Journal of Leukocyte Biology Volume 60, October 1996 433

Protein tyrosine phosphorylation in leukocyte activation

through receptors for IgGCarla Santana, Gino Noris, Bertha Espinoza, and Enrique Ortega

Department of Immunology, Institu.to de investigaciones Biom#{233}dica,s, Universidad Naci.onal Au.t#{243}noma de Mexico,

Cd. Universitaria, Mexico

Abstract: Membrane receptors for the Fe portion

of iinmunoglobulin G (IgG) antibodies (Fc’yRs) are

expressed on almost every type of hematopoietic

cells, where they mediate a wide variety of effector

functions. A high degree of structural heterogeneity

exists among FcyRs. The biological significance of

such heterogeneity is unknown, since the structural

diversity does not appear to be reflected in the

binding specificity nor in the effector functions thateach distinct receptor is able to mediate. Recent

work has emphasized the essential role of protein

tyrosine phosphorylation in the initiation of trans-

membrane signaling by these receptors. In this article

we review the role of protein tyrosine phosphoryla-

tion in signal transduction by the different types of

Fc’yRa in order to assess to what extent the structural

heterogeneity of this receptor family is related to

different activation pathways utilized by each of its

members. J. Leukoc. Biol. 60: 433-440; 1996.

Key Words: FcyR . transmembrane signaling . hematopoietic

cells

Membrane receptors for the Fe portion of immunoglobulin

G (IgG) class antibodies (FcyRs) are expressed on almost

every type of hematopoietic cells. Cross-linking of thesereceptors by aggregated IgG (in the forms of antigen-anti-

body complexes, opsonized cells, or bacteria, etc.), triggers

a very wide array of responses such as proliferation and

differentiation, cell cytotoxicity, secretion of cytokines and

inflammatory mediators, endocytosis, and phagocytosis,

etc. Heterogeneity of these receptors was inferred early in

their study, on the basis of differences in binding affinity

of IgG to cells expressing IgG receptors, and in the differ-

ent sensitivity of high- and low-affinity receptors to trypsin.

Later, the development of monoclonal antibodies (mAbs)

specific for the receptors and, more recently, the cloning

and structural characterization of the genes encoding them,

revealed a higher degree of heterogeneity than previously

recognized. Currently known receptors for the Fe portion of

IgG are classified into three groups: Fc’yRI, Fc’yRII, and

FcyRIII, each of them comprising several individual recep-

tor forms. The present classification is based on several

criteria, including molecular size, expression on different

cell types, recognition by distinct mAbs, and relatedness of

the genes encoding them. A thorough review of the struc-

ture of the genes, transcripts, and proteins of this family of

receptors can be found in references 1-3.

Despite the great progress achieved in defining the mo-

lecular structure of the receptors and the genes encoding

them, the functional and biological significance of the ob-

served heterogeneity among FcyRs is largely unknown.

Most of the heterogeneity among members of the FcyR

family is found in the transmembrane and cytoplasmic re-

gions, suggesting that it could be more related to the

mechanisms of signal transduction than to interacting with

different IgG subclasses. Indeed, the relative affinity of

binding of human IgG subclasses to each of the FcyRs

types follows very similar patterns: Fc’yRI, IgG3 IgGi >

lgG4 >> IgG2; FcyRII, IgG3 IgGi >> IgG2, IgG4;

and FcyRIII, IgG3 = IgGi >> IgG2, IgG4 [3, 4].It is still

unclear whether, in distinct cell types, specific effector

functions are mediated by only a particular class or isoform

of FcyRs. However, with the exception of modulation of B

cell activation by Fc’yRII bi and b2 isoforms, there is

ample evidence indicating that many effector functions can

be mediated by more than one particular type of Fey recep-

tor. This has been established for phagocytosis of opson-

ized particles and concomitant superoxide generation by

FcyRII and FcyRIII on human neutmphils [5], phagocy-

tosis of opsonized erythrocytes by murine FcyRII, and

FcyRIII transfected into RBL-2H3 cells [6], phagocytosis

mediated by each of Fc’yRI, Fc’yRII, or FcyRIII transfected

into COS-1 cells [7], and antibody-dependent cell cyto-

toxicity (ADCC) against target cells by distinct leukocytes

[8], etc.

The marked differences in the transmembrane and cyto-

plasmic regions of receptors that have similar binding spe-

cificities, and that mediate, to some extent at least, the

same effector functions, might suggest that distinct recep-

tors employ distinct biochemical transduction pathways.

It has been known for some time that FcyR cross-linking

induces several intracellular signals common to a variety

Abbreviations: EgG, immunoglobulin C; mAbs, monoclonal antibodies;

ITAM, immunoreeeptor tyrosine-based activation motifs; NK, natural

killer; IFN-y, interferon-y; GPI, glycosyl-phosphatidylinositol; P13-K,

phosphatidylinositol-3-kinase.

Correspondence: Dr. Enrique Ortega, Department of Immunology,

Instituto de Investigaciones Biom#{233}dicas, UNAM, Ap. Postal 70228, Cd.

Universitaria, D.F., CP 04510, Mexico.

Received January 22, 1996; revised April 23, 1996; accepted April

24, 1996.

434 Journal of Leukocyte Biology Volume 60, October 1996

of membrane receptors, such as the hydrolysis of mem-

brane inositol-phospolipids, and a rise in the cytoplasmic

concentration of Ca2+ ions. These biochemical events are

similarly induced by cross-linking of other membrane re-

ceptors involved in immune recognition and cell activa-

tion, like the antigen receptors of T lymphocytes (TCR)

and B lymphocytes (BCR), and the high-affinity receptor

for IgE (FcCRI). It is now clear that the initial biochemical

events generated by the later receptors involve the activa-

tion of distinct protein tyrosine kinases and the phospho-

rylation of several proteins, including chains that are

integral part of the receptors. A common sequence motif

(D/E-X2 -Y-X-X-L’I-X7-Y-X-X-LjI) has been found in the

cytoplasmic tails of several chains of these receptors.

These motifs, first identified by Reth [9], and designated

as immunoreceptor tyrosine activation motifs (ITAM), have

been shown to play an important role in cell activation

mediated by these receptors. The currently accepted model

proposes that cross-linking of these receptors induces the

phosphorylation of tyrosine residues within the ITAMs.

The phosphorylated tyrosines then serve as a point of at-

tachment for protein tyrosine kinases of the Src and

syk/ZAP7O families, which, upon binding to phosphory-

lated ITAMs, become activated [10-12]. Once activated

these kinases catalyze the phosphorylation and activation

of different cellular substrates, including phospholipase C

(PLC)-yl, PLC-’y2, MAP kinase, etc. (see below).

Recently, evidence has been accumulating indicating

that protein tyrosine phosphorylation is likewise playing an

important role in signaling by members of the FcyR family.

In this review, we examine comparatively the role of pro-

tein tyrosine phosphorylation in signal transduction by re-

ceptors of the FcyR family in order to ascertain whether the

structural heterogeneity in the cytoplasmic tails of these

receptors is reflected in a similar heterogeneity of

transduction mechanisms.

Fc’yRIII (CD16)

Human FcyRIII exists in two isoforms (FcyRIIIA and

FcyRIIIB), derived from two distinct but highly homolo-

gous genes. Both receptors are glycoproteins with an ex-

tracellular region of about 180 amino acids, containing two

Ig-like domains. FcyRIIIA (224 amino acids long) consists

of an extracellular IgG-binding region, a single transmem-

brane segment, and a cytoplasmic tail of 25 amino acids.

FcyRIIIA is expressed primarily in macrophages and natu-

ral killer (NK) cells, and interferon-y (IFN-y) induces its

expression in monocytes. FcyRIIIA is expressed in asso-

ciation with homo- or hetero-dimers of � and y chains,

which were originally described as subunits of the TCR

and FcERI, respectively. Both � and ‘y contain in their

�cy+oplasmic domains ITAM motifs (three in � and one in

y), and in -both cases they have been shown to play an

important role in signal transduction by the TCR and

FcERI [reviewed in ref. 13].

Human FcyRIIIB (203 amino acids long), in contrast, is

bound to the outer leaflet of the cell membrane via a gly-

cosyl-phosphatidylinositol (GPI) anchor, due to an amino

acid substitution (Ser at position 203 in FcyRIIIB vs. Phe

in Fc’yRIIIA), which creates a signal for attachment of a

GPI anchor. The FcyRIIIB isoform is expressed exclu-

sively on neutrophils and it is not known to associate with

any other polypeptide chain.

In macrophages, FcyRIIIA mediates several effector

functions, including phagocytosis of opsonized particles,

endocytosis of immune complexes, and ADCC. In NK

cells, FcyRIIIA is involved primarily in ADCC.

Signal transduction mechanisms of Fc’yRIIIA have been

more thoroughly studied in NK cells, partly because it is

the only IgG receptor expressed on these cells. Cross-link-

ing of CD16 on human NK cells with mAbs specific for this

receptor induces a rise in cytoplasmic Ca2+ ions and hy-

drolysis of inositol phospholipids, as well as the rapidphosphorylation in tyrosine residues of several proteins

[14-19]. Pretreatment of NK cells with tyrosine kinase

inhibitors abolishes all these immediate responses as well

as FcyRIII-mediated ADCC [15, 19-22], indicating that

tyrosine phosphorylation plays an important role in signal-

ing by this receptor.

Several observations suggest that the signaling capabil-

ity of FcyRIIIA is mediated by the associated y or � chains,

and that phosphorylation of the tyrosine residues within the

ITAM of these chains plays an important role in signaling.

Thus, cross-linking of CD16 induces the tyrosine phospho-

rylation of � and y chains [14, 18, 20, 23, 24]. In murine

macrophages, stimulation through FcyRIII also induces

phosphorylation of the y chains [25]. Furthermore, it has

been shown that upon transfection of murine FcyRIII or

FcyRIIbl into murine B cells, FcyRIII expression and sig-

naling was dependent on the co-transfection of ‘y chains

[26]. In this cell system, FcyRllbl failed to induce activa-

tion signals. The cytoplasmic tail of y was shown to be

essential for signal transduction, because chimeric recep-

tors composed of the extracellular portion of either Fc’yRIII

or FcyRIIbl joined to the transmembrane and cytoplasmic

domains of the y chains were able to induce cell activation.

Essentially the same results were obtained with chimeras

expressing the extracellular domain of human Fc’yRIIIA

and transmembrane and cytoplasmic tail of murine y trans-

fected into P815 murine mastocytoma cells [16]. Cotrans-

fection of y chains was also found to be required for

expression and signaling by FcyRIIIA transfected into

COS-1 cells [27]. In the later study, mutation of the tyro-

sines within the cytoplasmic tail of y abolished the ability

of the transfected receptors to induce activation.

In NK cells, CD16 has been found associated with �

or y-’y dimers. While it seems clear from the above that

signaling by FcyRIIIA is mediated by the associated y or

chains, it is unclear whether there are differences in sig-

naling mediated by any of the associated dimers. Vivier et

al. [20] showed that, upon engagement of CD16 in NK

cells, only �, but not a 12-kDa protein (presumably the ‘ychain) is phosphorylated. In contrast, in cultured human

Santana et at. Tyrosine phosphorylation in FcyR signaling 435

monocytes, cross-linking of CD1G induced tyrosine phos-

phorylation of y chains and very low phosphorylation of �

chains [24]. The differential signaling capabilities of y or

were further examined by Vivier et al. [28] using Jurkat

T cells transfected with intact CD16, or with chimeras

composed of the extracellular region of CD16 joined to the

transmembrane and cytoplasmic tail of � or ‘y. Intact CD16

was expressed in association with � dimers, whereas

CD16/T chimeras were expressed as (CD16/y)2 homodi-

mers or as CD16/y-� heterodimers. No stable transfectants

were obtained expressing CD16/�. Both CD16 and CD16/ytransfectants were able to induce Ca2+ mobilization, tyro-

sine phosphorylation of several proteins, and IL-2 produc-

tion, but with quantitative differences. CD16 transfectants

were more efficient in inducing Ca2+ mobilization, whereas

CD16/y transfectants showed a higher phosphorylation of

specific proteins and higher IL-2 production, suggesting

that y and � may preferentially stimulate distinct functions.

Other experiments by Kanakaraj et al. [29] showed that in

Jurkat cells expressing transfected Fc’yRIIIA in association

with homodimers of � or y, both complexes induce activa-

tion of phosphatidylinositol-3 kinase, although the recep-

tors expressing � chains did so with greater (9-fold)

efficiency. In contrast, Park et al. [27] showed that, al-

though FcyRIIIA transfected into COS-1 cells with either

y or � chains was able to mediate phagocytosis, y was

sixfold more effective than �. It is possible that although

both chains are able to mediate signal transduction, the

efficiency of � or ‘y chains for inducing activation signals is

dependent on the specific cell type.

Phosphorylation of tyrosine residues within the ITAM

motifs of yand � chains may serve as a point of attachment

for tyrosine kinases of the Src and syk/ZAP-70 families,

inducing their activation. Members of both families of ki-

nases have been shown to be involved in signaling by

FcyRIIIA; cross-linking FcyRIIIA in NK cells or trans-

fected Jurkat cells induces tyrosine phosphorylation and

activation of pS6lck and the physical association of p56lck

with Fc’yRIIIA complexes. There is also evidence of phos-

phorylation and activation of p72syk induced by Fc’yRIIIA

cross-linking in NK cells, macrophages, and cultured

monocytes (Table 1 and references therein). Moreover,

co-transfection of p72syk enhances phagocytosis by trans-

fected Fc’yRIIIA and FcyRl in COS-1 cells [7].

Among the proteins whose phosphorylation is induced

by FcyRIIIA cross-linking are PLC-yl and PLC-’y2 [14,

30, 31], which are responsible for generation of the second

messengers inositol triphosphate and diacyl glycerol, phos-

phatidylinositol-3-kinase (P13-K) [29], the proto-oncogene

p95 Vav, and a protein associated with p2lRas-GAP [32].

The roles played by each of these proteins in the effector

functions could at this time only be hypothesized.

The Fc’yRIIIB, which does not have transmembrane or

cytoplasmic domains but is anchored to the membrane via

a GPI moiety, has been shown to transduce activatory sig-

nals, although the mechanisms by which it does so are

unknown. In human neutrophils, which express both

FcyRH and FcyRIIIB, cross-linking of either receptor in-

duces tyrosine phosphorylation of similar sets of proteins

[33]. However, when FcyRIIIB was transfected in Jurkat T

cells, it failed to induce tyrosine phosphorylation and hy-

drolysis of inositol lipids [16], as well as activation of

P13-K [29], whereas transfected FcyRIIIA induced these

responses. This suggests that, in monocytes, signal

transduction by FcyRIIIB may involve an as-yet-unidenti-

fied protein, which is not present in T cells. No data is

available yet as to whether protein tyrosine kinases are

physically or functknally assodated to Fc’yRIIIR If they

are, this association might be indirect, involving an adapter

protein.

FCyRI (CD64)

Three highly homologous genes (1A, lB. and 1C), exist for

human FcyRI. From these, four transcripts have been iden-

tified, two of which encode proteins with no transmem-

brane domain (probably soluble forms). The two other

transcripts, FcyRIa and Fc’yRIb2, both of which have been

found in all cells expressing CD64, encode transmembrane

proteins. The FcyRIa transcript encodes a protein with anextracellular portion of 292 amino acids comprising three

Ig-like domains, a single transmembrane region, and a

cytoplasmic tail of 61 amino acids. Among the FcyRs, it is

the only one that has three Ig-like domains, and the pres-

ence of a third domain has been related to its higher affin-

ity for monomeric IgG [34]. FcyRIb2 transcripts lack the

coding region for the third extracellular Ig-like domain

and, if expressed, would encode a membrane-bound low-

affinity FcyRI. The expression and functionality of this

isoform of FcyRI has not been analyzed. Because all the

available mAbs react only with the FcyRIa isofoi-m, most

that is known about signaling through FcyRl refers to this

particular isoform.

Fc’yRI is expressed on monocytes and macrophages, and

its expression is up-regulated by IFN-y. IFN-y also induces

FcyRI expression in eosinophils and neutrophils. The

FcyRI is capable of mediating various different effector

functions, such as ADCC, phagocytosis, metabolic burst,

secretion of cytokines, etc. FcyRI has been found associ-

ated with a dimer of y subunits, which are also associated

with FcERI and FcyRIIIA [35-37]. In contrast to the later

receptors, membrane expression of FcyRI is not dependent

on its association with y chains, although some studies

suggest that FcyRI-y association is essential for signal

transduction [38, and below].

The essential role of the associated y chains for some

functions mediated by FcyRI has been analyzed in trans-

fected COS cells: two independent studies have shown that

FcyRI alone (without y subunits) is able to mediate endo-

cytosis through a tyrosine kinase-independent pathway,

but phagocytosis requires the co-transfection of y chains

and is dependent on tyrosine kinase activity [39, 40]. In

both studies it was shown that the cytoplasmic domain of

Fc’yRI is not required for mediating phagocytosis. A similar

dependency on the cytoplasmic domain of y chains was

Isoform Cells Activation of PTKs Association of PTKs Phosphorylated substrotes Refs.

FcyRI THP-1 several’� 42, 43, 44

FcyRl U937 PLC-yl 31

Fc’yRI U937 P13-K 46

FcyRI THP-1 PLC-yl, ‘y2, Vav, GAP 44

FcyRI THP-l PLC-yl 42

FcyRI U937 hck, MAPK 58

FcyRl THP-1, monocytes hck, lyn hck, lyn 59

FcyRI HL-60, THP-1 p72syk 44, 60,

FcyRl U937 p72syk p72syk 61, 62

FcyRI U937 associated y 5. 62

FcyRIIIA NK cells several0 17, 15, 19

Fc’yRIIIA NK cells associated � 14, 18,

20, 23

FcyRII1A monocytes associated y 24

FcyRlllA NK cells p56lck p56lck 14, 22, 23

63

FcyRIIIA NK cells p72syk p72syk 32, 64, 65

FcyRIIIA NK cells PLC-Ti, PLC-y2 30, 31

FcyRIIIA NK cells P13-K 29

FcyRIILA macrophages p72syk p72syk Vav, GAP 32

Fc’yRIla THP-1 several0 43, 44, 66

67

FcyRlla neutrophils several0 33 68

FcyRlIa THP-1 lyn, p72syk lyn, p72syk 66, 69

FcyRIIa neutrophils Fgr Fgr 70

FcyRIIa lymphoma line p59fyn 71

FcyRIIa platelets several,0

FcyRIl

72, 73

FcyRlla platelets p72syk p72syk 74

Fc’yRlla THP-i PLC-yi, �2, Vay,

GAP

44

1)937 P13-K 46

mitogen-activated protein kinase.

TABLE 1. Summary of Tvrosine Protein Phosohorvlation and Kinase Activation Induced by Fc’vRs

436

F#{231}��UIa

P13-K, phosphatidytinositol-3-kinase; GAP, GTPase activating protein; MAPK,

�The identity of the phosphorylated proteins was not determined.

reported for Fc’yRI transiently transfected into COS-7 cells

in order to mediate phagocytosis [41]. It is reasonable to

assume that the role of ‘y chains in FcyRI signal transduc-

tion is similar to its proposed role in signaling by FcERI

and Fc’yRIIIA: i.e., becoming tyrosine phosphorylated

upon receptor cross-linking and serving as point of attach-

ment for the binding and concomitant activation of protein

kinases. It has indeed been shown that cross-linking of

Fc’yRI on monocytic cells induces phosphorylation of the

associated y chains, and the activation of the Src family

tyrosine kinases hck and lyn, and of p72syk. Cross-linking

of Fc’yRI has also been shown to induce tyrosine phospho-

rylation of several proteins, among them PLC-yl, PLC-y2,

P13-K, Vav, GAP, etc. (Table 1 and references therein).

Several studies have examined in parallel the signaling

pathways activated by Fc’yRI and FcyRII on the same cells,

activating the receptors separately by means of mAbs spe-

cific for each one. In most of these studies, it is noteworthy

that similar phosphorylation patterns and biochemical

events have been found. Thus, stimulation of the monocytic

cell line THP-l, with mAbs specific for FcyRI or Fc’yRII,

induced a Ca2� signal and the phosphorylation of a similar

set of proteins [42-44]. Similarly, cross-linking either re-

ceptor on U937 cells induces hydrolysis of inositol phos-

pholipids and phosphorylation of PLC-yl [45], as well as

phosphorylation and increased enzymatic activity of P1-3

kinase [46].

FcyRII (CD32)

Human FcyRII is a transmembrane glycoprotein of 40

kDa, and is the receptor for IgG more widely distributed in

hematopoietic cells, being found in all cells expressing IgG

receptors except for NK cells. Three distinct but highly

homologous genes termed FcyRIIA, IIB, and IIC exist,

from which six different transcripts have been identified:

al, a2, bl, b2, b3, and c. There is heterogeneity in the

cytoplasmic domains of FcyRII: the Fc’yRIIal and FcyRIIc

have nearly identical transmembrane and cytoplasmic

tails, whereas the cytoplasmic domains of FcyRIIbl, b2,

and b3 are identical except for an insert of 19 amino acids

(encoded by a single exon), which is present in the mem-

brane-proximal region of Ilbi and 11b3, but not in 11b2. It

should be noted that the membrane expression of isoforms

11b3 and lIc in vivo remain speculative. FcyRIIa2 does not

contain a transmembrane region and thus is probably a

soluble isoform. Thus, at least three different cytoplasmic

domains can be found for FeyRil: al/c, bl/b3, and b2.

Sa,uana et al. Tyrosine phosphorylation in Fc’yR signaling 437

Cassel et al. [47] have analyzed the expression of human

FcyRII isoforms by analyzing transcripts found in human

hematopoietic cells. Megakaryocytic cells predominantly

contain FcyRIIa transcripts. In contrast, B cells do not

express FcyRIIa transcripts, but do express Fc’yRIIbl,

FcyRIIb2, and FcyRIIc transcripts. Myelomonocytic cells

contain mRNAs from all three FcyRII genes, including the

al, bl, b2, and c transcripts.

FcyRII has been shown to mediate phagocytosis, endo-

cytosis of Ag-Ab complexes, and ADCC in macrophages,

and a respiratory burst in neutrophils. In B cells (which

express only the b isoforms), co-cross-linking of FcyRII

with the BCR leads to diminished BCR signaling.

Although it has been reported that FcyRII on cultured

human monocytes can associate with y chains [37], the

association of FcyRII molecules with y or any other protein

has not been reproducibly found. In contrast to the cyto-

plasmic domains of FcyRI or FcyRIIIA, all three isoforms

of FcyRII contain in their cytoplasmic tail, tyrosines that

have been found to become phosphorylated upon receptor

aggregation. Moreover, these tyrosines have been shown to

be essential for mediating the effector functions (see be-

low). The FcyRIIa isoform contains the sequence D-G-G-

Y-M-T-L-X12-Y-L-T-L, which conforms to the consensus

ITAM motif except for a somewhat longer amino acid

stretch between the first and second tyrosine residues. The

bl and b2 isoforms contain a single (T/S)-X-X-Y-X-X-L

motif. This motif has been found to be essential for the

inhibition of BCR signaling, and thus the designation of

ITIM (immunoreceptor tyrosine-based inhibition motif) has

been proposed for this motif.

Several studies have comparatively analyzed the signal-

ing activities of different FcyRII isoforms in transfected

cells: Liu et al. [48] studied the induction of protein tyro-

sine phosphorylation by the a, bl, and b2 isoforms of hu-

man FcyRII transfected into mouse mastocytoma P815

cells, and found that only the a isoform was active. The

ability of different FcyRII isoforms to mediate phagocytosis

was also studied in transfected COS cells: FcyRIIa medi-

ated efficient binding and phagocytosis of IgG-coated

sheep red blood cells, whereas transfectants expressing

FcyRIIbl, Fc’yRIIb2, or a FcyRIIa mutant lacking the cy-

toplasmic tail, efficiently bind but did not ingest IgG-

coated erythrocytes [49]. Activity of the FcyRIIa isoform

was dependent on the integrity of both YXXL motifs [50].

Fc’yRIIa, but not the b isoforms, became tyrosine phospho-

rylated upon receptor activation. Interestingly, low levels

of phagocytosis were obtained with FcyRIIb2 and bi mu-

tants into whose cytoplasmic tail the FcyRIIa-isoform se-

quence YMTL, was introduced. The distinct activities of

FcyRIIa and FcyRIIb have also been analyzed upon trans-

fection into an FcR-negative B cell line IIA1.6 [51]. OnlyFcyRIIa was able to mediate phagocytosis of opsonized

bacteria and to trigger tyrosine phosphorylation. For both

these activities, the ITAM motif was critical. In contrast,

FcgRIIb isoforms induced tyrosine phosphorylation with

much slower kinetics, but only these receptors were capa-

ble of negatively regulating BCR-mediated signal

transduction, and their activity was dependent on an YSLL

(ITIM) motif. It has been recently shown that the modula-

tory effect of FcyRIIb1 is mediated by the recruitment and

activation of the tyrosine phosphatase P’l’PC 1, which binds

through its SH2 domain to the phosphorylated ITIM [52].

Cross-linking FcyRII has been shown to induce tyrosine

phosphorylation of several proteins, including the receptor

itself, and the association and activation of P’fKs of the Src

and syk families (Table 1 and references therein).

CONCLUSIONS AND PERSPECTIVES

Recent work summarized above has established the essen-

tial role that protein tyrosine phosphoiylation plays in the

initiation of transmembrane signaling by membrane recep-

tors of the FcyR family. With the exception of Fc’yRIIIB,

which do not have transmembrane and cytoplasmic do-

mains, all other membrane-bound Fc’y receptors appear to

employ a similar mechanism for signal transduction, in-

volving as a very early step, the phosphorylation of certain

tyrosine residues in the cytoplasmic domain of either the

IgG-binding chain or proteins associated with it. The criti-

cal tyrosines are part of either of two sequence motifs,

which have been called ITAM or ITIM (immunoreceptor

tyrosine-based inhibition motif) (Fig. 1). In both cases,

phosphorylation of the tyrosines within these motifs is nec-

essary for eliciting the functions associated with the recep-

tors.

The ITIM motif, which is found on Fc’yRIIbl and

FcyRIIb2 isoforms, is essential for the negative regulation

of BCR signaling in B cells. Mutation of the tyrosine or the

leucine of the ITIM motif eliminates the modulatory activ-ity [51]. Phosphorylation of the tyrosine mediates the asso-

ciation and concomitant activation of the protein

phosphatase PTP1C [52], which might dephosphorylate

Iga and Ig� subunits of the BCR, or kinases associated

with the complex, thus inhibiting its signaling activity.

ITAM motifs are found on the associated ‘yor � subunits of

the Fc’yRI and Fc’yRIIIA, and in the cytoplasmic tail of

Fc’yRIIa, and they play an essential role in signaling through

these receptors [53]. ITAM motifs are also found on the Igaand Ig�3 chains of the BCR, the y, 8, e, �, and TI chains of theTCR, and the y and f3 chains of the FccRI. Cross-linking of

the BCR, TCR, and FcERI, induces the phosphorylation oftyrosines within the ITAM motif, and this promotes the

association and concomitant activation of protein tyrosine

kinases of the Src and syk/ZAP-70 families. A similar

mechanism seems to operate upon cross-linking of FcyRI,

FcyRIIIA, and FcyRIIa, as both p72syk, as well as distinct

Src-family kinases have indeed been found associated to

FcyRs, and to be activated by FcyR aggregation (Table 1 and

references therein). The presence of a common signaling

motif on Fc’yRI, FcyRIIIA, and Fc’yRIIa, can explain the

observation that, when expressed on the same cell, structur-

ally distinct Fc’yR isoforms are able to mediate the same

functions. It seems improbable, however, that a family of

receptors, which are very homologous in their ligand binding

�‘ �Y,IY � viL � � V.

L LL V.

V

V

V

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4. Ravetch, J. V., Anderson, C. L (1990) FcyR family: proteins, transcripts andgenes. In Pc Receptors and the Action of Antibodies (H. Metzger, ed.)

Washington, DC, Am. Soc. Microbiol., 211-235.

5. Durden, D. L., Rosen, H., Michel, B. R., Cooper,J. A. (1994) Protein tymsinephosphatase inhibitors block myeloid signal transduction through the FcyRlreceptor. Exp. Cell Res. 211,150-162.

6. Dacron, M., Malbec, 0., Latour, S., Bonnerot, C., Segal, D. M., Fndman, W.

H. (1993) Distinct intracytoplasmic sequences are required for endocytosisand phagocytosis via murine FcTRII in mast cells. fat. I,nmunot. 5,1393-1401.

7. Indik, Z. K., Park, J. G., Pan, X. Q., Schreiber, B. (1995) Induction of

phagocytosis by a protein tyrosine kinase. Blood 85, 1175-1180.8. Segal, D. M. (1990) Antibody-mediated killing by leukoeytes. talc Receptors

and the Action of Antibodies (H. Metzger, ed.) Washington, DC, Am. Soc.

Microbiol., 291-301.

438 Journal of Leukocyte Biology Volume 60, October 1996

I:y2 ha Ilbi IIb2 IIIA:y2 IIIA:�2

Fig. 1. Schematic representation of members of the FcyR family and

associated chains. Only those receptors containing cytoplasmic domains

are included. The length of the cytoplasmic tails of all chains is propor-

tional to the number of amino acid residues it contains.The position of

tyrosine residues that are part of ITAM motifs is indicated, as well as the

YSLL sequence of the ITIM motifs of FcTRIlb isoforms. The solid

rectangles represent the predicted transmembrane domains.

domains, but whose cytoplasmic domains are structurally

different, would have evolved to mediate always the same

functions, and the question remains as to what is the func-

tional significance of the high degree of structural diversity

found among Fcyreceptors.

An attractive possibility is that, although FcyRI, FcyRIIa,

and FcyRIII isoforms use phosphorylated ITAM motifs as

docking points for effector cytoplasmic molecules, the com-

position of the signaling complexes assembled by the dis-

tinct receptors might be different. This may result from

differences in the amino acids near the consensus tyrosines

and leucines/isoleucines of the ITAM motif, which can

dictate a preferential association with distinct SH2-contain-

ing proteins (Src or syk kinases [54], or possibly other

effector molecules), as have been shown for the different sets

of proteins that bind to phosphorylated ITAM motifs of the

Igcx and Ig� chains of the BCR [55], and to phosphorylated yand f� chains of the FcERI [56]. The assembling of different

signaling complexes by each receptor isoform can result in

the triggering of different biochemical pathways.

Another important point to be noted is that the specific-

ity of the interactions of a particular ITAM-containing se-

quence with different SH2-containing proteins, does not

seem to be absolute. It has been shown that in cells defi-

cient of a given Src kinase, other members of the family

can substitute for it [57]. It is also evident from the data in

Table 1 that the y subunit can associate with both hck and

lyn in human monocytes, and the ITAM of Fc’yRIIa can

associate with at least three distinct Src family members:

fyn, lyn, and Fgr. This promiscuity in interactions of phos-

phorylated ITAMs with Src family kinases can endow thesystem with a certain degree of plasticity, by allowing the

coupling of the same receptor isoform to distinct activation

pathways on different cells, depending on the expression

levels of the various different proteins capable of interact-

ing with the phosphorylated ITAMs. Indeed, it has been

recently shown that p72syk, but not the related kinase

ZAP-70, stimulated FcyRIIIA mediated phagocytosis in

transfected COS-l cells, but ZAP-70 increased phagocy-tosis if coexpressed with the Src family kinase Fyn [54].

Despite the great progress achieved in recent years in the

molecular characterization of FcyRs, and in establishing the

role of tyrosine phosphorylation in signal transduction by

#{149}.,, these receptors, several questions remain unanswered, espe-

“ cially those related to the functional significance of the high

degree of heterogeneity found among this family of receptors.

The next step forward for understanding the initiation of

effector functions mediated by this family of receptors,

would depend on a full characterization of the composition

and pathways activated by the signaling complexes assem-

bled after receptor cross-linking, involving the IgG binding

chains, associated subunits, associated kinases and phos-

phatases, and substrates of them. Of special importance is

the realization that different signaling complexes might beassembled by the same receptor isoform under different

conditions, depending not only on the cell type involved, but

also probably on the activation or metabolic state of the cell.

It is not unreasonable to think that stimuli different from IgG

complexes, such as cytokines or hormones, might regulate

effector functions mediated by FcyRs by affecting the ex-

pression not only of receptor isoforms, but also of different

kinases and the substrates upon which they act.

ACKNOWLEDGMENTS

This work was supported by grants from the Direcci#{243}n

General de Asuntos del Personal Acad#{233}mico, UNAM

(1N205792 and IN204293), and from CONACyT (1072-N9201).

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