Cell surface receptors for the Fc portion of immunoglobulin confer on most cells of the immune system the ability to communicate with the humoral antibody response. These Fc receptors are known to be particularly important for the function of various effector cells, such as macrophages, since they are involved in mediating a variety of activities including endocytosis, antibodydependent cellular cytotoxicity, and triggering the release of potent inflammatory agents. Over the past few years, a considerable amount has been learned about the structure and functions of the Fc receptors expressed by murine and human cells, due to the availability of specific anti-receptor antibodies and the isolation of Fc receptor cDNA clones. In general, these receptors are transmembrane proteins whose extracellular domains contain two immunoglobulin-like regions and are thus members of the immunoglobulin gene family. Their domain structure consists of a glycosylated extracellular domain, a single membrane-spanning segment, and a relatively long cytoplasmic domain. The cytoplasmic tails exhibit a surprising degree of variation in length and amino acid sequence. This review summarizes some recent information concerning the structure and expression of the Fc receptors found on murine and human macrophages and lymphocytes. Particular attention is paid to the functional activities of these receptors, and the possible relationship between receptor function and receptor structure.

Macrophages, granulocytes, many lymphocytes, and certain epithelial cells express receptors for the Fc domains of immunoglobulin. While these Fc receptors (FcRs) can be associated with a range of cellular activities and exhibit binding specificities for a variety of immunoglobulin classes or subclasses, in general, one can view FcRs as forming a critical bridge between the humoral and cellular responses of the vertebrate immune system. By providing the means to bind the invariant Fc portion of antibody molecules, FcRs enable immune effector cells to interpret and respond to the highly variable and specific events involved in antibody-antigen recognition. Thus cells such as the macrophage, which are otherwise incapable of detecting antigens directly, are incorporated as important elements of the humoral response. In addition, B cell FcRs may even be involved in directly regulating the events leading to antibody secretion.

The fact that distinct FcRs have been described, which bind virtually all classes of immunoglobulin (Unkeless et al. 1981) itself suggests that considerable heterogenity must exist amongst this class of receptors. Only within the past 1-2 years, however, have we begun to appreciate the true extent of this heterogeneity. Macrophage-lymphocyte-epithelial cell FcRs are now known to belong to one of two large families, the first (and quantitatively more important) being a derivative of an ancestral immunoglobulin-like gene and the second being closely related to cellular lectins (lymphocyte IgE receptors). The structural heterogeneity also appears to extend to within individual classes of FcR specific for single immunoglobulin isotypes. Such heterogeneity must reflect significant functional and/or developmental differences, which were previously unexpected, for this family of invariant or ‘monotypic’ membrane receptors. In this brief review, we will summarize recent results, emphasizing those from our laboratory, that shed some light on the structure, functions, and cell biology of the IgG FcR found on murine and human mononuclear cells.

FcRs are widely distributed in nature on cells both in and out of the immune system, as well as on cells in and out of the animal kingdom. A survey of the receptors described to date is compiled in Table 1. A large number of receptors, distinguished biochemically by their different specificities for various forms of immunoglobulin (Ig), have been identified as being membrane-associated or secreted by a large number of mammalian cell types. These include receptors for IgA, IgM, IgD, IgE, and various subclasses of IgG found on cells of the immune system (mononuclear cells, granulocytes, B cells, some T cells, NK cells) and various transporting epithelia (mammary cells, hepatocytes, intestinal epithelia in newborn rodents). Certain FcR-negative cells, such as fibroblasts, can be induced to express FcR following infection with certain DNA viruses (Epstein-Barr virus, cytomegalovirus, herpes simplex virus (HSV) types 1 and 2, varicella-zoster virus). In the case of at least HSV, this receptor is encoded by a viral gene (glycoprotein E or gE) (ParaeZ al. 1980). Finally, various strains of Staphylococci and Streptococci express wellcharacterized FcR activities (protein A, protein G).

Table 1.

The family of Fc receptors

The family of Fc receptors
The family of Fc receptors

The IgG1/lgG2 Fc receptor

Mouse macrophages and lymphocytes express at least three biochemically distinct receptors for different IgG subclasses (Unkeless et al. 1981) (Table 1). At least on macrophages, the three receptor types can be expressed simultaneously. By far the best characterized is the receptor specific for immune complexes or aggregates of murine IgGl or IgG2b, designated FcRII. While the ligand-binding activity of this receptor is relatively resistant to inactivation by trypsin, it binds monomeric IgG poorly. Most importantly, this receptor is recognized specifically by the rat monoclonal antibody 2.4G2, a reagent that has allowed its identification and characterization as a 60K (K = 1037Mr) membrane glycoprotein (Unkeless, 1979; Mellman & Unkeless, 1980) as well as facilitating a variety of other structural and functional studies (see below). The IgGl/lgG2b FcR is widely distributed on cells of the immune system, being found on macrophages, lymphocytes and granulocytes. However, immunoprecipitation using 2.4G2 of the mature cell-surface form of the receptor from these diverse cell types has indicated the existence of considerable molecular weight heterogeneity on SDS-polyacrylamide gels (Mellman & Unkeless, 1980). On macrophages and many lymphocytes, the receptor is generally the most abundant, being expressed in amounts of up to 2-6×105 per cell (Mellman & Unkeless, 1980; Mellman et al. 1983; Mellman & Plutner, 1984). Functionally, macrophage FcRII is capable of mediating the endocytosis of soluble immune complexes and phagocytosis of large IgG-coated particles, and of triggering a variety of other important effector cell functions (see below).

Other murine IgG Fc receptors

Relatively little is known about the two remaining murine FcRs. The IgG2a receptor, also referred to as FcRI, is specific for monomeric IgG2a but can also bind aggregated immunoglobulin (Unkeless, 1977; Mellman & Unkeless, 1980). Its activity is sensitive to trypsin and has a much more restricted pattern of expression: FcRI has thus far only been detected on macrophages. Like the IgGl/lgG2b FcR, however, the IgG2a receptor is capable of mediating the phagocytosis of opsonized particles. A monoclonal antibody to murine FcRI has yet to be produced and the polypeptide(s) responsible for receptor activity has not been definitively identified. The structure of macrophage IgG3 receptor (FcRIII) also remains unidentified, and is characterized only as a trypsin-resistant molecule capable of mediating the phagocytosis of IgG3-sensitized particles (Diamond & Yelton, 1981).

Given its relative abundance, wide distribution, and the availability of specific antireceptor antibodies, most of what we know concerning the functional activities of FcR derives from studies of the murine IgGl/lgG2b receptor (mFcRII). As mentioned above, the receptor is associated with an impressive array of activities, all of which are critical to effector-cell function and all of which illustrate many important principles of membrane biology. These activities range from the clearance of antibody-antigen complexes via receptor-mediated endocytosis to the ligandtriggered transmission of signals across the plasma membrane, which result in alterations in secretion, exocytosis and cellular metabolism.

Fc receptor-mediated endocytosis

Unlike most other well studied receptors, the macrophage IgGl/lgG2b FcR is physiologically associated with two important forms of endocytosis: receptormediated pinocytosis of soluble antibody-antigen complexes and the phagocytosis of large IgG-coated particles. The internalization of soluble immune complexes proceeds via the standard pathway of receptor-mediated endocytosis described for a variety of cell surface receptors (Steinman et al. 1983; Helenius et al. 1983; Mellman et al. 1986, 1987). These events include binding to cell surface receptors, accumulation of the receptor—ligand complexes at clathrin-coated pits, internalization in coated vesicles and subsequent delivery of the ligand to acidic endosomes and finally to lysosomes for degradation (Mellman & Plutner, 1984; Ukkonen et al. 1986; Mellman et al. 1986).

Aside from the importance of this process to normal macrophage function, FcR-mediated endocytosis is of interest since the pattern of intracellular receptor transport after internalization appears to be regulated by the type of ligand bound. If the receptor is allowed to bind a monovalent ligand, namely Fab fragments derived from the anti-receptor antibody 2.4G2, the receptor-ligand complex is internalized, delivered to endosomes, and then rapidly returned (‘recycled’) back to the plasma membrane without requiring transit through lysosomes (Mellman et al. 1984).

On the other hand, if the ligand is multivalent, e.g. aggregated 2.4G2 Fab or polyvalent antibody—antigen complexes, the receptor is again internalized and delivered to endosomes, but its participation in the recycling portion of the pathway is rendered less efficient. The receptor-ligand complex remains intact (ligand binding to FcR is not disrupted by the slightly acidic pH of endosomes) and is transferred from endosomes to lysosomes where both ligand and receptor are degraded (Mellman & Plutner, 1984; Ukkonen et al. 1986). While the macrophage FcR in control cells turns over with a t1/2 of approximately 15-20 h, in cells exposed continuously to immune complexes the t1/2 is reduced to < 5 h. This results in a net decrease in the number of cell surface FcR and, presumably, in the ‘down-regulation’ of a macrophage’s responsiveness to subsequent exposure to ligand. Recovery of normal amounts of cell surface FcR requires new receptor synthesis. Given the role of FcR in triggering the release of inflammatory and cytotoxic agents by macrophages, this type of regulation may be important in attenuating macrophage function at sites of chronic inflammation. FcR-mediated phagocytosis also leads to the removal of receptors from the plasma membrane and intralysosomal receptor degradation (Mellman et al. 1983).

The importance of these events goes beyond the general implications for the regulation of macrophage function, i.e. the probable attenuation of FcR-triggered macrophage effector functions following immune complex endocytosis. As illustrated in Fig. 1, the finding that the pathway of intracellular transport of FcR, recycling or transport to lysosomes, can be regulated by the valency of the bound ligand suggests a possible mechanism governing membrane protein traffic in general. Indeed, many other receptors, such as the low density lipoprotein and transferrin receptors, which normally recycle rapidly during endocytosis can also be diverted to lysosomes if allowed to interact with artificial non-dissociating multivalent ligands (e.g. polyclonal anti-receptor antibody) (Mellman et al. 1987). It is thus conceivable that relatively simple alterations in the aggregation state or the degree of oligomerization of membrane receptors may influence their patterns of intracellular transport. However, it is not at all clear whether the signal generated by such ligand-induced changes in quaternary structure is manifested at the level of a receptor’s cytoplasmic, membrane-spanning, or extracellular domain.

Fig. 1.

Pathways of Fc receptor-mediated endocytosis in mouse macrophages. The lefthand panel illustrates the intracellular pathway taken by the receptor when bound to monovalent ligands (e.g. Fab fragment of 2.4G2). The receptor and ligand are internalized, delivered to endosomes and recycled intact back to the plasma membrane. The right-hand panel illustrates the pathway taken when the bound ligand is multivalent. Receptor and ligand are now targeted from endosomes to lysosomes and degraded. The diversion of FcR along the lysosomal pathway is probably not all or none, but reflects an alteration in the equilibrium distribution due to ligand valency (Mellman et al. 1984; Mellman & Plutner, 1984; Ukkonen et al. 1986).

Fig. 1.

Pathways of Fc receptor-mediated endocytosis in mouse macrophages. The lefthand panel illustrates the intracellular pathway taken by the receptor when bound to monovalent ligands (e.g. Fab fragment of 2.4G2). The receptor and ligand are internalized, delivered to endosomes and recycled intact back to the plasma membrane. The right-hand panel illustrates the pathway taken when the bound ligand is multivalent. Receptor and ligand are now targeted from endosomes to lysosomes and degraded. The diversion of FcR along the lysosomal pathway is probably not all or none, but reflects an alteration in the equilibrium distribution due to ligand valency (Mellman et al. 1984; Mellman & Plutner, 1984; Ukkonen et al. 1986).

Transmembrane signalling

In addition to endocytosis, binding of ligand to FcR generates a transmembrane signal, which triggers a variety of other events important for macrophage function. Typically, these include a localized reorganization of actin-containing microfilaments directly beneath the plasma membrane at the site of particle attachment (facilitating phagocytosis), the synthesis and release of bioactive lipids (i.e. prostaglandins and leukotrienes), the secretion of neutral proteases (e.g. plasminogen activator), and the release of hydrogen peroxide and other active oxygen intermediates that mediate antibody-dependent cellular cytotoxicity (ADCC) (Unkeless et al. 1981). These events occur rapidly and apparently do not require ligand internalization (Rouzer et al. 1980).

The mechanism of FcR-mediated transmembrane signalling remains unclear. As discussed further below, the receptor does not appear to be phosphorylated, either in the presence or absence of bound ligand, suggesting that kinase-like phosphorylation events are not involved. However, ligand binding to FcR can result in a depolarization response and a transient increase in cytosolic free Ca2+ (Young et al. 1984). Conceivably, a ligand-dependent alteration in transmembrane ion fluxes, as occurs in the case of receptors such as the acetylcholine receptor, could mediate the signal. That such changes in ion permeability may even be due to an intrinsic ligand-activated channel activity associated with the FcR itself has been suggested by experiments using isolated receptor protein reconstituted into artificial bilayers (Young et al. 1983). More recent data have shown, however, that at least some FcR-triggered functions, such as phagocytosis, can proceed without alterations in cytosolic free Ca2+ (DiVirgilio et al. 1988) and other functions (oxidative burst) are perhaps independent of changes in membrane potential (Pfefferkorn, 1984). In addition, the known structure of the receptor, which is a membrane protein with a single highly hydrophobic membrane-spanning segment (see below), places some constraints on the applicability of the ion channel concept.

Whatever the nature of the actual signal, it is clear that ligand binding does rapidly transmit information to cytosolic elements. One interesting manifestation of these events is an alteration in the pattern of protein myristylation which appears to correlate with macrophage secretory events (Aderem et al. 1986). The possible involvement of myristoylation in macrophage function is described elsewhere in this volume.

The function of the IgGl/lgG2b Fc receptor on lymphocytes

In contrast to macrophages, the function of the IgGl/lgG2b FcR on lymphocytes is poorly understood (Lydyard & Fanger, 1982; Fridman et al. 1984) although recent evidence suggests that it may be important in the regulation of T-and B-cell activation and proliferation, and in antibody secretion by B cells. Bijsterbosch & Klaus (1985) have shown that triggering of FcR-positive B cells to mature and proliferate could be stimulated by specific antigen or by the binding of surface IgM with the F(ab’)r fragment of an anti-IgM monoclonal antibody. However, when intact anti-IgM was used instead of the F(abc2, an additional signal was provided preventing the proliferative response necessary for terminal differentiation and antibody synthesis. The effect was believed to be mediated by the cross-linking of surface Ig with FcR (Phillips & Parker, 1984) and subsequent inhibition of the phosphatidyl inositol cascade (Bijsterbosch & Klaus, 1985).

Although lymphocyte membrane-bound FcRs are primarily associated with negative regulation (Lydyard & Fanger, 1982; Spiegelberg, 1981), these cells also appear to secrete one or more soluble FcRs or immunoglobulin-binding factors (IBFs) capable of positively or negatively influencing the immune response. Distinct IBFs have now been reported for all Ig isotypes and subclasses (see Table 1). The best studied are those synthesized and released from human or rodent FcR+ T lymphocytes (Fridman & Golstein, 1974; Fridman et al. 1984; Ishizaka, 1984). The synthesis and secretion of IgM and IgG antibodies are regulated, in part, by the secretion of soluble IBFs from FcR+ T cells (Fridman et al. 1984), while other IgE-binding T-cell factors selectively regulate the IgE response (Ishizaka, 1984; Spiegelberg, 1981). IgE-potentiating factors or IgE-suppressive factors are formed and released depending on the isotypic restriction of the membrane FcR, and on the exposure to additional T-cell factors affecting their ability to glycosylate (Ishizaka, 1984). In addition, a soluble FcR is secreted from stimulated B cells, which reacts with 2.4G2 and is thus antigenically similar to the cell-associated form of IgGl/lgG2b FcR; however, the function of this FcR is not known (Pure et al. 1984). B cell activation by lipopolysaccharide also results in the expression of a unique FcR epitope, recognized by monoclonal antibody 6B7c, which may correlate with the release of the soluble form of the receptor (Pure et al. 1987).

Given its involvement in a variety of activities such as endocytosis, transmembrane signalling, and secretion, all of which are critically important for macrophage function as well as being of general cell biological interest, understanding the structural features of the IgGl/lgG2b FcR has proved particularly important. We have approached this problem using both biochemical and molecular biological methods.

The IgGl/lgG2b FcR has been purified by immunoaffinity chromatography using 2.4G2-Sepharose, and used as immunogen to produce a variety of domainspecific anti-receptor monoclonal and polyclonal antibodies (Mellman et al. 1983; Green et al. 1985; Pure et al. 1987). These antibodies have permitted a preliminary characterization of receptor structure by immunoprecipitation of antigen from metabolically labelled cells. Using the J774 macrophage cell line, we found that the major receptor species was synthesized in the rough endoplasmic reticulum (RER) as a 53K precursor containing four asparagine-linked oligosaccharides, which were terminally glycosylated in the Golgi complex (Green et al. 1985). The TV-linked chains were tri-or tetra-antennary and contained internal lactosamine (GlcNAc-Gal) repeats; no O-linked sugar was detected (Howe et al. 1988). Microsome digestion experiments demonstrated that the receptor was a transmembrane protein, with a relatively large cytoplasmic tail, 10-15K in length, recognized by a particular anti-receptor monoclonal antibody designated C14 (Green et al. 1985). Thus far, the receptor does not appear to be phosphorylated either in the presence or absence of bound ligand (unpublished results).

Characterization of murine Fc receptor cDNA clones

Using amino acid sequence data derived from the purified J774 cell receptor and a monospecific anti-mFcRII antiserum, several cDNA clones encoding the murine IgGl/lgG2b FcR have been isolated and sequenced. One of these, designated pFcRI3, was found to contain the complete coding region as well as >90 % of the 5’ and 3’ untranslated regions (Lewis et al. 1986). As deduced from the nucleotide sequence, the structure of the FcR was in general agreement with the biochemical data (Fig. 2). The receptor was a transmembrane protein containing a single hydrophobic membrane-spanning segment. The extracellular domain contained four predicted sites for TV-linked glycosylation. Interestingly, the cytoplasmic domain of the receptor obtained from a P388D1 cDNA library, 47 amino acid residues, corresponding to approximately 6K in molecular weight, was somewhat shorter than predicted by the biochemical experiments using J774 cells (10-15K; Green et al. 1985). The cytoplasmic tail did not exhibit sequence homology with any other known protein; more specifically, there was no obvious kinase-like domain, which might be implicated in some of the receptor’s transmembrane signalling functions. This was consistent with our inability thus far to identify a phosphorylated form of mFcRII by immunoprecipitation.

Fig. 2.

Structure of the mouse macrophage-lymphocyte IgGl/lgG2b Fc receptor (mFcRII). The amino acid sequence was deduced from the cDNA clone pFcRI3, and the insertion in the membrane derived from biochemical determinations of receptor structure.

Fig. 2.

Structure of the mouse macrophage-lymphocyte IgGl/lgG2b Fc receptor (mFcRII). The amino acid sequence was deduced from the cDNA clone pFcRI3, and the insertion in the membrane derived from biochemical determinations of receptor structure.

Expression of pFcRI3 has been obtained in COS cells using a late SV4O replacement vector and found to yield a glycoprotein, which reacts with all available anti-receptor antibodies. Permanently expressing CHO cell lines have also been produced using a selectable vector containing a methotrexate-resistant dihydrofolate reductase cDNA (H. Miettinen et al., unpublished results). Most importantly, mFcRII is also functionally active even when expressed in fibroblast lines (see below).

The Fc receptor rs a member of the immunoglobulin gene family

The receptor’s extracellular domain was found to contain four regularly spaced cysteine residues which define two internal sequence repeats. Examination of the sequences directly surrounding these cysteines revealed significant homology to sequences associated with the V and/or C domains of immunoglobulin molecules, as well as other members of the immunoglobulin gene superfamily (Lewis et al. 1986; Barclay et al. 1987). Interestingly, however, the number of amino acids between the two domains was only 42-44, considerably shorter than that typically found in the constant and variable domains of immunoglobulin molecules (usually 70-80 residues). These truncated domains were quite similar to those found in at least one other immunoglobulin-like molecule, the neural cell adhesion molecule N-CAM (Hemperly et al. 1986). While the shortened domains presumably result from the deletion of ß strands from within the immunoglobulin fold, the functional significance of this arrangement is unknown.

Unique features of the Fc receptor signal sequence

The sequence of pFcRI3 indicated that mFcRII has an amino-terminal signal sequence which must be cleaved to generate the mature amino terminus determined by TV-terminal protein sequencing (Lewis et al. 1986). The signal sequence is atypical, however, since there are potential start sites for translation at positions -13, -29, and -39. Each of these initiator methionines begins a possibly functional signal and none is associated with any consensus nucleotide sequence thought to represent preferred start sites (Lewis et al. 1986). Cell-free translation of in vitro transcribed pFcRI3 mRNA (using pGEM vectors) has demonstrated that translation can, in principle, be initiated at any of the methionines (G. Healey, W. Hunziker et al., unpublished results). In reticulocyte lysates, initiation consistent with a start at Met-13 or (to a lesser extent) Met-29 occurs; in wheat germ lysates, initiation at Met-39 appears to be favoured. Irrespective of which initiator methionine is used, however, translated FcR is efficiently inserted into dog pancreas microsomes, glycosylated, and its signal cleaved to yield the same polypeptides. Thus, the FcR mRNA appears to encode three functional signal sequences, which nevertheless generate identical glycoproteins after translocation across the RER. While the biological significance of this observation is not known, it should not contribute to the observed size heterogeneity of mFcRII found in different cell types. In the case of the la invariant chain, which unlike the FcR is oriented with its amino terminus towards the cytosol, a similar existence of multiple start sites for translation does indeed result in the production of polypeptides of different lengths (O’Sullivan et al. 1987).

FcR immunoprecipitated with 2.4G2 or other anti-mFcRII reagents from a variety of cell types has demonstrated the existence of considerable size heterogeneity among the receptors expressed in cells of diverse origins. For example, labelled receptor from thioglycollate-elicited peritoneal macrophages behaves like a 47K protein on SDS-polyacrylamide gels, whereas receptor isolated from the S49.l T cell line is closer to 60K (Mellman & Unkeless, 1980). Much of this heterogeneity can be explained by cell type-specific differences in glycosylation; digestion of terminally glycosylated mFcRII from different sources with endoglycosidase F (which removes all N-linked oligosaccharides) almost always yields deglycosylated polypeptides of identical sizes (Lewis et al. 1986).

Three Fc receptor cDNAs

However, it is likely that some of the observed heterogeneity is due to the existence of other closely related FcR isoforms. Two additional murine FcR cDNAs have also been isolated using cDNA libraries constructed from J774 macrophages and S49.1 T cells (Ravetch et al. 1986). One of these, designated ß-1, is identical to the sequence encoded by pFcRI3 (hereafter referred to as ß-2) except for a 47 amino acid in-frame insertion in the cytoplasmic domain (Fig. 3). This predicts a cytoplasmic tail for ß-1 of some 94 amino acids, much closer in size to the 10-15K predicted from microsome digestion experiments using J774 cells (Green et al. 1985). Presumably, this insertion was the result of alternative splicing at the 3’ end of a single mFcRII gene.

Fig. 3.

The structure of three distinct murine Fc receptors deduced from cDNAs. The structures shown correspond to cDNAs encoding the ß-2 (identical to pFcRI3), the ß-1 and the α isoforms of the receptor.

Fig. 3.

The structure of three distinct murine Fc receptors deduced from cDNAs. The structures shown correspond to cDNAs encoding the ß-2 (identical to pFcRI3), the ß-1 and the α isoforms of the receptor.

The second cDNA, designated α, was isolated from the J774 library and appears to encode the product of a distinct gene (Ravetch et al. 1986). The extracellular domain of this sequence is 95 % homologous to the ß sequence, with isolated single amino acid changes (Fig. 3). The two immunoglobulin-like domains and four N-glycosylation sites remain intact. However, the putative membrane-spanning and cytoplasmic domains are totally non-homologous to either ß sequence, nor do they bear striking similarity to any other known protein.

Northern blot analysis of mRNA from various cell lines using cDNA probes that would selectively detect either α or ß sequences, has suggested that ß-specific mRNA is transcribed in virtually all mFcRII-positive cells (macrophages, B cells and T cells) while α -specific message can be detected in variable amounts only in macrophage cell lines (Ravetch et al. 1986; Lewis et al. 1986; T. Koch et al., unpublished results). Thus, while it is tempting to suggest that the acDNA encodes the monomeric IgG2a receptor (mFcRI), whose expression also appears to be limited to mouse macrophages (Table 1), this is far from clear. Expression of protein from transfected α cDNA has proved unsuccessful (Ravetch et al. 1986; H. Mietti-nen et al., unpublished results) and, in addition, no mFcRI-specific monoclonal antibodies are yet available. We have also been unable unequivocally to detect the presence of protein corresponding to α even in cells possessing α-specific mRNA (see below).

Differential expression of murine Fc receptors

In order to understand the functional significance of the heterogeneity predicted by the three FcR cDNAs, we have sought to determine the pattern of FcR expression at the protein level in a variety of receptor-positive primary cells and continuous cell lines (T. Koch, H. Plutner, G. Healey, W. Hunziker & I. Mellman, unpublished results). Using synthetic mRNAs translated in the presence of dog pancreas microsomes, we have obtained the in vitro expression of protein corresponding to each of the three receptor cDNAs. A xenogeneic rabbit antiserum to mFcRII, which recognizes extracellularly oriented determinants, was found to immunoprecipitate the ß-1, ß-2, and α translation products. Each protein differed, as expected, in molecular weight (ß-1 was 53K; ß-2 was 47K; α was 45K). In contrast, the rat monoclonal antibody (C14, see above; Green et al. 1985) directed towards the receptor’s cytoplasmic domain recognized ß-1 and ß-2 but not a, in accordance with the sequence differences between these receptor species (Fig. 3).

Having established that the three receptor isotypes could be distinguished on the basis of molecular weight and differential reactivity with anti-mFcRII antibodies, we next immunoprecipitated FcR from a variety of cell lines pulse-labelled with [35S]methionine for 10 min, conditions which would label only immature RER forms corresponding exactly to those obtained by cell-free translation in the presence of microsomes. In the lymphocyte lines (A20/2J B cells; S49.1 T cells), and in the J774 macrophage line, ß-1 was clearly the predominant receptor species synthesized although some lower molecular weight bands, including one corresponding in molecular weight to ß-2, were also observed as minor components. In P388D1 macrophages and in thioglycollate-elicited peritoneal macrophages, the predominant receptor species corresponded to ß-2. By digesting the labelled microsomes with proteinase K prior to immunoprecipitation (Green et al. 1985), we were also able to show that the two receptor types differed only in the lengths of their cytoplasmic tails. The protease-protected fragments (i.e. extracellular and membrane-spanning domains) immunoprecipitated from digested microsomes of cells expressing ß-1 or ß-2 were of identical molecular weights. Importantly, all labelled bands irrespective of molecular weight were immunoprecipitated with equal efficiency by both antibodies; none corresponded in mobility to the translation product derived from the α cDNA. Thus, although the macrophages examined expressed transcripts that apparently contained α -specific sequences, functional expression of an α protein could not be detected. It is important to note that by analysing the precursor RER forms of the various receptors, these experiments should have detected intracellular a, even if it failed to be transported to the cell surface or was rapidly degraded after synthesis.

Although it is too early to conclude that the a-specific transcripts are ‘sterile’, i.e. unable to program the translation of protein, it is clear that the predominant form of mFcRII expressed by mouse macrophages and lymphocytes corresponds to either ß-l or ß-2. Since macrophages and different macrophage cell lines were found to express either of the ß-type proteins, it is conceivable that the presumptive splicing events leading to the production of one or the other FcR isoform is developmentally regulated.

The degree of heterogeneity observed in the cytoplasmic domains of these receptors is both striking and unprecedented. It was also unexpected since the involvement of FcR in endocytic and transmembrane signalling events suggested a priori that there might have been considerable selection pressure to conserve cytoplasmically oriented sequences. Determining the functional consequences of these naturally occurring site-directed mutagenesis ‘experiments’ will be of considerable interest (see below).

Functional expression of mFcRII

As mentioned earlier, it has been possible to obtain both transient and permanent expression of mFcRII using ß-1 and ß-2 cDNAs transfected into COS or CHO cells (H. Miettinenet al., unpublished results). In the permanent CHO lines, cell surface expression is uniform and efficient (2-8× 105 per cell), approximating the quantities of receptor normally produced by macrophages. Importantly, the transfected receptor appears to be functionally active, at least with respect to endocytosis. When expressed in either CHO or COS cells, the ß-2 form of the receptor is capable of mediating the binding, internalization, and delivery to lysosomes of immune complexes in a fashion similar to that observed in macrophages. Using permanently expressing cell lines, we have even been able to document that the kinetics of endocytosis of the receptor and ligand also appear normal. By showing that receptor binding and endocytosis can occur in fibroblast cell lines, which normally do not express FcR, these observations demonstrate that the functional mFcRII activity does not require the involvement of any other macrophage-or lymphocyte-specific proteins.

In an attempt to identify which domains are important for the various functional activities of the receptor, we have already begun generating a series of mutant and hybrid receptors using site-directed mutagenesis. A permanent CHO cell line has been generated using one such mutant, from which the entire cytoplasmic tail (following the first cytoplasmic lysine) was deleted. Using both immunofluorescence and quantitative assays for immune complex uptake, it appears that this deletion substantially interferes with the ability of the mutant receptor to mediate internalization and/or delivery to lysosomes. Thus, the FcR’s cytoplasmic tail, although capable of considerable sequence heterogeneity, appears to be necessary for endocytosis. It is yet not clear, however, whether it is the initial internalization step or the subsequent transport from endosomes to lysosomes that is blocked in the mutant cell lines.

Given the unexpected heterogeneity observed among murine FcR, it has become of considerable interest to isolate and characterize cDNA clones encoding the homologous human FcR. Human monocytes, granulocytes, and lymphocytes are well known to express at least three distinct FcR types (Table 1) (Anderson & Looney, 1986). The first of these, hFCRI, is a 70K high-affinity receptor for monomeric IgG recognized by the monoclonal antibody 32 (Anderson, 1982; Cohen et al. 1983). It is found only on monocytes and macrophages and is thus thought to be directly homologous to mFcRI, i.e. the monomeric IgG2a receptor. HFCRII, like mFcRII, is more widely distributed and exhibits a low affinity for monomeric IgG. It is detected by two monoclonal antibodies (IV.3 and Ku67), which immunoprecipitate a narrow 40K glycoprotein (Looney et al. 1986; Vaughn et al. 1985). While there is no known human FcR homologous to the murine IgG3 receptor, a third hFcR (hFcR-N) has been identified on granulocytes and to a lesser extent on macrophages (Fleit et al. 1982). This receptor exhibits a very low affinity for IgG and is immunoprecipitated by several monoclonal antibodies as a heterogeneous glycoprotein band of 40-70K (Perussia et al. 1984; Fleit et al. 1982).

Using the ß-2 cDNA pFcRI3 as a probe, we have recently isolated and characterized a cDNA encoding a highly homologous FcR from the U937 hurnan monocyte cell line (Stuart et al. 1987). Expression of this cDNA (referred to as 16.2) by transfection in fibroblasts produces a 40K glycoprotein that reacts with the antihFCRII reagent IV.3. A monoclonal anti-peptide antibody specific to a synthetic peptide encoding a portion of the predicted amino acid sequence, recognizes IV.3-positive cells and not IV.3-negative cells.

The amino acid sequence deduced from this cDNA is strikingly similar to both the α and ß forms of murine FcR (Table 2). The extracellular domain of the human FcR is 70% homologous to both murine sequences. The two immunoglobulin-like domains are exactly conserved, although two of the four N-linked glycosylation sites found in the murine sequences have been lost by amino acid substitution. At the same time, however, the human receptor is somewhat chimeric in nature. Its predicted signal sequence, as well as the 5’ untranslated region of clone 16.2, are decidedly ‘α -like’, although the membrane spanning domain is 50% homologous to murine ß and thus completely distinct from α. Most interestingly, especially in view of the considerable sequence heterogeneity found amoung the murine FcR cDNAs, the cytoplasmic tail of the human FcR is completely unique, being unlike either α, ß or any other known protein sequence.

Table 2.

Sequence homology between mouse and human Fc receptors by domain

Sequence homology between mouse and human Fc receptors by domain
Sequence homology between mouse and human Fc receptors by domain

While the expression data and the sequence of the extracellular and membrane spanning domains indicate that the human receptor encoded by clone 16.2 is directly homologous to mFcRII, the differences in their cytoplasmic domains suggest that these two molecules may be functionally distinct. Presumably, hFCRII and mFcRII should mediate more or less identical activities in human and murine monocytes. Thus, the divergence in sequence may indicate that considerable sequence plasticity can be tolerated in the FcR cytoplasmic domain or that other hFcRII-like cDNAs have yet to be isolated. While further attempts to isolate such cDNAs from the U937 library have proved unsuccessful, we have recently identified two additional cDNAs from a human placental cDNA library (S. Stuart, N. Simister et al., unpublished results). Although originally screened to identify cDNAs corresponding to the syncytiotrophoblast FcR, which mediates the transcellular transport of maternal IgG, the deduced sequences obtained are strikingly similar to sequences characteristic of macrophage-lymphocyte FcR. Presumably, these cDNAs were derived from placental macrophages (e.g. Hofbauer cells) or lymphocytes as opposed to placental epithelial cells. One of these predicts a sequence, which is very similar to that encoded by 16.2, with only a few amino acid substitutions, except that it has a distinct 3’ untranslated region. The second, however, is directly homologous to the murine ß FcR sequences throughout, i.e. the cytoplasmic domains of the human and murine receptors are similar.

Further characterization of the expression of these molecules, as well as the analysis of the functional activities associated with them, will be needed to appreciate the significance of this extended but closely related family of membrane receptors. Such information will be of obvious importance to understand the functions of FcR on leukocytic cells. In addition, however, it will also provide some fundamental insights into the problems of receptor function in general, given the almost unprecedented variation in the cytoplasmic domains of this family of otherwise monotypic cell surface receptors.

This work was supported by grants from the National Institutes of Health (GM-29765; GM-33904 to IM). WH is a fellow of the Damon Runyon-Walter Winchell Cancer Fund. DV is the recipient of awards from the Leslie Warner and Argali Hull Foundations.

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