Proc. Nati. Acad. Sci. USAVol. 86, pp. 7566-7570, October 1989Immunology

Molecular structure and expression of the murine lymphocytelow-affinity receptor for IgE (FceRII)BERNHARD BETTLER*, HANS HOFSTETTER*, MANGALA RAOt, WAYNE M. YOKOYAMAt,FRANCOIS KILCHHERR*, AND DANIEL H. CONRADt§*Biotechnology Department, Ciba-Giegy Ltd., CH4002 Basel, Switzerland; tDivision of Molecular and Clinical Rheumatology, Department of Medicine,Johns Hopkins University School of Medicine, Baltimore, MD, 21205; and tLaboratory of Immunology, National Institute of Allergy and Infectious Diseases,National Institutes of Health, Bethesda, MD 20892

Communicated by William E. Paul, July 13, 1989

ABSTRACT The cDNA encoding the murine low-affinityreceptor for IgE (FcRII) has been isolated from a cDNAlibrary prepared from B cells activated with lipopolysaccharideand interleukin 4. It encodes a 37-kDa protein of 331 aminoacids with two potential N-linked glycosylation sites. Analogousto its human counterpart, there is no signal sequence and theputative transmembrane region is close to the amino terminus,indicating an inverse membrane orientation with the carboxylterminus at the cell exterior. The predicted murine Fc6RIIamino acid sequence demonstrates a 57% identity with itshuman counterpart. The murine sequence has an additionalinternal repeat motif of 21 amino acids giving four repeats ascompared to three in the human sequence. Furthermore, themurine FcRII is truncated at the carboxyl terminus and theArg-Gly-Asp sequence, a common recognition site of integrinreceptors, which is found in the reverse configuration in humanFc6RII, is missing. B cells activated with interleukin 4 andlipopolysaccharide have an increased amount ofFcRII mRNAas compared with resting or lipopolysaccharide-stimulated Bcells. Con A-activated normal T cells, the TH-2 cell line D1O,as well as the macrophage cell line J774 have no detectableFc6RII mRNA. Expression analysis using transiently trans-fected COS cells revealed that recombinant murine Fc6RIIbinds anti-Fc6RII as well as mouse and rat IgE but does notbind human IgE or mouse IgG. FcRH expressed in COS cellshas a molecular mass of 45 kDa whereas the Fc6RII from B-celllines is a 49-kDa protein.

The low-affinity receptor for IgE (Fc6RII is found on a widevariety of hematopoietic cell types (for review, see ref. 1).Interest in the FcRII has come from two directions. (i) Itsligand-binding capacity (IgE) and the classic correlationbetween elevated IgE and elevated B-cell FcRII levels (2)have long suggested an involvement in IgE biosynthesismechanisms. In addition, interleukin 4 (IL-4), which isclearly important in both the human and murine systems withrespect to IgE production (3, 4), is the agent primarily respon-sible for the FceRII increases seen in parasite infections (5).The most definitive indication ofinvolvement of FceRII in theregulation of IgE synthesis has been described in man. Therecombinant soluble Fc6RII fragment (sFc6RII) releasedfrom the surface of cells transfected with the human FceRIIhas been shown to potentiate IgE synthesis (6). Anti-FcRIImonoclonal antibodies (3, 7) and IgE immune complexes (8)suppress IgE synthesis by human peripheral blood lympho-cytes and/or the IgE-secreting plasmacytoma (U266). (ii) Thedemonstrated identity between the human FceRII and theB-cell differentiation antigen CD23 (9) has led to studiesindicating that the FcRII, and especially the sFcRII, is

involved in B-cell growth and differentiation (for review, seeref. 10).The murine system provides an attractive model to inves-

tigate the biological function of the Fc6RII by correlating invitro and in vivo studies. The Fc6RII is dramatically inducedon activated murine B cells in the presence of IL-4 (11, 12).However, no activity of anti-Fc6RII or sFcRII with respectto IgE synthesis has as yet been reported in the murinesystem, in contrast to the human studies mentioned above.The human Fc6RII was cloned independently by three groups(13-15). Herein we report the cloning and characterization ofa cDNA encoding the murine FcRII.$ Sequence analysisindicates that, analogous to its human counterpart, the mu-rine FcRII is a type II membrane protein with homology tothe C type animal lectin family.

MATERIALS AND METHODSIsolation of cDNA Clones Encoding Murine Fc6RII. B cells

were purified from BALB/c mice and were stimulated withlipopolysaccharide (LPS; 5 ,ug/ml) and recombinant IL-4(1000 units/ml; a gift from W. Paul, National Institutes ofHealth; see ref. 16 for unit definition) as described (11). After3 days the cells were centrifuged through Ficoll and the viablecells were collected. Poly(A)+ mRNA was isolated fromthese cells using the method described by Badley et al. (17).A cDNA library was made in Agtll using a cloning kit fromAmersham according to the manufacturer's directions. Thefinal nonamplified library had a phage titer of 2 x 106 totalplaques.The cDNA library in Agtll was screened at 420C under

low-stringency conditions [35% (vol/vol) formamide/5 xSSPE (lx SSPE = 0.18 M NaCl/10 mM sodium phosphate,pH 7.4/1 mM EDTA)/1 x Denhardt's solution (0.02% bovineserum albumin/0.02% Ficoll/0.02% polyvinylpyrrolidone)/0.1% NaDodSO4/herring sperm DNA (100 gg/ml)] with the32P-labeled 454-base-pair (bp) Dde I-Nco I fragment of thehuman cDNA. A total of 3 x 105 plaques were screened. Thefilters were washed at room temperature for four 15-minperiods with 2x SSC (lx SSC = 0.15 M NaCI/0.015 Msodium citrate, pH 7)/0.1% NaDodSO4 and for two 30-minperiods with the same buffer at 550C. Positive plaques wererescreened and phage DNA from 11 independent singleplaques was purified. The cDNA insert was recovered withEcoRI from the isolated phage DNA and ligated in eitherorientation in Bluescript phagemids. The complete sequenceof one clone was determined and partial sequences were

Abbreviations: Fc6RII, low-affinity receptor for IgE; sFcRII, sol-uble Fc8RII; IL-4, interleukin 4; LPS, lipopolysaccharide.§Present address and to whom reprint requests should be addressedat: Department of Microbiology and Immunology, Medical Collegeof Virginia, Box 678, MCV Station, Richmond, VA, 23298-0678.$The sequence reported in this paper has been deposited in theGenBank data base (accession no. M27150).

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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obtained from other independently isolated clones to confirmthe sequence data. The Exo/mung bean nuclease deletion kit(Stratagene) was used to produce unidirectional nested de-letions in the cDNA. Single-stranded template DNA wassequenced by the dideoxynucleotide chain-terminationmethod (18) and the T7 polymerase Sequenase system (Unit-ed States Biochemical). Where needed, oligonucleotide prim-ers were synthesized to complete first- and second-strandsequence. The additionally isolated cDNAs were analyzed byrestriction enzyme cleavage. This analysis indicated that allcDNAs were identical with the sequenced one.

Northern Blot Analysis. Poly(A)+ mRNA was isolated asdescribed (17) or total RNA was isolated by the guanidineisothiocyanate method. Poly(A)+ (1-2 ,g) or total RNA (10,g) was electrophoresed on a 1.2% agarose/glyoxal gel,transferred to GeneScreen, and hybridized with the 32P_labeled 1.2-kilobase (kb) Fc8RII cDNA probe labeled by therandom hexamer priming method (19).

Expression Analysis of Murine cDNA. The cDNA clone(initially 2.1 kb) was shortened by Exo/mung bean nucleasedigestion at its 3' end to position 1176. The truncated 1.2-kbcDNA containing the large open reading frame was unidi-rectionally inserted into the CDM8 expression vector (20).After expansion and plasmid isolation COS cells were trans-fected (20 ,ug per 5 x 106 cells) by electroporation with eitherCDM8 alone or CDM8 containing the 1.2-kb insert in thecorrect orientation. The transfected COS cells were exam-ined after appropriate immunofluorescent staining using aFACSCAN (Becton Dickenson). Dead cells were excludedby propidium iodide staining and light scatter parameters; 104cells were analyzed per group. Alternatively, binding anal-ysis utilized 1251I-labeled mouse IgE (2 ,ug/ml; specific activ-ity, 107 cpm/,g) and a 100-fold excess of the respective

inhibitors; incubation was for 1 hr at 37°C. Mouse and rat IgEwere purified as described (11); human IgE (PS) was a kindgift of Kimishige Ishizaka (Johns Hopkins University). Freeand cell-bound IgE were separated on a phthalate oil mixture,as described (11). 125I-labeled FceRII or sFc8RII was pre-pared from surface-labeled (lactoperoxidase) FceRII cDNA-transfected COS cells or Fc8RII+ hybridoma cells [01.2A2cells (21)]. Fc6RII-immunoactive material was isolated byanti-Fc6RII (monoclonal antibody B3B4) (22) affinity chro-matography from either detergent (Nonidet P-40)-solubilizedcells (see ref. 23 for protease inhibitors used) or from cell-freesupernatants. After low-pH elution (22), the labeled materialwas reprecipitated with either B3B4 or a control rat IgG2apreparation and subsequently examined by gradientNaDodSO4/PAGE (23).

RESULTSCloning of the Murine FcRII cDNA. Based on the assump-

tion that the IgE-binding domain (24) of the human FcRIIwould be the most homologous with the murine FcRII, aprobe from this region (corresponding to amino acids 148-300) was used to screen a mouse cDNA library prepared fromLPS/IL-4-activated B cells. The largest FcRII cDNA ob-tained was 2.1 kb long and its sequence is shown in Fig. 1.This putative murine FcRII cDNA consists of a 164-bp 5'untranslated region, a continuous open reading frame of 993bp, and a relatively long 3' untranslated region of954 bp. The3' terminal sequence of the cDNA is almost perfectly ho-mologous With the 130-bp B1 repetitive sequence (41). Thissequence is ubiquitous in the mouse genome and often foundat the 3' end of mRNA. No common Poly(A) addition signal(AATAAA) nor a poly(A) tail was present in the murine

1

101 GAGTGACAAGTGCCTTGGCAGGTAGTGCACGCCTCATCACTGAAAGGATCCAAACAAGACTGCCATGGAAGAAAATGAATACTCAGGATACTGGGAACCTM E E N E Y S G Y W E P

201 CCTAGAAAGCGTTGCTGCTGTGCAAGACGTGGGACACAGCTCATGTTGGTGGGGCTGCTGAGCACAGCAATGTGGGCTGGCCTGCTGGCCCTGCTTCTTCP R K R C C C A R R G T Q L M L V G L L S T A M W A G L L A L L L L

301 TGTGGCACTGGGAAACGGAGAAGAATCTAAAACAGCTGGGAGACACTGCAATTCAGAATGTCTCTCATGTTACCAAGGACTTACAAAAATTCCAGAGTAW H W E T E K N L K Q L G D T A I Q N V S H V T K D L Q K F Q S N

-CHO-401 TCAATTGGCCCAGAAGTCCCAGGTTGTTCAGATGTCACAAAACTTGCAAGAACTCCAAGCTGAACAGAAGCAAATGAAAGCTCAGGACTCTCGGCTCTCC

Q L A Q K S Q V V Q M S Q N L Q E L Q A E Q K Q M K A Q D S R L S501 CAGAACCTGACCGGACTCCAGGAGGATCTAAGGAACGCCCAATCCCAGAACTCAAAACTCTCCCAGAACCTGAACAGACTCCAAGACGATCTAGTCAACA

Q N L T G L Q E D L R N A Q S Q N S K L S Q N L N R L Q D D L V N I-CHO-

601 TCAAATCCCTGGGCTTGAATGAGAAGCGCACAGCCTCCGATTCTCTAGAGAAACTCCAGGAAGAGGTGGCAAAGCTGTGGATAGAGATACTGATTTCAAAK S L G L N E K R T A S D S L E K L Q E E V A K L W I E I L I S K

701 GGGAACTGCATGCAACATATGTCCCAAGAACTGGCTCCATTTCCAACAGAAGTGCTACTATTTTGGCAAGGGCTCCAAGCAGTGGATCCAGGCCAGGTTCG T A C N I C P K N W L H F Q Q K C Y Y F G K G S K Q W I Q A R F

A C S D L Q G R L V S I H S Q K E Q D F L M Q H I N K K D S W I G L901 TCCAGGATCTCAATATGGAGGGAGAGTTTGTATGGTCGGACGGGAGCCCTGTGGGTTATAGCAACTGGAATCCAGGGGAGCCCAATAACGGGGGCCAGGG

Q DL N M E G E F V W S D G S P V G Y S N W N P G E P N N G G Q G1001 TGAGGACTGTGTGATGATGCGGGGATCCGGCCAGTGGAACGACGCCTTCTGCCGCAGCTACTTGGATGCATGGGTGTGTGAGCAGCTGGCAACATGTGAG

E D C V M M R G S G Q W N D A F C R S Y L D A W V C E Q L A T C E

1101 ATATCTGCCCCCTTAGCCTCTGTGACTCCAACAAGGCCCACCCCAAAAAGTGAACCCTGACAAACTTCTGCTCACACTCTTCTGGATTTCTCCTCTACCTI S A P L A S V T P T R P T P K S E P *

12011301140115011601170118011901

20012101

CACCTCCTATCAAGTTCCCCACTTTCTCCCCCTCGGCATCAGAGAACAGGGGTTCCCTTTCCCCAGGATCTGGGATGAGTCCTCCCATCAAGTTTGCATCAGTGGTCCCAGCACTCCGACCCTCCTTGGAGGCTGCACCAGGTGTGCTCCTGGTGCGGGAGGTATTGAAGGAACTCTAAACAGCTCCAGCAAGGCGAGCCTGGCTCTGTCTGGTAGGCCTGGCCCTTCTCTCCCATTCCTTCTACCTTACTAAAAGCTGTTAGAGAACAGTCCTAAAGCTAGCCCCCAAGGTCTATTCCCTTATTTGGCCACTTCCTCCTCCTGAGGCTGACTACAAGGTCCAGCTATCCAAGTACTGAAGTCTAACATCAAAAGCCCCCTTTGTCTCACCTAAGTAGCAATGCCCAATCAAAATACACCATCACATCATAGCCCAGTCTAACAGACCGCCCTTTTTCTCTTCATAAAATTACACCTGCAACCAGGCGTAGTGGTGCAGGCCTTTAGTCCCAGCACTTGGGAGGCAGAGACAAGCGAATTTCTGAGTTCGAGGCCAGCCTGGTCTACAAAGTGAGTTCCAGGACAGCCAGGGCTACACAGAGAAACCCTGG

FIG. 1. Nucleotide and predicted amino acid sequence of mouse FcRII cDNA. Nucleotide numbering is given at the left. The predictedtransmembrane region is underlined; sites of potential N-linked glycosylation are indicated by -CHO-. The termination signal is marked by anasterisk. The single-letter amino acid code is used.

CCTGTCTTCAACACACTAGCCTGAGCTACCTTATCCAAGTGCTCCACATATTCCAGAAGGAGAAGGACAGACTTCAAGTTCAAATCACTTCCAGAGCTGT

GCTGGTGGATGCCAGCTCCTGCCAGCTACCCAGAAACCCTCTCCAGCTCTCCAGCTAAGCTGGCCATCCCATTCCATCTGCCTTCCTCAAACCTGGGCCC

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1 2 3 4 5 6 7 8

FIG. 2. Northern blot analysisof Fc8RII mRNA. Poly(A)+mRNA was electrophoresed on aglyoxal/1.2% agarose gel, blotted,and probed with 32P-labeled cDNAfragments. mRNA is from the fol-lowing sources. Lanes: 1, IL-4-induced 01.2A2 cells (2 ,g); 2,01.2A2 (2 ,g); 3, LPS/IL4acti-vated B cells (1 jig); 4, resting Bcells (1 ,ug); 5, LPS-activated Bcells (1 ,ug); 6 resting B cells (re-peat); 7, D10 cells (1 ,ug); 8, J774cells (1 ,ug). RNA ladder markersizes (in kb) are shown on the left.

FcERII cDNA isolated. The reason for this is not clear;possibly the cDNA library was incompletely methylated,resulting in cleavage of the EcoRI site that is in the 3' trailerand subsequent loss of the 3' end. Northern blot analysisindicates that a nearly full-length clone must have beenisolated since the mature mRNA is 2.4 kb (see below).The predicted initiation codon is enbedded in an initiation

consensus sequence (GCCATGG) (25). The translated aminoacid sequence, also shown in Fig. 1, indicates a protein of 331amino acids with a calculated molecular mass of 37 kDa. Asin the case of the human sequence, there is no signal peptideand the 21-bp hydrophobic segment from amino acid 26 toamino acid 46 presumably serves as an internal signal se-quence resulting in an inverse membrane orientation of themurine FcRII, with a carboxyl-terminal extracellular do-main and an amino-terminal cytoplasmic orientation. Theextracellular domain contains two sites for N-linked glyco-sylation at amino acids 65 and 114.Northern Blot Analysis of Murine FcERII mRNA. Fig. 2

shows the analysis ofFcRII mRNA isolated from B cells andFc6RII+ hybridoma cells in the presence and absence of IL-4;the latter is known to induce Fc8RII biosynthesis (5). Thesame amount of Fc6RII mRNA was present when comparingmRNA from the FceRII' hybridoma cells (2 ug) and B cells

(1 ,g). Clearly, IL-4 induces a higher level of FcRII expres-sion. Poly(A)+ mRNA from D10.G4.1 (D10), a TH2 T-cellline (26), and the macrophage cell line J774 was examined onthe same blot (lanes 7 and 8). No FcERII mRNA was detected.

Expression of the Murine FcRII cDNA. A large portion ofthe 3' untranslated region was deleted and the resulting1.2-kb cDNA was cloned into the CDM8 expression vectorand used to transfect COS cells. Fluorescence-activated cellsorter analysis of viable COS cells 48 hr after transfectionrevealed that =30% of the cells bound B3B4 (Fig. 3A), ananti-murine B-cell Fc6RII antibody (22). In contrast, COScells transfected with the original CDM8 vector did not bindmonoclonal antibody B3B4. Transfected COS cells had theexpected specificity of the FceRII, in that binding of mouseIgE was blocked by mouse and rat IgE but not by human IgE.Also B3B4, but not normal mouse IgG, blocked binding ofmouse IgE (Fig. 3B). In addition, mouse and rat IgE blockedthe binding of '25I-labeled B3B4 (data not shown). Thus, thedata demonstrate that the cDNA sequence shown in Fig. 1encodes a molecule with the properties expected of murineFceRII.The biochemical properties of the recombinant FcRII

were examined. COS cells (48 hr after transfection) weresurface radiolabeled, one-half were detergent-solubilized,and the intact FcRII was isolated by affinity chromatogra-phy. The other half of the cells were put back into culture;after 6 hr, the cell-free supernatant was collected; and thesFcERII (22) was isolated by B3B4 affinity chromatography.Analysis by NaDodSO4/PAGE of the FceRII immunopre-cipitated from transfected COS cells identified a 45-kDa band(Fig. 3C, lane 1) as compared to the 49-kDa band (lane 2)obtained from the FcRII' hybridoma cells. When the FceRIIimmunoreactive material isolated from COS culture super-natants (lane 4) was compared to sFc6RII from the hybridomacells (lane 3), recombinant sFc8RII was found to be slightlysmaller in molecular mass and may be more susceptible toproteolysis than the respective protein produced by thehybridoma cells (see Discussion). Fc8RII or sFc6RII were notisolated using control IgG2a affinity columns as no bandswere visible by NaDodSO4/PAGE (data not shown).Comparison of the Murine and Human Fc6RII Amino Acid

Sequences. The murine and human FcRII protein sequencesare related; a 57% identity (identical residues) or a 74%identity (conservative substitutions) was obtained by usingthe GAP alignment program (Fig. 4A) (27). The probe from thehuman cDNA used to isolate the murine cDNA has an overall

B

Medium

MouseIgERatIgE

HumanIgEAnti-FcRIIMouseIgG

lo 102 103Fluorescence intensity

I

=4

m~~~~~~E

2 4 6CpM X 10-4

C

66

30--

22

.......

1 2 3 4

FIG. 3. Expression of murine Fc6RII. (A) Transfected COS cells (48 hr after transfection) were incubated with B3B4 and, after washing,incubated with fluorescein isothiocyanate-labeled goat anti-rat immunoglobulin. Viable cells were examined by fluorescence-activated cell sorteranalysis. COS cells were transfected with either CDM8 containing the FcR insert (solid line) or with CDM8 alone (dots). (B) Binding of125I-labeled mouse IgE to 106 COS cells transfected with the FcRII expression vector in the presence or absence of a 100-fold excess of theindicated competitor. 1251-labeled IgE binding is shown (cpm x 10-4). (C) Fc8RII expressing COS cells or FcRII hybridoma cells were surfaceradiolabeled with lactoperoxidase. Fc6RII immunoreactive material was isolated by affinity chromatography and analyzed on a 10-18% gradientNaDodSO4/polyacrylamide gel. Lanes: 1, intact FcRII from COS cells; 2, intact FcRII from 01.2A2 hybridoma cells; 3, sFc6RII fromhybridoma cells; 4, sFc8RII from COS cells. Molecular mass markers are shown (in kDa) on the left.

9.5-7.5 -

4.4 -

2.4

1.4

A

6

r.ct

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AMURINE 1 MEENEYSGYWEPPRKRCCCARRGTQLMLVGLLSTAMWAGLLALLLLWHWETEKNLKQL

il 1 11111 11111 1I 11 I Hi1 l1111111 li1HUMAN 1 MEEGQYSEIEELPRRRCC--RRGTQIVLLGLVTAALWAGLLTLLLLWHWDTTQSLKQL

MURINE 59 GDTAIQNVSHVTKDLQKFQSNQLAQKSQVVQMSQNLQELQAEQKQMKAQDSRLSQNLT1 111i 111IIHiII 11 11i 11 1111111

HUMAN 57 EERAARNVSQVSKNLESHHGDQMAQKSOSTQISQELEELRAEQQRLKSQDLELSWNLN

GLQEDLRNAQSQNSKLSQNLNRLQDDLVNIKSLGLNEKRTASDSLEKLQEEVAKLWIE111 111~l~I11 1111 1

GLQADLSSFKSQE---------------------LNERNEASDLLERLREEVTKLRME

MURINE 175 ILISKGTACNICPKNWLHFQQKCYYFGKGSKQWIQARFACSDLQGRLVSIHSQKEQDFI 11 11 I 11111111 111 111111 1111111iii

HUMAN 152 LQVSSGFVCNTCPEKWINFQRKCYYFGKGTKQWVHARYACDDMEGQLVSIHSPEEQDF

LMOHINKKDSWIGLODLNMEGEFVWSDGSPVGYSNWNPGEPNNGGOGEDCVMMRGSGO11111 1III III 111111111111

HUMAN 210 LTKHASHTGSWIGLRNLDLKGEFIWVDGSHVDYSNWAPGEPTSRSQGEDCVMMRGSGR

MURINE 291 WNDAFCRSYLDAWVCEQLATCEISAPLASVTPTRPTPKSEP111111 11111 1iii

HUMAN 268 WNDAFCDRKLGAWVCDRLATCTPPASEGSAESMGPDSRPDPDGRLPTPSAPLHS

H V T K D

Q M S Q N

K L S Q N

Q V S K N

Q I S P E

E L S W nN

L

L

L

L

L

L

L

Q K F Q S N Q

Q E L Q A E Q

T G L Q E D LN R L Q D D L

E S H H G WD[QE E L R A E Q

N G L A D] LL-EEFiIRA

L A Q Ks QK Q M K A QR N A Q S QV N I K S L

M A Q K S QQ -RL K S QS S F K S QI

FIG. 4. (A) Alignment of mu-rine and human amino acid se-quences of FceRII. Identical resi-dues are indicated and dots repre-sent gaps to maximize homology.The single-letter amino acid codeis used. (B) Repeated structure inthe mouse and human FcRII pro-tein. In human receptor gene, ex-ons 5-7 each encode a repeatedmotif (27). In the mouse receptor,a fourth repeated motif is present,probably encoded by an additionalexon in the mouse gene. Onlythose amino acids fully encodedwithin the corresponding humanexons are shown.

homology of 70%, including a stretch of 18 nucleotides ofperfect identity with the murine cDNA, thus explaining thesuccess that was obtained using this probe. As was reportedin the human FceRII genomic sequence (28), exons 5, 6, and7 code for highly related 21-amino acid repetitive sequences.The murine sequence has an additional repetitive sequencebetween residues 129 and 150 that suggests that an additionalrepetitive exon will be found in the murine gene. Comparisonof these sequences in the mouse and human is shown in Fig.4B and their highly related nature is indicated by outliningamino acids that appear in at least four of the sequences. Themurine predicted amino acid sequence terminates 13 residuesbefore the human counterpart. To confirm that this termina-tion was not a cloning artifact, four additional cDNA cloneswere sequenced through this same region and identical se-quences were obtained. In addition, the 3'-terminal 269nucleotides were sequenced in one of these clones and again,identical sequences to those reported in Fig. 1 were obtained.Yokota et al. (29) described evidence that there are twospecies of the human FcRII termed Fc8RIIa and Fc6RIIb.Comparison of the amino-terminal residues indicates that themurine FceRII reported here corresponds to the humanFceRIIa species.

DISCUSSIONAnalysis of the cDNA sequence for the murine FceRIIreveals that due to an additional repetitive sequence themurine cDNA codes for a protein of 331 amino acids,compared to 321 amino acids for the human. Interestingly,the murine sequence terminates just before the inverted RGD(Arg-Gly-Asp) sequence (Asp-Gly-Arg, position 309 in thehuman sequence). Confirmation ofthis early termination wasseen in that other cDNA clones had identical sequences.Since this inverted sequence has been reported to alsobehave as a target sequence for adhesion receptors in somesystems (30), its existence in the human Fc6RII led to thesuggestion that this sequence was involved in the biologic

function of FceRII (10). The absence of a similar sequence inthe mouse casts doubt on that hypothesis since sequencesimportant for biological function would be expected to beconserved.The human FcRII and now also its murine counterpart

have extensive homology with C-type animal lectins (28).Several other cloned membrane proteins in the hematopoieticsystem contain domains that exhibit this lectin homology.These include Elam-1 (31), Mel-14 (32), and GMP140 (33), allof which are adhesion molecules potentially controlling lym-phocyte migration. Additionally, two lymphocyte type II

membrane proteins share significant homology with the mu-rine Fc6RII. These molecules are the Lyb-2 antigen (34),which is a mouse B-cell differentiation antigen, and Al (35),a murine cell-surface disulfide-linked dimeric antigen presenton some T-cell lymphomas. A closely related form of the Almolecule, termed YE1/48, has also been described (36).The murine FceRII, analogous to the human, releases a

soluble fragment from the cell surface and this releaseappears to be a major catabolic breakdown pathway for thisprotein (5). Amino-terminal sequencing of the released mu-rine fragment has not yet been performed; thus the exact siteof cleavage is unknown. However, the existence of oneN-linked carbohydrate complex on the murine sFceRII (37)suggests that the cleavage site is between amino acids 67 and114, the two positions found that could potentially haveN-linked carbohydrate attached.The expression data indicated that the transiently infected

COS cells bound both anti-FcRII (B3B4) and rodent (mouseand rat) IgE. No inhibition of mouse IgE binding by mouseIgG or human IgE was obtained, indicating that the FceRIIexpressed on COS cells had the same specificity as wasreported for the murine B-cell FceRII (38). The immuno-precipitation results show that the Fc6RII expressed in COScells is slightly lower in molecular mass than the FcRII fromhybridoma cells (45 vs. 49 kDa), indicating that carbohydrateprocessing is different in the COS cells and/or only one of theN-linked sites is used. Analysis of the sFceRII (Fig. 3C)

MURINE 117

HUMAN 11511 11 11

MURINE 233

BMURINE

MURINE

MURINE

MURINE

HUMAN EXON 5

HUMAN EXON 6

HUMAN EXON 7

Vv

s

IsiT

L

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indicates that most likely the latter hypothesis is true. Themaximum fragment size (35 kDa) is essentially the same aswas seen with the FceRII from tunicamycin-treated hybrid-oma cells (37), suggesting that the N-linked site at position114 is not used by the COS cells. In addition, the Fc6RIIexpressed in COS cells is somewhat more susceptible tobreakdown than is the hybridoma FcRII, especially whenthe sFcRII is examined. (Fig. 3C, lanes 3 vs. 4). Previousstudies using tunicamycin showed that the complete absenceof N-linked carbohydrate makes the Fc6RII much moresusceptible to degradation at the cell surface (37). Thus,overall, the results are compatible with our hypothesis (37)that N-linked carbohydrate, and specifically the N-linkedcarbohydrate at position 114, is important for Fc6RII stabil-ity. This observation may be very important with regard tothe biologic activity of the sFc6RII. Results in the humansystem indicate that only the complete sFcERII, and not itsfurther breakdown products, is biologically active with re-spect to IgE synthesis modulation (6). Since studies to datein the murine system have not demonstrated biologic activityof the sFcRII (11, 12), clearly, the conditions that result insFc8RII degradation are worth further analysis.The Northern blot analysis demonstrates that the full-

length mRNA is 2.4 kb; thus, the cDNA insert analyzed musthave been near full length. In addition, the expected [as seenin man (13)] increase in FceRII mRNA when IL-4 was presentwas observed. This is in good agreement with earlier studiesfrom this laboratory demonstrating an increased FcRIIsynthetic rate in the presence of IL-4 (5). No mRNA was seenwith Con A-activated normal T cells (data not shown) or witha TH2 cell line or with the one macrophage line examined.Mathur et al. (39) found that CD8' T cells from IgE plasma-cytoma bearing mice expressed FcRII, and later studiesdemonstrated (40) a 45-kDa protein that bound to polyclonal,but not monoclonal, anti-FcRII. These studies indicate thatthe T-cell FcRII is different; this can now be more preciselyanalyzed if T-cell FceRII mRNA is found. As stated earlier,the amino-terminal amino acid sequence corresponds to theFceRIIa form described in man (29). Restriction mapping ofthe other cDNA clones isolated did not give any evidence fora FceRIIb form in the mouse.The cloning of the murine Fc6RII will now allow more

detailed studies on the structure and function of this inter-esting protein. The species-specificity observed, that thehuman FceRII does not bind rodent IgE and conversely themurine Fc8RII does not bind human IgE, will allow the siteof IgE interaction to be more precisely determined usingcross hybrids between human and mouse FcRII sequences.In addition, the function of the FcRII in IgE synthesis andcell interactions can be further explored by transfection ofcell lines and transgenic technology.

We gratefully acknowledge J. L. Crespo for technical advice, Ch.Albisser for oligonucleotide synthesis, R. Van Dusen for technicalassistance, and S. Thakur for preparation of the manuscript.

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