Y77) or CH3 (Y49, Y98) domains of IgG1 (A) and that of CHI (ZB8), CH2 (FIOf, G7c, A55) and CH3 (x3a8, 0F3) domain-specific monodonal antibodies (B). As control, IgGi-specific monoclonal antibody

UL512) was

used. Human blood lymphocytes as effector cells were pretreated with different amounts of synthetic peptides at 37#{176}C for 30 mm, and the anti-D IgG-sensitized target erythrocytes were added (A); or anti-D IgG-sensitized human red blood cells were pretreated with different dilutions of monoclonal antibody containing ascitic fluids for 30 mm at room temperature, and added to the effector cells (B). Effector:target cell ratio was 10:1. The ADCC tests were carried out overnight, and specific 51Cr release from the target cells was measured in the supernatants of the samples. The % ADCC inhibition was calculated by taking the values of untreated control samples as 100%.

characterized as stable types being maintained in a cellattached state. In a similar manner, Fc-1RIII and FcyRII on resting B cells also possess TABLE

2. Fine specificity

FcyRI

Released Fc7RIH Fc7RIII

of Fcy

receptor binding

U937

resting soluble

binding

Investigated cell types

Receptor

Fc.yRII

two active

B cells

Although

sites Number of binding sites

Domain specificity

monocytes

One

N-proximal of CH2

B cells

Two

CH2

One

CH3

Two

CH2

Resting Activated

K cells

sites.

the contact residues of the Fc domains are not yet mapped, it was demonstrated that FcyRIIs also express one specific binding site for CH2 and a second one for CH3 domains (51).

B cells

and

and

Fine specificity end

CH3

CH3

References

Leu234-Ser239 Gly316-Lys338

44

Not

tested

51

Not

tested

49, 50

Lys274-Arg3Ol (G1y237 and

His268?)

45, 46

Ser408-Arg416

3280 Vol. 4 December 1990 The FASEBJournal GERGELY AND SARMAY www.fasebj.org by Kaohsiung Medical University Library (163.15.154.53) on September 05, 2018. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNumber

FcyRIIs expressed on B cells play an important role n regulating antibody production. The cross-linking of hese receptors with the antigen-binding slg results in nhibition of the antibody production of B cells (52). cRs or FcR-like molecules released from various cells s Ig binding factors (IBF) are also known as modulaors of the humoral response and antibody isotypes reviewed in ref 53). One might suppose that expression and release of these receptors are integral elements of mechanisms regulating B cell antibody production. Therefore it was important when a correlation was found between B cell activation and Fc’yRII expression and release (51). Resting B cells express Fc7RII in stable form. In the very early phase of B cell activation, a transient increase in expression of functionally altered Fc-yRIIs (with decreased ligand binding capacity) occurs. This was followed by down-regulation of the receptors

as a result

of a complex

series

of events.

At

first, the down-regulation may result from receptor release accompanied by a decreased rate of FcR expression. Later, although receptors are expressed, they shed easily from the cell membrane. The shed receptor is monomeric, with one active CH3 domain-specific binding site. Finally, probably in the G2 phase of the cell cycle, increased expression and normal binding capacity (CH2and CH3-specific active binding sites) reappear. These observations agree with recent findings by Amigorena et al. (54), who found that Fc7RII expression on mouse B cells occurs in the late G1 phase of the cell cycle. In contrast to the resting B cells, activated B lymphocytes have a tsypsin-like protease activity. Moreover, the release of FcR could be inhibited by serine protease inhibitors. Therefore, we propose that FcR release from activated B cells results from proteolytic cleavage. At least two sites sensitive for trypsin-like serine proteases (Arg 57-Phe 58, Arg 133-Ser 134) were identified while examining cleavage sites on the predicted primary sequence of human Fc-1RII. The cleavage at these sites might not interfere with the biological activity (Fc binding, IBF) of the released receptors because the molecule is held

together

by intrachain

disulfide

bridges

(G.

J. Gergely,

of FcyRII

low-affinity

to the

release

of the

cleavage

THE BIOLOGICAL BINDING SITES

SIGNIFICANCE

OF TWO

resting

of the second, findings have

B

CH2-specific, also shown

not equivalent

cells.

The

that

released

form

functionally:

binding site (57, the two binding

only

is able

to transfer

signals

The

expression

of two binding

in the world ent domains,

of

in the

58). Our sites are

the CH2-specific case

site

of Fc7RIII.

sites is not unknown

of receptors: on CD2 termed T111A and

molecules two T111B, appear

differto be

responsible for the interaction with LFA-3. T1IIA represents the active site for LFA-3 triggering function whereas T11i might define a site through functional signals are mediated (59). According to our observations, Fc-1RIII

on K cells in stable

form,

whereas

the Fc-yRII molecule on B cells in stable or labile forms. There showing that stable receptors

other

membrane

components

which

and/or

no

is expressed

depending

cycle, both dence

on the cell

can be expressed is no direct eviare anchored to

interact

with each

other. Recent findings by Ra et al. (60) raised the possibility of such an interaction, which may explain the expression of the second active binding site as well. They investigated whether subunits of FcRI, like /3- and ‘y-

chains, could be associated with FcR other than FceRI. Biosynthetic labeling and gene transfer experiments have revealed y-chains associated with mouse Fc’yRIIa. Because human Fc-yRIII in NK cells is the human homolog

of mouse

Fc-yRIIa,

with

the a-chain

of FceRI,

they each share the same eight consecutive residues in the transmembrane domains. Ra et al. (61) predicted that

human

FcyRIII

would

also

be found

to associate

with -y-chains. By using the polymerase chain reaction method, they could detect a message for the -y-chains in human

NK

cells.

Moreover,

Lanier

et

al.

(61)

have

shown that CD16 specifically associates with the CD3 homodimer on the membrane of human NK cells. On the basis of such findings, we suppose that the interacof Fc’yRIII

with

chains) may result in the specific

FccRII (CD23) from B cells (55), and it may play an important role in the regulation of antibody production in both cases.

on

Fc’yRII is monomeric and has only one, CH3 specific, binding site. However, the in vitro cross-linking of these receptors (either by means of treatment with transglutaminase or after the interaction of released FcR and actomyosin complexes) resulted in the appearance

S#{225}r- tion

may, Z. Rozsnyay, I. Szabo, A. Biro, and unpublished results). This type of proteolytic is analogous

expressed

associated

alter the expression

binding

site.

chains

(such

as ‘y- or

conformation of the of the second, CH2 The

interaction

FcR and domain-

of Fc7RIII

and

the associated chain (or chains) may be responsible both for binding of the IgG CH2 domain and transduction of signals. This would be in good agreement with our findings showing that only the binding of the CH2 domain of the target cells sensitizing IgG antibody to the corresponding binding site can induce killing signals.

In

the

early

days

of FcR

research,

Spiegelberg

Ct

al.

(56) predicted that in the case of K cell-mediated ADCC, the CH3 domain of the sensitizing antibody might be responsible for high-affinity binding, whereas the CH2 domain could play the role of triggering site. In our studies, various approaches were used to map the interacting sites on IgGFc, and all of these approaches have shown that two active binding sites are present on the stable form of FcyRIII and on FcyRII

The support

authors and

thank

Drs.

Michael

Sela

and

Israel

Pecht

for their

advice.

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