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RAPID COMMUNICATION
Substitutions and Deletions in the Cytoplasmic Domain of the Phagocytic
Receptor FcyRIIA: Effect on Receptor Tyrosine Phosphorylation
and Phagocytosis
By Marilyn A. Mitchell, Min-Mei Huang, Paul Chien, Zena K. Indik, Xiao Qing Pan, and Alan D. Schreiber
FcyRllA in the absence of other Fc receptors or receptor
subunits induces the ingestion of IgG-coatedcells. The cytoplasmic domain of FcyRllA contains two Y-x-x-L sequences
similar t o those in other Ig gene family receptors plus an
additional tyrosine residue not in a Y-x-x-L motif. Upon
cross-linking, FcyRllAis phosphorylated on tyrosine and the
cytoplasmic tyrosines, Y275 (Yl), Y282 (Y21, and Y298 (Y3).
may be important for its phagocytic activity. Because COS1 cells can serve as a model for examining molecular structurea involved in phagocytosis, substitutions and deletions
were introduced into the cytoplasmic domain of FcyRllA and
examined in COS-l cell transfectants for their effects on
phagocytosis and tyrosine phosphorylation. Disruption of a
single cytoplasmic Y-x-x-L motif by substitution of tyrosine
Y2 or Y3 by phenylalanine or by removing the threonine
and leucine residueswithin the motif inhibited
phagocytosis
50% t o 65%. Tyrosine phosphorylation of FcyRllA also was
inhibited, although t o a greater extent by the substitution
of Y3 than of Y2. Replacement of the N-terminal first cyto-
plasmic domain tyrosine, Y1, which is not within a typical
Y-x-x-L, by itself did not inhibit phagocytosis, but replacement of Y1 in mutants lacking Y2 or Y3 virtually eliminated
phagocytic activity and receptor tyrosine phosphorylation.
Thus, at least two cytoplasmic tyrosines, including at least
one typical single Y-x-x-L motif, are required for phagocytosis by FcyRIIA. The data suggest that there is a close but
not a simple relationship between phosphorylation of the
FcyRllA cytoplasmictyrosines and FcyRIIA-mediated phagocytosis. Y3 appears t o be particularly important because its
removal by truncation or replacement with phenylalanine
inhibits both tyrosine phosphorylation and phagocytosisin
parallel. Alterations in the 12 residue proline-containing sequence between the two Y-x-x-L motifs also reduced phagocytic activity and tyrosine phosphorylation. Thus, the specific structure of the FcyRllA cytoplasmic domain accounts
for its ability t o stimulate phagocytosis in the absence of
other subunits.
0 7994 by The American Society of Hematology.
P
cytoplasmic domains of the y , 6, and subunits of the Tcell receptor, the y and p chains of the FceRI mast cell
receptor, and the IgMa (MB-1) and Igp (B29) subunits of
the B-cell receptor." However, these receptors are not involved in phagocytosis and, therefore, it is not certain that
similar structures in FcyRIJA would be involved in signalling for phagocytosis. In contrast to other Ig family receptor
molecules, 12 amino acid residues rather than 6 or 7 amino
acids separate the two Y-x-x-L sequences in FcyRIIA. Furthermore, FcyRIIA has an additional upstream tyrosine at
Y275 that is not in a typical Y-x-x-L context. Because of
these differences in the structure of FcyRIIA and its different
function from Ig gene family receptors in nonphagocytic
cells, we studied whether modification of the cytoplasmic
domain in the region of the conserved motif affects the ability of FcyRIIA to induce receptor phosphorylation and to
mediate phagocytosis.
HAGOCYTOSIS of IgG-coated cells, an essential component of the host defense system, is mediated by receptors for the constant region of IgG expressed on the surface of hematopoietic cells. There are three classes of human
Fcy receptors, distinguishable from one another on the basis
of size, structure, ligand binding, and cellular distribution.
The extracellular regions of the Fcy receptors are highly
conserved and functional differences are likely due to the
divergent sequences within their cytoplasmic domains. Although recent studies have enriched our understanding of
the structure of Fcy receptors,"' the mechanisms by which
these molecules transmit extracellular signals to cells remains largely unknown.
Because hematopoietic cells express multiple classes of
Fcy receptors,'" the individual contribution of each Fcy
receptor class is difficult to assess. Transfection of receptor
cDNA into COS-l cells, a cell line that lacks endogenous
Fcy receptors, but has the capacity for phagocytosis, has
proved valuable for analyzing individual Fcy receptor
phagocytic function."" Human FcyRIIA expressed in
transfected COS-l cells efficiently induces the phagocytosis
of IgG-sensitized cells6 and cross-linking of FcyRIIA in
monocytes, hematopoietic cell lines, platelets, and transfected COS-1 cells leads to induction of tyrosine phosphorylation of the receptor itself." The other phagocytic Fcy
receptors, FcyRI and FcyRIIIA, in contrast to FcyRIIA,
require the cytoplasmic domain of an associated y-subunit
to induce phagocytosis.'"'
A conserved cytoplasmic motif containing tyrosines (D/
E-7x-DIE-x-x-Y-x-x-J. - 6 / 7 x - w ) has been implicated
in signal transduction in several Ig family receptor molecules.12"4 These individual Y-x-x-L tyrosine activation
motifs (TAMS) are generally found in pairs in a larger conserved sequence. The sequence E-8x-D-x-x-Y287.-x-x-L12x-Y798-x-x-L in thecytosplasmictail
of human
Fc~RIIA".'~is closely related to this motif found in the
Blood, Vol 84.
No 6 (September 15). 1994: pp 1753-1759
MATERIALS AND METHODS
Culture and transfection. COS-l cells were maintained in Dulbecco's modified Eagle's medium (DMEM) containing glucose (4.5
From the Departments of Medicine and Microbiology, University
of Pennsylvania School of Medicine, Philadelphia, PA.
Submitted May 23, 1994; accepted July 7, 1994.
This manuscript is dedicated to the memory of Drs Ira Goldstein
and John Parker.
Supported by National institutes of Health Grant No. AHHL22 193.
Address reprint requests to Alan D. Schreiber, MD, University of
Pennsylvania Cancer Center, 7 Silverstein Bldg, 3400 Spruce St,
Philadelphia, PA 19104.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
0 I994 by The American Society of Hematology.
0006-4971/94/8406-0040$3.00/0
1753
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1754
MITCHELLET
AL
mg/mL), glutamine (2 mmol/L), streptomycin (100 U/mL), penicillin
kinase. COS-l cells transfected with wild-type or mutant FcyRIIA
(l00 ,ug/mL),and10% heat-inactivated fetal calf serum. Cells at
were lysed with RIPA buffer. After clarification, the supernatant
70%to80% confluence were transfected with full-length human
was aliquoted for coimmunoprecipitation and analyzed as previously
Fcy receptor cDNA in the SV40-based vector pKC4 obtained from
described'' using anti-Src MoAb 327" and anti-FcyRII MoAb IV.3.
Dr Mark Hogarth (University of Melbourne, Melbourne, Australia).
The immune complexes were adsorbed to Pansorbin (Calbiochem,
Full-length human FcyRIIA cDNAI5 was provided by Dr Hogarth.
La Jolla, CA), incubated with [y3*P]ATP to allow autophosphorylaTransient transfection of COS-l cells was performed in complete
tion, and examined by 7.5% SDS-PAGE. The gels were washed
media containing 10% Nu-Serum instead of fetal calf serum (Collabwith 1.0 N KOH at 55°C for 2 hours to remove serindthreonine
orative Research, Bedford, MA), diethyl aminoethyl (DEAE)-dexphosphorylation and developed by autoradiography.
tran (1 mg/mL), chloroquine chloride (100 pmol/L) and2.5 pg
RESULTS AND DISCUSSION
plasmid DNA per milliliter of transfection media. After 4 hours at
37"C, thetransfection media was replaced with 10% dimethyl sulfoxFigure 1 illustrates the sequence of the cytoplasmic doide (DMSO) in phosphate-buffered saline for 2 minutes atroom
main and conserved motif of human FcyRIIA. FcyRIIAhas
temperature. The cells were then washed, overlaid with fresh media
three cytoplasmic tyrosines Y275, Y282, and Y298, which
for further incubation, and analyzed after 48 hours at 37°C.
are designated Y1, Y2, and Y3, respectively. Figure 1 also
Flow cytometry. Cell samples incubated with anti-FcyRII monoshows
the modifications introduced in FcyRIIA to assess
clonal antibody (MoAb) IV.3I6 for 30 minutes at 4°C were washed,
the function of the three cytoplasmic tyrosines and other
labeled with fluorescein isothiocyanate (FITC)-conjugated goat antiamino acid residues within the conserved motif. Overlap
mouse F(ab'), IgG (TAGO, Inc, Burlingame, CA) for 30 minutes at
4"C, and then washed and fixed with 4% paraformaldehyde. Isotype
extension PCR" was used to substitute phenylalanine in
controls were used for all reactions, and fluorescence was measured
place of tyrosine for eachof the tyrosine codons individually
on a FACSTAR cytometer (Becton Dickinson, Mountain View, CA).
and in all combinations of two. In addition, termination coIn each experiment, 15% to 30% of wild-type and mutant transfecdons were introduced at appropriate positions to produce
tants expressed FcyRIIA.
receptor molecules truncated at positions 268, 280,285,290,
Binding and phagocytosis of IgG-sensitized red blood cells (EA).
and 303. These mutations produced molecules from which
Sheep RBCs were sensitized with rabbit antisheep RBC antibody
no
(A303), only one (A285 and A290), two (A280), or all
by incubation with an equal volume of the highest subagglutinating
three (A268) tyrosines were deleted, as shown in Fig 1 . In
concentration of rabbit antisheep RBC antibody (Cappel Laboraaddition, two single TL (theoninefleucine) deletions (A284tories, Malvern, PA) at 37°C for 1 hour as previously de~cribed.6~~.~~"
285 and A300-301) were made adjacent to Y2 and Y3 and
COS-l cells were incubated with washed EA at 37°C for 30 minutes.
Unbound EA were removed by washing and the plates were stained
five amino acids were deleted from the 12 residues between
with Wright-Giemsa. The percentage of cells binding RBCs was
the two Y-x-x-L sequences.
determined by counting in a coded fashion those cells binding 5 or
To examine functional differences among the FcyRIIA
more sensitized RBCs. To assess phagocytosis, parallel groups of
mutants, we translated COS-l cells with each mutant cDNA.
cells were briefly exposed to a hypotonic solution to remove adherent
The effect of truncation and replacement mutations on
EA.6.8-9.'6
The cells were then stained with Wright-Giemsa and the
phagocytosis by FcyRIIA is shown in Fig 2. Replacement
number of COS-l cells with one or more internalized EA was deterof the first tyrosine (which is not within a typical Y-x-x-L
mined in a blinded fashion. Phagocytosis was expressed as phagomotif) by phenylalanine (Y,F) did not reduce phagocytosis,
cytic index, ie, the number of internalized EAper 100 FcyRIIA
whereas substitution of the second or third tyrosines (both
expressing COS-l cells as determined by flow cytometry.
within Y-x-x-L motifs) with phenylalanine inhibited phagoConstruction of FcyRIIA mutants. Two-step overlap extension
cytosis by an average of 65% (Fig 2). Replacement of any
polymerase chain reaction (PCR)" was used to construct FcyRIIA
cDNA deletion and substitution mutants. Truncations of the receptor
combination of two tyrosines by phenylalanine resulted in
were produced by inserting a termination codon at appropriate sites.
86% to 96% loss of phagocytic activity, although slight acTyrosine (Y) codons were replaced by phenylalanine (F) or lysine
tivity was retained by mutant Y lF/Y3F. There was no differ(K) codons. The mutant FcyRIIA receptor DNA fragments were
ence among the FcyRIIA mutants in their ability to bind
ligated into the vector pKC4" at the Sal I, EcoRI restriction sites
IgG-sensitized cells (data not shown). These data suggest
and cloned into Escherichia coli. Sequence analysis was performed
that Y2 and Y3, which are within Y-x-x-L motifs, andor
to verify the sequence.
the
structure of these domains are particularly important for
Tyrosine phosphorylation of wild-type ormutant FcyRIIA in COSthe
phagocytic activity of FcyRIIA. Significant phagocytic
I cell transfectants. The COS-l cell transfectants were activated
activity is retained with only one Y-x-x-L sequence in the
at 37°C with IgG sensitized RBCs (EA) and lysed directly on plates
presence of the intact upstream Y1 tyrosine, and the inat 4°C with RIPA buffer (1% Triton X-100, 1% sodium deoxychocreased inhibition of phagocytosis observed in the double
late, 0.1% sodium dodecyl sulfate [SDS], 158 mmol/L NaC1, l0
mmoVL Tris-HC1, pH 7.2, 5 mmoVL NaEGTA, 1 mmoVL phenylmutants containing Y1F compared with the single mutants
methylsulfonyl fluoride, 1 mmoVL sodium orthovanadate and aproY2F or Y3F suggests that, although Y 1 is
not within a typical
tonin). The lysates were immunoprecipitated with polyclonal phosY-x-x-L motif, it also contributes to the efficiency of phagophotyrosine antisera UP28." The immunoprecipitates were separated
cytic function.
by 7.5% SDS-polyacrylamide gel electrophoresis (SDS-PAGE),
In addition to phenylalanine replacement of tyrosine, we
transferred to a nitrocellulose filter, and probed with antiphosphotyalso substituted lysines at positions Y 1 and Y2. The substiturosine MoAb 4G10 and horseradish peroxidase-conjugated antition of lysine at Y1 or Y2 resulted ina greater loss of
mouse IgG (Bio-Rad, Richmond, CA).'' The phosphorylated bands
phagocytic activity than the corresponding phenylalanine
were detected using enhanced chemiluminescence reagents (ECL;
substitution ( P < .01). The small amount of phagocytosis
Amersham, Int, Amersham, UK) and visualized with Kodak XARassociated
with Y 1FN3F was reduced further with Y 1 lysine
5 film (Eastman Kodak, Rochester, NY).
in place of Y 1F(not shown). These results suggest that the
In vitro phosphorylation of wild-type or mutant FcyRllA by Src
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TYROSINE PHOSPHORYLATION AND PHAGOCYTOSIS
1755
EX
representsFig 1. Schematic
tion- of FcyRllA with^ external
(EX), transmembrane (TM), and
cytoplaamk (CY)domains. Solid
lines repreaent amino acid (AA)
residues that correspond to the
wild-type shown on the top
line.
Point mutations are designated
as wild-type amino acid, position number,and
substiiuted
residue (for example, Y,F indicates that the tyrosine at position 275 is replaced by phenylalanine). Dotted linearepresent
deleted sequences starting from
the indicated residuenumber.
Two-step overlap PCR" was
used to obtain all FcyRllA mutations.
CY
TM
Y1K
Y2F
- ~Y2K
Y3F
YlFlY2F
YlF/Y3F
Y2F/Y3F
A268
K
F
K
F
-F
F
F
........................................
..............................
..........................
...................
........
A280
A285
A290
A303
A284-285
.....
MOO-301
A287-291
W)
215
F
F
F
270
W)
282
c131
298
311
FcyRIIA......ETNNDXETADGG~PRAFTDDDKNWPNDHVNSNN
tyrosines at 275 (Yl) and 282 (Y2) play a structural role in Deletion
of the carboxy terminal eight amino acids that are
addition to their possible roles as substrates for phosphorylabeyond the most distal Y-x-x-L (A303) reduced phagocytotion (see below).
minimally.
sis only
These results that
suggest
the amino
The results with the truncation mutations were consistent
terminal structure of the cytoplasmic domainonlyslightly
with those obtained with the replacement mutations (Fig 2).
influences the signal for phagocytosis. Deletion of Y2and
400
T
T
-
T
FoyRllA Mutants
a
a
a
Fig 2. Phagocytosis by wild-type and mutant FcyRllA in transfected COS-l cells. The phagocytic index (EAper 100 kyRIlA-expressing
transfected COS-l cells) is shown for each mutant receptor. The reauks f SEM from 4 to 8 experiments are given. Nomenclatureis as shown
in Fig 1. Wld-type FcyRllA ingeatedan average of 3.6 EA (phagocytic index, 356 & 25). Electron m i c r m p y has determined that the EA are
contained within intrecellular vacuoles.'.'' Unsensitiied E do not bind to FcyRllA transfectanta and COS-l cells transfected with a FcyRllA
mutant lacking the cytoplasmic tail did notphagocytose EA, although they avidly bound EA on the cell surface."'6
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1756
Y3 (A280) or all three cytoplasmic tyrosines (A268) eliminated phagocytic activity. Furthermore, truncating FcyRIIA
at A285 or A290, which removes Y3 and preserves Y2, also
substantially inhibited phagocytosis, in agreement with a
previous report." Thus, all truncations removing Y3 substantially inhibited phagocytosis.
To establish the importance of the two nontyrosine amino
acids in the two Y-x-x-L cytoplasmic motifs containing tyrosine, Y2 (YMTL) and Y3 (YLTL), in transmission of a
phagocytic signal by FcyRIIA, we examined the effect of
deleting T284L285 (A284-285) at
Y2
and T300L301
(A300-301) at Y3 while preserving the tyrosine residues Y2
and Y3. These two deletions decreased the phagocytic signal
transmitted by FcyRIIA (Fig 2), even though the tyrosine
residues in each Y-x-x-L were present. These results, similar
to that observed with Y2F and Y3F, suggest that the tyrosine
must be held in the appropriate conformation in the Y-x-xL sequences for full phagocytic function.
FcyRIIA also is uniqueamongthe
Ig gene family of
receptor proteins in that there are 12 rather than 7 residues
between the two Y-x-x-L sequences, and in that this intervening stretch of amino acids contains two pro line^."^'^
Although the three-dimensional structure of FcyRIIA is unknown, this proline-containing intervening sequence would
likely form a nonhelical structure within the cytoplasmic tail.
To explore whether this structure must be intact for FcyRIIA
phagocytic function, we constructed a mutant FcyRIIA
(A287-291) in which 5 amino acids including the two prolines were deleted from this sequence (Fig 1). This mutant
contains a 7 amino acid proline-free region between the
two Y-x-x-L sequences and is thus similar to the consensus
sequence of other Ig gene family receptor^.'^"^ The phagocytic function of this mutant was decreased to 30% of wildtype (Fig 2), indicating that the 12 amino acid proline
containing sequence in wild-type FcyRIIA contributes to
phagocytic efficiency.
Because phosphorylation of tyrosine residues in one or
both Y-x-x-L sequences may be involved in the activation
of intracellular signaling events, wild-type and mutant
transfectants were also examined for induction of tyrosine
phosphorylation of FcyRIIA (Fig 3). COS-l cell transfectants were stimulated with IgG-sensitized RBCs (EA) and
immunoprecipitated with antibodies to phosphotyrosine. Immunoblots were probed with antibodies to phosphotyrosine.
Activation of FcyRIIA elicited a strong phosphorylation response in COS-l cells transfected with wild-type FcyRIIA
(Fig 3, lane 4). The effects on FcyRIIA tyrosine phosphorylation were different for each Y to F replacement mutant
(Table 1) and are thus somewhat discordant from the effects
on phagocytosis (see below). All single tyrosine mutants
showed reduced induction of tyrosine phosphorylation; however, severe reduction in tyrosine phosphorylation was observed for Y3F (Fig 3, lanes 5 through 7). The time course
of tyrosine phosphorylation of Y1F and Y3F was also delayed (data not shown).Little or no tyrosine phosphorylation
was observed for any of the double tyrosine replacements
(Fig 3, lanes 8 through 10). Truncation mutants in which Y3
and one or two of the more proximal cytoplasmic tyrosines
were eliminated showed no tyrosine phosphorylation.
The data suggest that there is a close, but not a simple
MITCHELL ET AL
relationship between phosphorylation of the FcyRIIA cytoplasmic tyrosines and FcyRIIA-mediated phagocytosis. Substitution of F for Y3 severely impairs phosphorylation of
FcyRIIA and reduces phagocytosis by FcyRIIA by about
two-thirds, but substitution of F for Y2, which permits substantial phosphorylation of the receptor, causes a reduction
in phagocytosis comparable to Y3F (Fig 2 and Table 1). In
addition, the mutant YlF, in which phosphorylation was
somewhat reduced, mediated phagocytosis to a similar extent
as the wild-type receptor (Figs 2 and 3 and Table 1). Furthermore, introduction of the additional mutation Y IF into mutant Y2F or Y3F completely eliminated both FcyRIIA tyrosine phosphorylation and phagocytosis. Although the
tyrosines in the Y-x-x-L motifs are more critical for
FcyRIIA-mediated phagocytosis than Y 1, this tyrosine (not
in a typical Y-x-x-L context) may serve as a phosphorylation
site when one of the downstream tyrosines is absent.
These studies do not preclude the possibility of additional
interactions in the FcyRIIA cytoplasmic domain and among
the FcyRIIA cytoplasmic tyrosines that might add further
complexity to the pattern of tyrosine phosphorylation. For
example, the slight reduction in tyrosine phosphorylation
caused by Y2F could indicate that Y2 need only be minimally phosphorylated in vivo for phagocytic activity, as long
as Y3 is intact and can be phosphorylated. Thus, for Y2F
mutants, the contribution of Y2 could be masked if there is
a compensatory increase in phosphorylation at Y 1 and/or
Y3. It is likely that Y2 also contributes to receptor function
by providing structural stability or an important conformation to the receptor, because Y2K did not function as well
as Y2F.In addition, tyrosine phosphorylation was diminished in the mutant containing the 5 amino acid deletion
A287-291 between the two Y-x-x-L sequences. The extent
of inhibition of phosphorylation compared with wild-type
FcyRIIA approximated the diminution of phagocytosis observed with this mutant receptor (Fig 4 and Table l), indicating that this sequence and/or the conformational relationship
it imparts to the two Y-x-x-L regions is important for both
FcyRIIA tyrosine phosphorylation and phagocytosis.
Receptor tyrosine phosphorylation wassignificantly reduced in the mutants with deletion of the YMTL or YLTL
threonine and leucine 284-285 and 300-301, respectively. In
addition, the time course of tyrosine phosphorylation of these
mutants was delayed (data not shown) and phagocytosis was
impaired (Fig 2). These results suggest that optimal phagocytosis and tyrosine phosphorylation do not occur within a
structurally altered Y-x-x-L sequence even if
all
three
FcyRIIA cytoplasmic tyrosines are present.
We previously reported that the protein tyrosine kinase
Src phosphorylates FcyRIIA in vitro." Therefore, FcyRIIA
transfectants were coimmunoprecipitated with anti-Src and
anti-FcyRIIA to expose the wild-type and mutant forms of
FcyRIIA to Src kinase activity (Fig 5). The individual tyrosine mutants Y 1 F, Y2F, and Y3F (Fig 5, lanes 2 through 5 )
and the double tyrosine mutantYIFTY2F (Fig 5, lane 6 )
displayed about half the level of phosphorylation of wildtype receptor. Phosphorylation in the other double tyrosine
mutants was more severely reduced (Fig 5, lanes 7 and 81,
similar to the results with the antiphosphotyrosine immunoblots (Fig 3). Y2F/Y3F, the mutant lacking the tyrosines
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1757
TYROSINEPHOSPHORYLATION AND PHAGOCYTOSIS
l 2 3 4 5 6 7 8 9 1 0
E A : - + - + + + + + + +
MW
kDa
1--""-w---~
10172 Fig 3. Tyrosine phosphorylation in COS-l cells transfected
with wild-type
or
mutant
FcyRIIA.
Lanes
1 and 2, sham
transfectants; lanes 3 and 4.
wild-type FcyRIIA; lane 5,Y1F;
lane 6, Y2F; lane 7 Y3F; lane 8,
YlF/Y2F; lane 9, Y2F/Y3F; and
lane
YlF/YOF.
10,
Molecular
weight markers (MW)
are shown
in kilodaltons (kDa). Tyrosine
phosphorylation of FcyRllA (40
kD) is seen in lanes 4 through 7.
-.
3FcyRIIA
44-
29-
in both Y-x-x-L sequences, is very weakly reactive to Src
kinase in vitro and is not phosphorylated on tyrosine in intact
cells. The results with the double replacement Y IFN2F indicate that Y3 is phosphorylated to a significant extent in vitro
and is consistent with the in vivo data inwhich selective
substitution of Y3 (Y3F) results in substantial diminution in
receptor phosphorylation. Thus, these data suggest a potential role for Src or another yet unidentified Src-like tyrosine
kinase in vivo, because the activity in vitro to some extent
mimics the pattern found with intact cells.
These observations suggest a possible mechanism by
which FcyRIIA mediates a phagocytic signal. At least two
cytoplasmic tyrosines are required for phagocytosis, but one
of the two tyrosines, Y 1, can be located upstream from the
conserved consensus motif and need not be a part of a Yx-x-L sequence. It is likely that this tyrosine plays a structural role in the function of the receptor, because phenylala-
nine at Y 1 functions almost as well as tyrosine. Although it
is not a component of a typical Y-x-x-L motif, Y1 (YETA)
may provide a binding site for a kinase or substrate involved
in receptor function. Y3 is likely substantially phosphorylated during receptor activation and appears to be particularly
1 2 3 4 5 6 7 8 91011
EA:
- + -++++++++
TIME
O S 0
0
S1530605lS30@0
(MW
Q
Table 1. Effect of Mutations onFcyRllA Phagocytosis
and Tyrosine Phosphorylation
Mutant FcyRllA
Phagocytosis (%l
Tyrosine
Phosphorylation
Y1F
Y1K
Y2F
Y3F
Y 1FN2F
Y 1FN3F
Y2FN3F
A207-291
A284-285 (Y2-ATL)
A300-301 (Y3-ATL)
92
++
34
36
4
14
4
30
42
51
++I+++
]FcyRllA
ND
+
0
0
0
++
+
+
Values are the percentage of wild-type FcyRIIA. Wild-type FcyRllA
gave ++++ FcyRllA tyrosine phosphorylation. Results from four to
six experiments for each mutant are shown.
Abbreviation: ND, not determined
Fig 4. l i m e course of tyrosinephosphorylation of wild-type
FcyRllA and the mutant FcyRllA A287-291.Lanes 1 and 2, sham
transfectana; lanes 3 through 7, wild-type FcyRllA at 0, 5, 15, 30,
and 60 minutes, respectively; lanes 8 through 11, mutant A287-291,
deletion of PRAPT from thesequence separating YP-x-x-L and Y3-x30, and 60 minutes, respectively. For wildx-L (see Fig l), at 5, 15,
type FcyRIIA, a strongly phosphorylated bandappears at 5 minutes
and persists for 60 minutes (bracket). For mutant A287-291, phosphorylation ispresent at 5 minutes, but does not reach the intensity
of wild-type FcyRIlA. Mutant A287-291 is a smaller molecule than
wild-type FcyRllA and migrates more rapidly than the wild-type
FcyRllA on thegel.
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1758
1 2 3 4 5 6 7 8
I
I
MITCHELL ET AL
I
Fig 5. In vitro kinase phosphorylation of mutant
FcyRllA receptors by Src kinase. Lane 1, wild-type
FcyRIIA; lane 2, Y1F; lane 3, Y2F; lane 4, Y2K; lane 5,
Y3F; lane 6, YlF/YZF; lane 7,Y2F/Y3F; and lane 8,
YlF/Y3F. The positions of the phosphorylated Src
and wild-type or mutant FcyRllA are indicated.
1
important because its removal by truncation or by replacement with phenylalanine inhibits both tyrosine phosphorylation and phagocytosis in parallel. However, replacement of
Y3,with Y1 andY2 remaining intact, still allows some
phagocytosis. Y2 may thus play roles in both receptor phosphorylation and structural stability, whereas YI may function as a “back up” phosphorylation site when one of the
other tyrosines in a Y-x-x-L motif is not available.
The fact that phagocytosis can occur with only one intact
Y-x-x-L consensus sequence distinguishes human FcyRIIA
from other Ig receptor family proteins, including the phagocytic receptor FcyRIIIA. Current studies suggest that both
Y-x-x-L sequences must be phosphorylated for signal transduction20-23I’ n other Ig family receptors. Similarly, FcyRIIIA, which mediates phagocytosis through an associated y
subunit, requires two intact y chain Y-x-x-L sequences for
both tyrosine phosphorylation and phagocytosis when a murine y subunit is employed.’ The requirements for phagocytosis by the human y chain, which contains an additional
cytoplasmic upstream tyrosine not within a typical Y-x-x-L
sequence, are unknown.
Most other Ig family receptors, including the y subunit
of FcyRIIIA, have 6 to 8 amino acid residues not including
prolines between the Y-x-x-L sequences>12.13whereas
FcyRIIA has 12 amino acids (including 2 prolines) between
its two Y-x-x-L sequences.”.I5 For FcyRIIA, these prolinecontaining sequences also are important for optimal phagocytosis. This structure appears to be important for FcyRllA
tyrosine phosphorylation as well as for phagocytosis. Disruption of the conformation between the two Y-x-x-L sequences
may also be partially responsible for the effect of the deletion
of threonine and leucine 283-284.
There are some similarities between human and murine
FcyRII and some observations on the role of cytoplasmic
domains of murine FcyRIIB have been reported. Although
mice lack FcyRIIA, FcyRIIB2 has been shown to mediate
immune complex endocytosi~?~
whereas FcyRIIB 1 does not
have this activity. Murine FcyRIIB2 contains two cytoplasmic tyrosines. When these tyrosines were replaced by
alanines, endocytosis was not
There are several
differences between these studies and our own. Although
phagocytosis and endocytosis are related processes, fundamental differences appear to exist between them. For example, endocytosis involves localization of crosslinked receptors to clathrin-coated pits, whereas clathrin is not
necessarily involved in phagocytosis. Furthermore, although
FcyRIIB2 undergoes endocytosis when it is cross-linked by
a n t i b ~ d i e s , ~it~ .does
~ ’ not induce the phagocytosis of IgGcoated RBCs.“ Therefore, it is likely that there are cytoplasmic sequences responsible for phagocytosis that are distinct from those required for endocytosis.
We also examined whether cytochalasin D, a potent inhibitor of actin polymerization and FcyRIIA mediated phagocytosis, alters tyrosine phosphorylation of FcyRIIA (Fig 6).
Whereas cytochalasin D inhibited phagocytosis by FcyRIIA
transfectants: it didnot alter tyrosine phosphorylation of
FcyRIIA. The levelof tyrosine phosphorylation wasthe
same with and withoutcytochalasin D treatment. In contrast,
FcyRllA
SHAM -CD
+CD
EA:
L
1
2
3
4
5
Fig 6. Cytochalasin D does not inhibit tyrosine phosphorylation
ofFcyRItA. COS-l cells were sham-transfected (lanes 1 and 2) or
transfected with FcyRllA (lanes 3, 4, and 51. Cells were incubated
with 10 pg/mL cytochalasin D (lane 5) or buffer (lanes l through 4)
for 15 minutes at room temperature, followed by stimulation with
EA for 30 minutes at 37°C (lanes 2, 4, and 5). Antiphosphotyrosine
immunoblots were then performed.
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
TYROSINEPHOSPHORYLATION AND PHAGOCYTOSIS
we have observed that genestein and tyrphostin 23, which
alter tyrosine kinase activity, reduce the phosphorylation of
FcyRIL4 and inhibit the phagocytosis of EA.'6 Thus, it is
likely that tyrosine phosphorylation and phagocytosis are
serially ordered reactions and that tyrosine phosphorylation
of FcyRIIA occurs before the actin polymerization required
for phagocytosis.
The structural differences between the cytoplasmic domains of FcyRIIA on the one hand and the T-cell and Bcell receptors on the other may explain in part how the
former cells induce phagocytosis while the latter cells induce
T-cell and B-cell activation events. These differences in the
receptor structures likely involve intermediate signal transducing elements in phagocytic cells that interact with known
components of the phagocytic machinery, eg, actin and actinbinding proteins. The unusual 12 amino acid sequence containing two prolines in FcyRIIA may be structurally important in this regard. Furthermore, in contrast to FcyRIIA,
the other twophagocytic
Fcy receptors, FcyRIand
FcyRIIIA, require a subunit to mediate a phagocytic signa1.8"0 Characterization of those elements that interact with
cytoskeletal components are currently under study.
Mutations in the cytoplasmic domains of the FcyRIIA
receptor have provided important information about the sequences in its structure that are required for phagocytosis.
However, it willbe necessary to obtain a detailed threedimensional structure to completely understand the effect of
these modifications on the interactions with other components of the pathway for phagocytosis. In addition, peptide
analysis will be necessary to precisely define the extent of
phosphorylation of each cytoplasmic tyrosine. Further studies to more precisely define the structural requirements of
the FcyRIIA cytoplasmic domain in signal transduction are
in progress.
ACKNOWLEDGMENT
We thank Dr Joan Brugge for her thoughtful advice and suggestions.
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1994 84: 1753-1759
Substitutions and deletions in the cytoplasmic domain of the
phagocytic receptor Fc gamma RIIA: effect on receptor tyrosine
phosphorylation and phagocytosis [published erratum appears in
Blood 1994 Nov 1;84(9):3252]
MA Mitchell, MM Huang, P Chien, ZK Indik, XQ Pan and AD Schreiber
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