1998 91: 2800-2809
Thomas, Ramón Vilella, Jo di Vi es and An oni Gayá
Miguel Angel de la Fuen e-Ga ci?a, Josep Ma ia Nicolás, John H. F eed, Edua d Palou, And ew P.
Lymphocy es
Hema opoie ic Lineages and Is In ol ed in Signal T ansduc ion on
CD148 Is a Memb ane P o ein Ty osine Phospha ase P esen in All
h p://bloodjou nal.hema ologylib a y.o g/con en /91/8/2800. ull.h ml
Upda ed in o ma ion and se ices can be ound a :
(3153 a icles)Hema opoiesis and S em Cells A icles on simila opics can be ound in he ollowing Blood collec ions
h p://bloodjou nal.hema ologylib a y.o g/si e/misc/ igh s.xh ml# epub_ eques s
In o ma ion abou ep oducing his a icle in pa s o in i s en i e y may be ound online a :
h p://bloodjou nal.hema ologylib a y.o g/si e/misc/ igh s.xh ml# ep in s
In o ma ion abou o de ing ep in s may be ound online a :
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In o ma ion abou subsc ip ions and ASH membe ship may be ound online a :
Copy igh 2011 by The Ame ican Socie y o Hema ology; all igh s ese ed.
20036.
he Ame ican Socie y o Hema ology, 2021 L S , NW, Sui e 900, Washing on DC
Blood (p in ISSN 0006-4971, online ISSN 1528-0020), is published weekly by
Fo pe sonal use only. a Ha a d Lib a ies on Augus 27, 2013. bloodjou nal.hema ologylib a y.o gF om
CD148 Is a Memb ane P o ein Ty osine Phospha ase P esen in All Hema opoie ic
Lineages and Is In ol ed in Signal T ansduc ion on Lymphocy es
By Miguel Angel de la Fuen e-Ga cı´a, Josep Ma ia Nicola´s, John H. F eed, Edua d Palou, And ew P. Thomas,
Ramo´n Vilella, Jo di Vi es, and An oni Gaya´
E idence is p esen ed showing ha a p o ein y osine phos-
pha ase di e en om CD45 is p esen on he memb ane o
human hema opoie ic cells. The molecule ecognized by he
monoclonal an ibody 143-41, which has been classi ied as
CD148 in he VI In e na ional Wo kshop on Leukocy e Di e -
en ia ion An igens, was immunopu i ied and sequenced.
Thesequenceob ained omN- e minusaswellas om wo
di e en CNB -diges ed pep ides showed a close iden i y
wi h a p e iously desc ibed y osine phospha ase named
HPTP-h/DEP-1. CD148 is p esen on all hema opoie ic lin-
eages, being exp essed wi h highe in ensi y on g anulo-
cy es han on monocy es and lymphocy es. In e es ingly,
whe eas i is clea ly p esen on pe iphe al blood lympho-
cy es, i is poo ly exp essed on di e en lymphoid cell lines
o T and B o igin. When his p o ein y osine phospha ase
was coc osslinked wi h CD3, an inhibi ion o he no mally
obse edcalciummobiliza ionwas obse ed.Thisinhibi ion
co ela es wi h a dec ease in phospholipase C-g(PLC-g)
phospho yla ion and is simila o he one obse ed wi h
CD45. In addi ion, i is shown ha he c osslinking o he
CD148 alone is also able o induce an inc ease in [Ca21]i. This
inc ease is abolished in he p esence o genis ein and by
coc osslinking wi h CD45. These da a, oge he wi h he
induc ion o y osine phospho yla ion on se e al subs a es,
including PLC-g, a e CD148 c osslinking, sugges he in-
ol emen o a y osine kinase-based signaling pa hway in
his p ocess. In conclusion, he da a p esen ed show ha
CD148 co esponds o a p e iously desc ibed p o ein y o-
sine phospha ase HPTP-h/DEP-1 and ha his molecule is
in ol edin signal ansduc ion in lymphocy es.
1998by The Ame icanSocie y o Hema ology.
THE PHOSPHORYLATION o y osine esidues o p o eins
is a c ucial e en in he egula ion o cellula p ocesses,
including hose o p oli e a ion and di e en ia ion. The le el o
p o ein phospho yla ion is mainly he esul o he an agonis ic
unc ions o p o ein- y osine kinases (PTKs) and p o ein-
y osine phospha ases (PTPs).1,2 Thus, he ac i a ion and inac i-
a ion o bo h enzymes a e ele an in de e mining he unc-
ional s a e o a g ea a ie y o in acellula molecules.
In ecen yea s an inc easing numbe o PTPs ha e been
desc ibed. Cu en ly o e 40 PTPs ha e been epo ed.3They
ha e been subca ego ized4in o h ee g oups: (1) ecep o -like
PTPs, (2) in acellula PTPs, and (3) dual speci ic PTPs. The
common s uc u al ea u es o he ecep o -like PTPs include an
ex acellula domain o a iable leng h and composi ion, a
single memb ane-spanning egion, and one o wo in acellula
ca aly ic domains.
The in e ac ion o T cell ecep o (TCR) wi h he app op ia e
an igen o i s s imula ion wi h an i ecep o an ibodies induces a
signal ansduc ion cascade ha leads o he exp ession o a
numbe o genes and e en ually o e ec o unc ions. One o he
ea lies biological e en s a e lymphocy e s imula ion is he
ac i a ion o PTKs, which esul s in y osine phospho yla ion o
a ious cellula p o eins. Recen ly i has been obse ed ha he
apid, and gene ally ansien , y osine phospho yla ion e-
sponse is he esul o a complex and s ill poo ly unde s ood
kinase cascade in ol ing a leas h ee amilies o PTKs: s c,
syk, and csk (see Zenne e al5 o e iew).Al hough leukocy es
exp ess a wide a ie y o PTPs in he cy oplasm, hei p ecise
ole emains unknown in mos o he cases.6Among he PTPs,
CD45 is he only memb ane PTP ha has been desc ibed o be
in ol ed in he p ocess o signal ansduc ion,7modula ing he
esponse o an igen ecep o engagemen in bo h T8and B
lymphocy es.9
In his epo we p esen e idence ha he ecen ly desc ibed
CD14810 co esponds o a p e iously desc ibed memb ane PTP,
HPTPh/DEP-1.11-13 This molecule is p esen on all hema opoi-
e ic lineages and, in addi ion o being able o ansduce signals
by i sel , i is also able o modula e he signal ansduc ion
h ough he TCR/CD3 complex in a manne simila o CD45.
MATERIALS AND METHODS
Cells. Blood samples we e ob ained om heal hy adul dono s.
Pe iphe al blood mononuclea cells (PBMCs) we e isola ed by cen i u-
ga iono e Ficoll-Hypaque (Pha macia LKB,Uppsala, Sweden) densi y-
g adien sedimen a ion. The ollowing cell lines we e g own in RPMI
plus 10% e al cal se um (FCS): CEM, HPB-ALL, HSB2, JURKAT,
MOLT-4, RAJI, KM3, NAMALWA, RAMOS, NALM-6, K562, U937,
and HL-60.
Monoclonal an ibodies (MoAbs). The ollowing MoAbs we e p o-
duced in ou labo a o y and asc ibed o hei CDs h ough one o he
In e na ional Wo kshop on Human Leukocy e Di e en ia ion An igens
(WLDA): CRIS-7 (CD3, IgG2a), 72-5D3 (CD45, IgG2a), and 111-5A1
(CD41, IgG1). MoAbs we e pu i ied om asci ic luid by p o ein A
a ini y ch oma og aphy. The 143-41 (IgG1) hyb idoma was p oduced
in acco dance wi h a p e iously desc ibed me hod14 a e immuniza ion
o BALB/c mice wi h PBMCs ha had p e iously been s imula ed wi h
phy ohemagglu inin (PHA) o 3 days. The ollowing phycoe y h in
(PE)-labeled MoAbs we e used: HD-37 (CD19), Leu 4 (CD3), and
mouse IgG2a con ol (Bec on Dickinson, San Jose, CA).
Immuno luo escence assay. The 143-41 MoAb was labeled wi h
luo escein ollowing con en ional echniques. Cells we e washed wi h
immuno luo escence bu e (phospha e-bu e ed saline [PBS] con ain-
ing 0.02 mmol/L sodium azide and 1% bo ine se um albumin [BSA])
F om he Se ei d’Immunologia, Se ei de Medicina In e na, Hospi-
al Clı´nic, Ba celona, Spain; he Na ional Jewish Cen e o Immunol-
ogy and Respi a o y Medicine, Den e , CO; and he Depa men o
Pa hology, Ana omy and Cell Biology, Thomas Je e son Uni e si y,
Philadelphia, PA.
Submi ed Ap il 14, 1997; accep ed Decembe 9, 1997.
Suppo ed by G an No. 96/0788 om Fondo de In es igacio´n
Sani a ia. M.A.d.l.F.-G. is a ecipien o a p edoc o al ellowship om
Hospi al Clı´nic i P o incial.
Add ess ep in eques s o An oni Gaya`, MD, Se ei d’Immunologia,
Hospi al Clı´nic, Villa oel 170, Ba celona 08036, Spain.
The publica ion cos s o his a icle we e de ayed in pa by page
cha ge paymen . This a icle mus he e o e be he eby ma ked ‘‘ad e -
isemen ’’ in acco dance wi h 18 U.S.C. sec ion 1734 solely o indica e
his ac .
1998 by The Ame ican Socie y o Hema ology.
0006-4971/98/9108-0021$3.00/0
2800
Blood,
Vol 91, No 8 (Ap il 15), 1998: pp 2800-2809
Fo pe sonal use only. a Ha a d Lib a ies on Augus 27, 2013. bloodjou nal.hema ologylib a y.o gF om
and incuba ed wi h speci ic MoAb o iso ype-ma ched con ol MoAb
o 30 minu es on ice in immuno luo escence (IF) bu e con aining 5%
abbi se um. Fo wo-colo analysis he simul aneous combina ion o
143-41 luo escein iso hiocyana e (FITC)-conjuga ed MoAb wi h a
PE-conjuga ed MoAb, was used. Samples we e un on a FACScan low
cy ome e (Bec on Dickinson). Whe e applicable, di e en cell popula-
ions (eg, lymphocy es, monocy es, and neu ophils) we e iden i ied
based on 2-dimensional ligh sca e cha ac e is ics.
Su ace bio inyla ion, immunop ecipi a ion, speci ic glycosidases
ea men , and immunoblo ing. Adul human pe iphe al blood mono-
nuclea cells we e isola ed om heal hy dono s by cen i uga ion o e
Ficoll-Hypaque. Cells we e p epa ed o su ace bio inyla ion by
washing wice in PBS and esuspending a 5 3107cells/mL in PBS
con aining 200 µg/mLSul o-NHS-Bio in (Pie ce, Rock o d, IL). Label-
ing was allowed o p oceed o 30 minu es a 4°C and was quenched by
incuba ion o 15 minu es a oom empe a u e wi h an equal olume o
RPMI 1640 medium supplemen ed wi h 10% FCS. Cells we e hen
washed h ee imes in cold PBS and lysed. A e 20 minu es on ice,
pos nuclea ex ac s we e added o CNB -ac i a ed Sepha ose 4B
(Pha macia LKB) ha had been p e iously coupled o 143-41 an ibody
and blocked wi h 2% BSA (Sigma Chemical Co, S Louis, MO). A e
60 minu es a 4°C, immunop ecipi a es we e washed i e imes in lysis
bu e (0.5% Nonide P-40, 10 mmol/LT is-HCL, pH 7.40, 150 mmol/L
NaCl, 1 mmol/L EDTA, 1 mmol/L EGTA, 1 mmol/L NaF, 20 mg/mL
egg whi e ypsin inhibi o , 1 mg/mL leupep in, 1 mg/mL peps a in, 1
IU/mLap o inin, and 1 mmol/Lphenylme hyl sul onyl luo ide [PMSF])
con aining 0.05% sodium dodecyl sul a e (SDS). Fo deglycosyla ion,
samples o immunop ecipi a es we e washed and, a e boiling, esus-
pended in he co esponding bu e s p epa ed acco ding o manu ac u -
e ’s ins uc ions o Neu aminidase (150 mU/50 mL; Boeh inge -
Mannheim, Mannheim, Ge many), O-Glycosidase (1.5 mU/50 mL,
Boeh inge -Mannheim) and ecombinan N-glycosidase F (0.7 mU/100
mL, Boeh inge -Mannheim). O e nigh diges ions a 37°C we e usually
used; howe e , o Neu aminidase ea men a sho e diges ion ime (2
hou s) was used. The glycosidase- ea ed p o eins we e un on 5%
sodium dodecyl sul a e-polyac ylamide gel elec opho esis (SDS-
PAGE) and ans e ed o Immobilon-P (Millipo e Co p, Bed o d, MA)
in 39 mmol/L glycine, 48 mmol/L T is-base, 1.3 mmol/L SDS, and 5%
me hanol o 2 hou s a 60 V. A e incuba ing he il e s wi h blocking
solu ion (10% non a milk p o ein in PBS) o 2 hou s a 4°C, blo s we e
incuba ed wi h s ep a idin-pe oxidase (Sigma Chemical Co) a 1:2,000
in blocking solu ion o 1 hou a 20°C. Fil e s we e washed again wi h
0.1% Tween-20 in PBS, and he Wes e n blo s we e de eloped using
subs a e solu ion (0.6 mg/mL diaminobenzidine, 0.1% hyd ogen
pe oxide (30%), and 0.3% (w / ol) CoCl2).
Immunoa ıni y pu i ica ion and p o ein sequencing. The molecule
ecognized by 143-41 MoAb was pu i ied ollowing p e iously de-
sc ibed p ocedu es15 wi h modi ica ions. Bu y coa s om no mal
heal hy dono s we e ob ained and e y h ocy es sepa a ed by sedimen a-
ion in PBS-2% Dex an-500 (Pha macia LKB). Leukocy e- ich supe -
na an s we e cen i uged, and abou 20 3109whi e blood cells we e
ob ained and washed wice in PBS. Pelle s we e dis up ed in lysis bu e
o 30 minu es on ice. Insoluble ma e ial was emo ed by cen i uga ion
a 50,000g o 30 minu es a 4°C and he supe na an was p eclea ed by
passing i sequen ially h ough columns o CNB -ac i a ed Sepha-
ose-4B beads alone and CNB -ac i a ed Sepha ose 4B beads coupled
wi h polyclonal mouse Ig o i ele an IgG1 MoAb. The esul ing lysa e
was hen applied o a column o CNB -ac i a ed Sepha ose 4B beads
(15 mL) de i a ized wi h MoAb 143.41. The a ini y column was
washed ex ensi ely wi h modi ied lysis bu e con aining 0.05% NP40
and hen wi h wo column olumes o PBS 0.5 mol/LMgCl2. The bound
ma e ial was elu ed wi h 4 mol/L MgCl2in PBS and he sample
concen a ed o 150 µL using a Cen ip ep 30 memb ane (AMICON,
Be e ly, MA) while he bu e was changed o PBS. Cyanogen b omide
clea age o he p o ein was pe o med acco ding o a p e iously
desc ibed p o ocol15: 400 mg o p o ein we e esol ed wi h SDS-PAGE
(5%) and elec oblo ed on o ni ocellulose memb ane. A e ans e , a
band isualized by Ponceau S (Sigma Chemical Co), was excised,
ans e ed o a sc ewcap mic o ial, and incuba ed in 300 µL o 0.150
mol/L CNB (Pie ce Chemical Co) in 70% o mic acid ( ol/ ol) (E.
Me ck, Dams ad , Ge many) o 4 hou s in he da k, a oom empe a-
u e.A e clea age, memb ane agmen was d ied comple ely wi h N2
and washed wi h 200 µL wa e and d ied again. The p o ein agmen s
we e edissol ed in Laemmli’s sample bu e con aining 5% me cap o-
e hanol, and he SDS-PAGE o he sepa a ion o he pep ides was 20%
ac ylamide (200:1, ac ylamyde:bis), 10% ( ol/ ol) glyce ol, 0.75 mol/L
T is pH 9.3, and 0.1% SDS. The gel was aged 2 days, and he unning
bu e con ained 0.1 mmol/L hioglycola e. A e he un, he p o eins
we e blo ed on o poly inylidine di luo ide memb ane (P oBlo ; Ap-
plied Biosys ems, Inc, Fos e Ci y, CA) wi h ans e bu e (48 mmol/L
T is pH 9, 39 mmol/L T icine, 1.3 mmol/L SDS, and 20% me hanol).
SDS-PAGE molecula weigh s anda ds (Bio-Rad Labo a o ies, Rich-
mond, CA) we e used. The memb ane was s ained wi h AmidoBlack
(0.1% in 40% me hanol, 1% ace ic acid), washed ho oughly in H2O,
and d ied. Two bands we e cu ou and s o ed in mic o ials illed wi h
N2.NH
2
- e minal sequence analysis o he in ac p o ein and he wo
p o ein agmen s was pe o med on an Applied Biosys ems 470A/
120Amic osequence .
Phospha ase assay. Subs a e p epa a ion and PTP assay we e
pe o med wi h he Malachi e G een Phospha ase Assay (Ups a e
Bio echnology Inc, Lake Placid, NY) as desc ibed in he p oduc
manual.A ini y pu i ied 143-41 and CD50 molecules (kindly p o ided
by D C. Vila dell, H. Clinic, Ba celona, Spain) we e dilu ed in assay
bu e (10 mmol/L T is-HCl, pH 7.4) and added o mic o i e wells wi h o
wi hou he subs a e phosphopep ide (2 mmol/L, TSTEPQpYQPGENL)
allowing enzyme eac ion o p oceed o 30 minu es. One hund ed
mic oli e s pe well Malachi e G een solu ion was added, and a e
incuba ion o 15 minu es he abso bance a 620 nm was de e mined
wi h a Ti e ek Mul iscan enzyme-linked immunoso ben assay eade
(Flow Labo a o ies, Rock ille, MD). The assay was pe o med in he
p esence o absence o 10-mmol/L sodium o ho anada e (Sigma
Chemical Co).
T ans ec ion o COS cells. A ull-leng h cDNA(hp21) encoding he
human p o ein- y osine phospha ase h(HPTP-h)12 was he kind gi o
D s H. Honda and H. Hi ai, Facul y o Medicine, Uni e si y o Tokyo,
Japan. To examine whe he MoAb 143-41 ecognizes HPTP-hgene
p oduc , COS-7 cells we e ans ec ed by lipo ec ion. B ie ly, 1 3106
cells in log phase we e washed wice wi h PBS and incuba ed in
se um- ee Dulbecco’s modi ied Eagle’s medium con aining 30 µg/mL
DOTAP (N-[1-(2,3-Dioleoyloxy)p opyl]-N,n,n- ime hylammonium
me hylsul a e; Boeh inge Mannheim GmbH, Mannheim, Ge many)
and 5 µg o he pSSRa exp ession ec o wi h hp21 inse , o 5 µg o he
pSSRa plasmid alone (mock ans ec ion) a 37°C o 6 hou s. A e 2
days, cells we e s ained wi h 143-41 MoAb and FITC-labeled goa
an imouse Ig and analyzed on a FACScan low cy ome e (Bec on
Dickinson).
Analysis o [Ca2
1
]i.[Ca21]iwas measu ed in indi idual lympho-
cy es basically ollowing he me hod desc ibed by Wachol z and
Lipsky.16 B ie ly, pe iphe al blood lymphocy es (PBL) we e esus-
pended a a inal concen a ion o 30 3106cells/mL in RPMI
supplemen ed wi h 10% FCS. Cells we e loaded wi h u a-2 ace oxy-
me hyl es e ( u a-2/AM; 2 mmol/L; Calbiochem, San Diego, CA) by
incuba ion o 25 minu es a 37°C, wi h gen le shaking. A e u a-2
loading, lymphocy es we e incuba ed wi h MoAbs (CRIS-7: 10 mg/mL
and 72-5D3, 143-41,111-5A1: 40 mg/mL) o 30 minu es a 4°C. A e
washing, u a-2-loaded PBL (106cells) we e pla ed on he cen e o a
25-mm glass co e slip coa ed wi h Cell-Tak (Collabo a i e Biomedical
P oduc s, Bed o d, MA) in 50 mL o RPMI medium wi hou FCS. The
cells we e incuba ed o 20 minu es a 37°C unde an a mosphe e o 5%
CO2/ai , and washed wi h incuba ion bu e composed o 121 mmol/L
CD148 INDUCES [CA21]iINCREASE ON LYMPHOCYTES 2801
Fo pe sonal use only. a Ha a d Lib a ies on Augus 27, 2013. bloodjou nal.hema ologylib a y.o gF om
NaCl, 10 mmol/L HEPES, 5 mmol/L NaHCO3, 4.7 mmol/L KCl, 1.2
mmol/L KH2PO4, 1.2 mmol/L MgSO4, 2 mmol/L CaCl2, 10 mmol/L
glucose, and 0.01% BSAa pH 7.4 o emo e una ached and non iable
cells. Co e slips wi h a ached lymphocy es we e ans e ed in o an
open low chambe (1 mL, olume) moun ed on he hea ed s age o a
Nikon Diapho -300 in e ed epi luo escence mic oscope. The s age, 40
3 luo imme sion objec i e (Nikon) and chambe we e main ained a
37°C. Ca21mobiliza ion was induced by he c osslinking o cell su ace
molecules a e addi ion o a second s ep sa u a ing amoun o
polyclonal a ini y pu i ied goa -an imouse an ise um (GAM; Tago Inc,
Bu lingame, CA). Cells we e conside ed o espond when [Ca21]i
inc eased mo e han 100% o he basal le el. Fluo escence images we e
ob ained by a CCD CH250 came a (Pho ome ics, Tucson, AZ) and
we e digi ized, s o ed, and analyzed in an Apple-MacIn osh 840AV
compu e (Apple Compu e s Inc, Cupe ino, CA). Images we e col-
lec ed al e na ely a exci a ion wa eleng hs o 340 and 380 nm (10 nm
bandwid h il e s) o exci e he Ca21-bound and Ca21- ee o ms o his
a iome ic dye, espec i ely. The emission wa eleng h was 510 nm
(120 nm bandwid h il e ). The in eg a ion ime o each image was 100
ms, and indi idual pixels we e binned in o 2 32 supe pixels a ead ou
om he cha ge coupled de ice de ec o o imp o e signal o noise. To
minimize pho obleaching, a compu e -con olled shu e was used o
limi he exposu e o he cells o exci a ion ligh . [Ca21]i alues we e
calcula ed on a single-cell basis om he 340- o 380-nm luo escence
a ios a each ime poin as desc ibed p e iously.17,18 All images we e
checked o mo emen a i ac s, and a e e ence poin was used o
ob ain ue co egis a ion o he 340- and 380-nm images. A he end o
each expe imen , cells we e exposed o ionomicyn (10 mmol/L) and
MnCl2 o 20 minu es. This ea men quenches he luo escence o he
in acellula Ca21-sensi i e u a-2, lea ing he esidual luo escence a
each wa eleng h because o cell au o luo escence and any Ca21-
insensi i e o ms o he dye. The esidual luo escence was measu ed
o e he same egion o each cell as he Ca21-dependen luo escence.
S a is ics. S anda d s a is ical me hods om SPSS S a is ical Analy-
sis Sys em V4.01(SPSS, Chicago, IL) we e used. Pai ed wo- ailed
- es s we e used o analyze he di e ences be ween condi ions in each
expe imen . All a iables we e exp essed as mean 6s anda d e o
(SE), and a signi icance le el o P,.05 was used.
Cell s imula ion and y osine phospho yla ion analysis. Cells (50 3
106) we e incuba ed wi h he di e en MoAbs (10 µg) o 15 minu es a
4°C ollowed by he addi ion o c osslinking abbi an imouse Igs (5
µg). Incuba ion was e mina ed a e di e en pe iods o ime by he
addi ion o 1 mL o ice-cold s op bu e (50 mmol/L HEPES, 150
mmol/L NaCl, 100 mmol/L NaF, 10 mmol/L EDTA, 10 mmol/L
Na4P2O7, 2 mmol/L sodium pe anada e, 2 mmol/L PMSF, 10 mg/mL
ap o inin, 10 mg/mL peps a in, 1 mg/mL leupep in, 100 mmol/L PAO).
Cells we e pelle ed and lysed wi h s op bu e con aining 1% NP-40.
P o eins we e sepa a ed by SDS-PAGE and ans e ed o ni ocellulose
memb anes. Phospho y osine-con aining p o eins we e p obed wi h
an iphospho y osine MoAb PY-20 om San a C uz Bio echnology
(San a C uz, CA) and ho se adish pe oxidase-conjuga ed abbi an i-
mouse and isualized by luo og aphy wi h enhanced chemilumines-
cence eagen (Ame sham, Buckinghamshi e, UK).
Immunop ecipi a ion o phospholipase C-
g
1. Fo immunop ecipi a-
ion expe imen s, cell lysa es o 25 3105s imula ed cells we e p epa ed
as desc ibed p e iously. P eclea ed lysa es we e incuba ed o e nigh
wi h 1 µg o an i–PLC-g1 MoAb (UBI Inc, Lake Placid, NY).
Immunop ecipi a es we e eco e ed by incuba ion wi h 20 µLo P o ein
A-Sepha ose beads o 120 minu es a 4°C and washed h ee imes in
lysis bu e . The p o eins we e hen elu ed and dissol ed by boiling o 5
minu es in Laemmli sample bu e and subsequen ly esol ed by
SDS-PAGE. Wes e n blo analysis was hen pe o med using PY20
an iphospho y osine an ibody (UBI Inc), bio inyla ed goa an imouse
(Sigma Chemical Co), and a idin-pe oxidase (Sigma Chemical Co) as
desc ibed abo e. Nex , memb anes we e s ipped o p ima y an ibody
wi h s ipping bu e (100 mmol/L 2-ME, 2% SDS, T is-Cl 65 mmol/L,
pH 57.5) washed and ep obed wi h an i–PLC-g1 MoAb (UBI Inc).
RESULTS
Pheno ypic and immunochemical cha ac e iza ion o CD148.
Wi h he aim o p oducing MoAbs de ining new memb ane
p o eins, se e al MoAbs we e ob ained in ou labo a o y. One
o hem, MoAb 143-41, de ined a molecule p esen on pe iph-
e al blood cells ha shows i s highes exp ession on g anulo-
cy es, being p esen a in e media e in ensi y on monocy es and
lymphocy es (Fig 1A, le ). I s exp ession on ed blood cells and
pla ele s was e en lowe han he one obse ed on hose cells
(Fig 1A, igh ). This molecule was de ec ed on bo h T and B
lymphocy es as de e mined by double immuno luo escence
wi h FITC-labeled 143-41 and PE-labeled CD3 and CD19 (Fig
1B). The eac ion o 143-41 MoAb wi h di e en hema opoie ic
cell lines was also es ed (Fig 2). Thus, CD148 showed a clea
eac ion wi h cell lines o myeloid o igin (K562, U937, and
HL-60) and wi h some B-cell lines (Raji, KM3, Nalm-6),
whe eas his was weakly exp essed on Namalwa and Ramos,
also o B-cell o igin. Su p isingly, and in con as wi h he clea
exp ession obse ed on CD31lymphocy es, i was no de ec ed
Fig 1. Flow cy ome ic analysis o human hema opoie ic cell
lineages. Whole blood was s ained wi h luo esceina ed-CD148 as
desc ibed in Ma e ials and Me hods and analyzed on a FACScan low
cy ome e . (A) E y h ocy es and pla ele s we e analyzed a e selec -
ing cell popula ions by side sca e and o wa d size. In pa allel, a e
washing, e y h ocy es we e lysed by incuba ing wi h lysis bu e and
he di e en leukocy e popula ions we e selec ed on basis o cell
sca e and o wa d size cha ac e is ics. His og ams o luo escence
o simul aneously s ained lymphocy es, monocy es, and g anulo-
cy es ha e been supe imposed. (B) PBL we e ob ained om no mal
heal hydono sbyFicoll-Hypaqueg adien densi ycen i uga ionand
lymphocy e popula ions we e analyzed by wo colo luo escence by
using luo esceina ed 143-41 MoAb and come cially a ailable PE-
labeled CD3 and CD19 MoAb. The app opia e nega i e con ol FITC-
and PE-labeled MoAbs we e used o es ablish he ma ke posi ion.
The s aining in ensi y o PE-labeled cells is shown in he e ical axis
wi h143-41–FITC s aining on he ho izon alaxis.
2802 DE LA FUENTE-GARCI
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Fo pe sonal use only. a Ha a d Lib a ies on Augus 27, 2013. bloodjou nal.hema ologylib a y.o gF om
on T-cell lines (CEM, HPB-ALL, Ju ka , and MOLT-4) wi h he
excep ion o HSB-2.
Al hough his MoAb was analyzed du ing he IV WLDA i
could no be clus e ed. Du ing he wo k o he VI WLDA held
ecen ly in Kobe, ano he MoAb (A3) wi h a simila pa e n o
eac ion was de ec ed. In basis o he compa a i e analysis o
his an ibody wi h ou MoAb 143-41, a new clus e o
di e en ia ion could be de ined: CD148.10
To u he cha ac e ize his molecule a ho oughly biochemi-
cal cha ac e iza ion was pe o med. The molecule was immuno-
p ecipi a ed, and be o e and a e diges ion wi h neu aminidase,
O-glycanase, and N-glycanase i was subjec ed o elec opho e-
sis in educing condi ions and analyzed by immunoblo ing, as
desc ibed in Ma e ials and Me hods. As can be obse ed in Fig
3, CD148 appea ed be o e ea men as a b oad band wi h an
appa en molecula weigh o 240 kD. T ea men o pu i ied
CD148 wi h he a ious glycosidases led o al e a ions in he
elec opho e ic mobili y o he p o ein. Thus, a e ea men
wi h N-glycosidase F, he molecula weigh o CD148 was
shi ed o a much smalle size in Wes e n blo analysis,
indica ing ha he molecule ecognized by 143-41 was a
glycop o ein con aining Asn-linked ca bohyd a e and ha he
molecula weigh o his molecule was, o he mos pa ,
modi ied by he N-glycosyla ion. O-Glycanase ea men also
a ec ed he elec opho e ic mobili y o he CD148 by dec eas-
ing i s appa en molecula weigh unde educing condi ions. In
addi ion, a sligh dec ease in he elec opho e ic mobili y was
obse ed a e ea men wi h neu aminidase sugges ing he
exis ence o sialic acid esidues. The eac ion o CD148 wi h
Maackia amu ensis and Sambucus nig a L bio inyla ed lec ins,
con i med his p esence and showed ha hey a e linked in bo h
a(2-3) and a(2-6) o galac ose (da a no shown).
P o ein sequencing and iden i ica ion as HPTP-
h
/DEP-1.
CD148 was pu i ied by immunoa ini y ch oma og aphy om
leukocy e memb anes and subjec ed o N- e minal sequencing.
A e diges ion o CD148 wi h CNB , se e al pep ides we e
ob ained, wo o hem being subjec ed o N- e minal sequenc-
ing. The sequences ob ained a e shown in Fig 4. A comple e
homology o hese sequences wi h he ecen ly desc ibed PTP
HPTP-h12 o DEP-113 was de ec ed. As can be seen in Fig 4, in
all he posi ions in which a clea sequence was ob ained, an
iden i y o sequence was obse ed. In bo h CNB -de i ed
pep ides i was obse ed ha he sequence s a ed a e a
me hionine esidue in he sequence o he PTP, in ag eemen
Fig 2. Exp ession pa e ns o CD148 on di e en cell lines o
myeloidand lymphoid o igin.The 143-41 MoAbwas assayed o hei
binding o cell lines by i s incuba ing he cells wi h a sa u a ing
amoun o an ibody and, a e washing, cells we e incuba ed wi h a
FITC-labeled goa an imouse an ibody (—). Cells we e also s ained
wi h a con ol iso ype-ma ched an ibody. The s aining in ensi y o
FITC-labeled cells is shown on he ho izon al axis wi h he numbe o
cellson he e ical axis.
Fig3. Immunochemicalcha ac e iza iono CD148molecule.Adul
human PBMCs we e su ace bio inyla ed and CD148 molecule was
immunop ecipi a edby using 143-41–coupled CNB -ac i a ed Sepha-
ose 4B. Sample aliquo s we e subjec ed o ea men wi h
N-endoglycosidase F (lane 3), O-endoglycosidase (lane 2), and neu -
aminidase (lane 1) as desc ibed in he Ma e ials and Me hods. Nex ,
samples we e analyzed on a 5% SDS-polyac ylamide gel unde
educing condi ions be o e (lane 4) and a e glycanase ea men
(lanes 1, 2, and 3), ollowed by elec opho e ic ans e o p o eins
on o Immobilon-P. A e blocking and incuba ing wi h s ep a idin-
pe oxidase, he Wes e n blo s we e de eloped using diaminobenzi-
dinewi h cobal enhancemen .
Fig 4. Iden i y be ween CD148 N e minal and pep ide sequence
and deduced HPTP-h/DEP-1 p o ein sequence om he cDNA. Num-
be ing o HPTP-h/DEP-1 amino acid posi ions a e om Honda e al12
and O
¨s man e al.13 Assignmen o X in he CD148 sequence ep e-
sen snoniden i iable signal in he sequenceanalysis.
CD148 INDUCES [CA21]iINCREASE ON LYMPHOCYTES 2803
Fo pe sonal use only. a Ha a d Lib a ies on Augus 27, 2013. bloodjou nal.hema ologylib a y.o gF om

wi h he exis ence o a poin o clea age o CNB . F om he
N- e minal sequence da a we ob ained, i should be no ed ha
he ma u e N e minus co esponds o Ala 36 and no o Th 39
(HPTP-h)12 o Gly 37 (DEP-1).13
To con i m ha he molecule ecognized by 143-41 MoAb
co esponds o he p e iously desc ibed p o ein y osine phos-
pha ase HPTP-h/DEP-1 wo di e en app oaches we e unde -
aken. Fi s , hp21, a ull leng h HPTP-hcDNA was used o
ans ec COS-7 cells. When s ained wi h 143-41 MoAb,
whe eas mock- ans ec ed COS-7 cells we e nega i e (Fig 5A),
he COS-7 cells ans ec ed wi h he HPTP-hcDNA showed a
clea posi i e eac ion (Fig 5B). In addi ion, he phospha ase
ac i i y o CD148 was analyzed by es ing i s abili y o elease
phospha e g oups om a y osine phospho yla ed pep ide. As
can be obse ed in Fig 6 a clea y osine phospha ase ac i i y
was obse ed when immunopu i ied CD148 was es ed. This
ac i i y was clea ly diminished in he p esence o sodium
o ho anada e, a PTP inhibi o . No PTP ac i i y was de ec ed
when a simila ly immunopu i ied molecule, CD50, was es ed
in he same assay.
Ca2
1
mobiliza ion a e CD3, CD148, and CD45 c osslink-
ing. P e ious s udies had shown ha CD3-induced Ca21
mobiliza ion is modula ed by he y osine phospha ase ac i i y
o CD45 when bo h molecules we e coc osslinked.7Based on
he ac ha CD148 was iden i ied as a PTP exp essed on he
memb ane o lymphocy es, we e alua ed whe he CD148 could
modi y he ac i a ion induced by CD3 c osslinking. The e o e,
PBL we e loaded wi h u a-2/AM, and Ca21mobiliza ion a e
cell s imula ion was analyzed by compu e -aided luo escence
imaging. Figu e 7 depic s he mean changes o [Ca21]iincluding
all PBLpopula ions a e c osslinking he CD3 molecule. These
da a a e no malized o he basal alues be o e he addi ion o
a ini y pu i ied GAM, and Ca21changes we e measu ed in
indi idual cells. Mean [Ca21]iinc eased om a baseline o
71 64 nmol/L o 163 615 nmol/L a e addi ion o he CD3
c osslinking agen . In ag eemen wi h p e ious esul s,7we
obse ed a signi ican dec ease in he CD3-induced esponse
because o CD45 coc osslinking. Mo eo e , when CD3 was
coc osslinked wi h CD148, we also obse ed a clea educ ion
in he mean peak [Ca21]i esponse (P,.05; n 55 expe imen s;
Fig 7), sugges ing ha he PTP ac i i y o CD148 could
modula e signals ansduced h ough he CD3 complex, simi-
la ly o CD45. In e es ingly enough, when CD148 alone was
c osslinked, we obse ed an inc ease in he mean [Ca21]i,
eaching a mean peak o 130 68 nmol/L, which was delayed
and signi ican ly lowe (P,.05, n 56 in bo h expe imen s; Fig
7) when compa ed wi h CD3 c osslinking s imula ion. In
addi ion, o de e mine he ole o y osine phospho yla ion in
[Ca21]iinc ease induced by CD148 s imula ion we es ed
whe he he phospha ase ac i i y o CD45 could abolish his
e ec when bo h molecules we e coc osslinked, as has been
desc ibed in o he y osine kinase-media ed ac i a ions.19 As
can be obse ed in Fig 8, he [Ca21]iinc ease induced by
CD148 c osslinking was inhibi ed when CD45 was co-
c osslinked wi h CD148.
Because he mean [Ca21]ikine ics may be in luenced ei he
by he pe cen age o esponding cells, appea ance o unsynch o-
nized esponses, o a iable in ensi y esponse,20 indi idual cell
measu emen s o he [Ca21]ichanges a e CD3, CD45, and
CD148 c osslinking we e also e alua ed (Fig 8). Fo he
analysis o he single cell esponses, we conside ed a esponse
signi ican when he Ca21mobiliza ion induced by c osslinking
was a leas wice he [Ca21]io he basal le el. In his sense,
an i-CD3 induced a apid and signi ican (wi hin 1 minu e)
[Ca21]iinc ease in 65% o cells, wi h hese cells exhibi ing a
mean peak [Ca21]io 414 610 nmol/L. This was ollowed by
smalle [Ca21]ioscilla ions, especially in hose cells wi h a
highe [Ca21]ipeak (poly opic esponse). The asynch onous
[Ca21]ioscilla ions obse ed a he single cell le el a e espon-
sible o he sus ained phase o he [Ca21]ichanges iden i ied in
cell suspension expe imen s. The educ ion in he mean [Ca21]i
esponse obse ed a e CD31CD45 coc osslinking was basi-
cally caused by a dec ease in he immedia e Ca21mobiliza ion,
a ec ing an a e age o 69% o he esponding cells. As may
also be obse ed in Fig 8, c osslinking o CD45 alone did no
induce any signi ican modi ica ion o he [Ca21]ile els. Wi h
espec o he inhibi o y e ec o CD148 on CD3-induced
Fig 5. Immuno luo escence analysis o mock- ans ec ed COS-7
cells(A)o COS-7cells ans ec edwi hHPTP-hcDNA(B). COS-7cells
we e ans ec ed wi h hp21 clone encoding HPTP-ho plasmid only
and s ained wi h 143-41–FITC as desc ibed in he Ma e ials and
Me hods.
Fig 6. PTP ac i i y o pu i ied CD148 molecule. A ini y pu i ied
CD148 molecule was incuba ed wi h a y osine phospho yla ed
syn e hic pep ide (TSTEPQpYQPGENL). The amoun o ee phos-
pha e eleased as ino ganic phospha e in he absence o p esence o
anada e was measu ed by he Malachi e G een Phospha ase Assay
(UBI) and i is shown as nmol/L concen a ion. A ini y pu i ied CD50
moleculewas used as a nega i econ ol.
2804 DE LA FUENTE-GARCI
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Fo pe sonal use only. a Ha a d Lib a ies on Augus 27, 2013. bloodjou nal.hema ologylib a y.o gF om
s imula ion, we obse ed a dec ease in he immedia e esponse
in 35% o he cells.
Indi idual esponses o CD148 c osslinking exhibi ed a
synch onized, mo e han wo old inc ease in [Ca21]iin 39% o
he cells (Fig 8). This inc ease, which was delayed (1 minu e) i
i was compa ed wi h he apid esponse obse ed a e CD3
c osslinking, was signi ican ly di e en om he le els ob-
se ed in he p esence o an iso ype ma ched con ol MoAb
(111-5A1, CD41; da a no shown). Likewise, no esponse was
obse ed a e adding GAM o he sample. The inhibi o y e ec
o CD45 coc osslinking was obse ed o a ec 80% o CD148
esponding cells, a ec ing especially he ea ly phase o he
esponse (Fig 8). These da a sugges ha he c osslinking o
CD148 could induce Ca21mobiliza ion h ough y osine phos-
pho yla ion p ocesses. To be e cha ac e ize his poin , he
esponse o CD148 c osslinking in he p esence o a known
p o ein y osine kinase inhibi o , genis ein, was analyzed. The
da a p esen ed in Fig 9Ashow ha genis ein clea ly inhibi s he
Ca21mobiliza ion induced by CD148 c oslinking. When indi-
idual cell measu emen s o he [Ca21]ichanges we e analyzed
a e CD148 c osslinking in he absence (Fig 9B) o p esence o
genis ein 75 µmol/L (Fig 9C) i was con i med ha he
inhibi ion obse ed in he p esence o genis ein was eal and no
caused by he induc ion o unsynch onized esponses. The
inhibi o y e ec o genis ein a ec ed 70% o CD148 espond-
ing cells, mainly du ing he ea ly phase o he esponse
(Fig 9C).
Induc ion o y osine phospho yla ion by CD148 c osslink-
ing. The esul s o he CD45 coc osslinking and genis ein
expe imen s led us o in es iga e whe he speci ic y osine
phospho yla ion e en s we e associa ed wi h an i-CD148–
igge ed Ca21mobiliza ion. Thus, cells p e iously coa ed wi h
CD3, CD148, o bo h we e lysed in SDS sample bu e a
a ious imes a e addi ion o abbi an imouse an ise um and
y osine-phospho yla ed p o eins we e de ec ed by immunoblo -
ing. As shown in Fig 10, and as i has been exhaus i ely
desc ibed, a apid inc ease in he phospho y osine con en o
se e al p o eins was obse ed a e CD3 c osslinking (Fig 10A,
lanes 2 and 3). In addi ion, phospho yla ion o se e al sub-
s a es a e CD148 c osslinking was also e iden (Fig 10A,
Fig 7. Calcium mobiliza ion induced a e CD3, CD148, and
CD31CD148 c osslinking. Pe iphe al blood lymphocy es we e loaded
wi h u a-2, and [Ca21]iwas measu ed as desc ibed in he Ma e ials
and Me hods. Cells we e incuba ed 30 minu es a 4°C wi h he
di e en MoAbs (CD3, 10 mg/mL; 143-41, 50 mg/mL; CD45, 50 mg/mL)
andadhe ed o Cell-Takcoa edco e slips. A e es ablishing baseline
alues, a sa u a ing amoun o GAM was added o p ewa med
samples (a ow). [Ca21]iwas measu ed e e y 5 seconds o 15
minu es on a single-cell basis in a compu e -aided luo escence
imaging. The a e age cu e o a minimum o 200 indi idual cells o
each condi ion o a leas ou independen expe imen s a e shown.
Calciumchangesa e exp essedas hepe cen age change om basal.
Fig 8. Fluo escence imaging o [Ca21]i esponses om indi idual
cellsa e CD3, CD148,and CD31CD148 c osslinking. Cells p e iously
loaded wi h u a-2 (2 mmol/L), we e incuba ed wi h he co espond-
ing an ibody (CD3, 10 mg/mL; 14341, 40 mg/mL; CD45, 40 mg/mL) o
30 minu es a 4°C. A e washing, cells we e pla ed on Cell-Tak–
coa edglassco e slips.A e es ablishingbaseline alues, sa u a ing
amoun o polyclonal GAMwas added op ewa med samples.[Ca21]i
was measu ed e e y 5 seconds o 15 minu es in e e y cell. Calcium
changesa e exp essed as he pe cen ilechange o basal. A o al o 20
cells ep esen a i eo eachcondi iona eshown.Eachline ep esen s
anindi idual cell.
CD148 INDUCES [CA21]iINCREASE ON LYMPHOCYTES 2805
Fo pe sonal use only. a Ha a d Lib a ies on Augus 27, 2013. bloodjou nal.hema ologylib a y.o gF om
lanes 4 and 5). These esul s a e in ag eemen wi h hose ha
ha e been p esen ed p e iously and sugges ha he Ca21
mobiliza ion induced by CD148 c osslinking could be media ed
by y osine phospho yla ion e en s.
On he o he hand, and aking in o accoun he y osine
phospha ase ac i i y o CD148, we we e in e es ed o know
whe he he CD148-induced inhibi ion o CD3-media ed Ca21
mobiliza ion was caused by speci ic dephospho yla ion e en (s).
The pa e n o p o ein y osine phospho yla ion a e ac i a ion
o human lymphocy es wi h CD3 c osslinking was modi ied
when CD148 was coc osslinked o his ecep o (Fig 10A, lanes
6 and 7). Thus, a selec i e inhibi ion o some subs a e was
de ec ed, which could be ela ed wi h he phospha ase ac i i y
o CD148. Taking in o accoun he p e iously es ablished
ela ionship be ween calcium mobiliza ion and y osine phos-
pho yla ion o phospholipase C g(PLC-g) we we e in e es ed
in analyzing he phospho yla ion s a us o his enzyme a e cell
s imula ion. Cells coa ed wi h CD3, CD148, o bo h we e
s imula ed wi h abbi an imouse Ig and a e cell lysis a
di e en imes, PLC-gwas immunop ecipi a ed. The immuno-
blo ing wi h an an iphospho y osine an ibody (Fig 10B) showed
ha , whe eas a e s imula ing wi h CD3 and CD148 an
inc ease in y osine phospho yla ion o PLC-gwas obse ed, a
clea dec ease was de ec ed when bo h CD3 and CD148 we e
coc osslinked. By ep obing wi h an an i–PLC-gi was ob-
se ed ha he same quan i y o PLC-gwas loaded on each
lane. These esul s a e in ag eemen wi h he da a p o ided by
Ca21mobiliza ion analysis.
In addi ion, i was obse ed ha co igge ing o CD3 wi h
CD148 esul ed in an inc ease in p o ein y osine phospho yla-
ion in some o he p o eins. This inc ease in he phospho yla ion
pa e n was pa icula ly e iden on a 56-kD subs a e.
DISCUSSION
Du ing ecen yea s he e has been an inc ease in he
desc ip ion o new PTPs,3al hough he majo i y o hese PTPs
ha e been mainly ela ed o ac i i ies o he cen al ne ous
sys em. In con as , al hough he numbe o PTKs in ol ed in
lymphocy e signal ansduc ion has also g own, his has no
been he case o he numbe o PTPs in ol ed in his p ocess.
In addi ion o he cy oplasmic PTPs in ol ed in lymphocy e
signaling, jus one memb ane PTP, CD45, is known o in luence
he signaling p ocess a e an igen ecep o engagemen (see
S euli21 o e iew). In his pape , we ha e p esen ed e idences
showing ha in addi ion o CD45, he e is ano he PTP on he
Fig 9. E ec o Genis ein on calcium mobiliza ion induced by
CD148 c osslinking. Pe iphe al blood lymphocy es we e loaded wi h
u a-2, and [Ca21]iwas measu ed as desc ibed in he Ma e ials and
Me hods. Cells we e incuba ed 30 minu es a 4°C wi h 50 mg/mL o
CD148 and adhe ed o Cell-Tak–coa ed co e slips. A e es ablishing
baseline alues, a sa u a ing amoun o GAM was added in he
absence o p esence 0 75 mmol/L Genis ein o p ewa med samples
(a ow). [Ca21]iwas measu ed e e y 5 seconds o 15 minu es on a
single-cell basis in a compu e -aided luo escence imaging. (A) The
a e age cu e o a minimum o 200 indi idual cells o each condi ion
o a leas ou independen expe imen sa eshown.Calciumchanges
a e exp essed as he pe cen age change om basal. Fluo escence
imaging o [Ca21]i esponses om 20 indi idual cells, a e CD148
c osslinking, in he absence (B) o he p esence (C) o genis ein (75
mmol/L).Each line ep esen s an indi idualcell.
Fig 10. P o ein y osine phospho yla ion induced by CD148
c osslinking.(A)Cellswe eincuba edwi hou (lane1)o wi h(lanes2
o 7) 10 mg o he di e en monoclonal an ibodies o 15 minu es a
4°C ollowed by he addi ion o c osslinking abbi an imouse Igs.
Incuba ion was e mina ed a e 1 minu e (lanes 2, 4, and 6) and 5
minu es (lanes 3, 5, and 7). P o eins we e esol ed by SDS-PAGE
ollowed by an iphospho y osine immunoblo ing. (B) PLC-g1 was
immunop ecipi a ed om cells s imula ed du ing 1 and 3 minu es
and analyzed by Wes e n blo wi h an an iphospho y osine an ibody
and,a e s ipping, wi h an an i–PLC-g1.
2806 DE LA FUENTE-GARCI
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Fo pe sonal use only. a Ha a d Lib a ies on Augus 27, 2013. bloodjou nal.hema ologylib a y.o gF om
lymphocy e memb ane ha could be able o modula e he
signaling p ocess a e CD3 c osslinking. Thus, we ha e
obse ed ha he coc osslinking o he molecule ecognized by
he MoAb 143-41, oge he wi h CD3 inhibi s he subsequen
[Ca21]iinc ease. Ou esul s show ha his molecule is a
memb ane p o ein y osine phospha ase iden ical o a ecen ly
desc ibed HPTPcalled HPTP-h12 o DEP-1.13 This phospha ase
is p esen on he memb ane o all he hema opoie ic cells and i
has been classi ied as CD148 du ing he las VI WLDA.10
Wi hin he lymphoid cells, i is p e e en ially exp essed on B
cells, memo y T lymphocy es, and ma u e hymocy es (A.
Gaya´, unpublished obse a ions). In con as , i s exp ession on
hema opoie ic cell lines is he e ogeneous. Thus, whe eas i is
clea ly exp essed on myeloid cell lines and on he majo i y o
B-cell lines es ed i is absen om he majo i y o T-cell lines
analyzed. Al hough his pa e n o dis ibu ion could seem
con adic o y he e a e o he molecules ha display a simila
pa e n o dis ibu ion. Thus, CD26, which is clea ly p esen on
all ma u e T lymphocy es is absen om T-cell lines wi h he
excep ion o HSB-2.22
We ha e p esen ed se e al pieces o e idence showing ha
CD148 is iden ical o HPTP-h12 and DEP-1.13 Fi s , he
sequences we ob ained by p o ein sequencing and he p e i-
ously published cDNAsequences o HPTP-h12 and DEP-113 a e
iden ical. These sequences include an N- e minal agmen o
18 amino acids as well as wo di e en CNB -de i ed pep ides
o 18 aa. We conside hose sequences as iden ical a all o he
esol ed posi ions, and he non esol ed posi ions can be
explained by echnical p oblems inhe en in he sequencing
p ocess. The mos in e es ing aspec o hese da a is he
de e mina ion ha he N- e minal esidue o he ma u e p o ein
co esponds o an Ala and no o he p e iously p oposed Th 12
o Gly.13 Second, when COS-7 cells we e ans ec ed wi h a
plasmid (hp21) con aining a HPTP-hencoding cDNAclone,12 a
clea eac ion wi h 143-41 MoAb was obse ed. Thi d, by using
an immunopu i ied p epa a ion o CD148 molecule, ob ained
om leukocy e memb anes, i was possible o de e mine i s
capaci y o elease ee phospha e g oups om a y osine
phospho yla ed pep ide, hus, con i ming i s y osine phospha-
ase ac i i y. The immunochemical cha ac e iza ion o CD148
molecule con i med ha his molecule con ains bo h O- and
N-linked ca bohyd a es. F om he analysis wi h glycosidases i
could be deduced ha he majo pa o ca bohyd a es a e
N-linked. This is in ag eemen wi h he 34 po en ial si es o
N-linked glycosila ion de e mined om he cDNAsequence10,11
and he da a p esen ed by Honda e al12 on N-glycosidase F
ea men o HPTP-h. Conce ning he appa en molecula
weigh o HPTP-h, di e ences ha e been desc ibed among
se e al cell lines11,12 a ying om 250 kD (HL60) o 230 kD
(F-36P) o 220 kD (F-36E). Taking in o accoun ha CD148
molecule was ob ained om a he e ogeneous popula ion o
cells, he b oad band a ound 240 kD we ha e obse ed bo h by
immunop ecipi a ion and immunoa ini y pu i ica ion could in-
clude all he o ms p e iously men ioned, sugges ing he
exis ence o a ce ain le el o he e ogenei y in he exp ession o
his phospha ase among se e al cellula lineages.
HPTP-ho DEP-1 is a ecen ly desc ibed ecep o PTP, he
ex acellula po ion o which is composed o 813 o 1012 FNIII
domains, whe eas he in acellula segmen con ains a single
PTP domain spanning amino acids 1060 o 1296. The e o e, i
joins an expanding g oup o such ecep o s classi ied as ype III
PTPs3 ha includes PTP-b,23 PTP-U2,24 GLEPP1,25 and SAP-
126 om humans and DPTP10D,27 DPTP99A,28 and DPTP4E29
om D osophila, wi h HPTP-hbeing he unique ype III PTP
exp essed on hema opoie ic cells. These enzymes a e cha ac e -
ized by a simila o ganiza ion o hei ex acellula segmen s,
which consis o a epea ed a ay o FNIII mo i s, and a single
in acy oplasmic phospha ase domain. The FNIII epea s, in
addi ion o being in ol ed in adhesion p ocesses,29 a e also
ound in he ex acellula egions o he ecep o s o in e leu-
kin-2 (IL-2), IL-4, IL-6, g anulocy e-mac ophage colony-
s imula ing ac o , p olac in, e y h opoie in, and g ow h ho -
mone.30 The DEP-1 molecule has been implica ed in con ac
inhibi ion o cell g ow h because i is up egula ed in dense cell
cul u es al hough i s up egula ion is ini ia ed be o e sa u a ion
densi y is eached. In addi ion, i has been desc ibed ha he
exp ession le el o HPTP-hwas al e ed when he HL-60 cells
we e exposed o di e en ia ing compounds such as dime hyl
sul oxide and 12-O- e a decanoyl pho bol 13-ace a e, sugges -
ing ha his gene migh be in ol ed in he di e en ia ing
p ocesses o g anulocy e o monocy e/mac ophage lineages in
hese cells.11
The CD45 molecule, he p incipal p o ein y osine phospha-
ase p esen on he memb ane o hema opoie ic cells, is capable
o egula ing signal ansduc ion and unc ional esponses,7-9
because in T lymphocy es, CD45 c osslinking inhibi s inosi ol
phospha e p oduc ion, calcium lux, and p oli e a ion.31 The
abili y o CD45 o modula e signals ansduced by CD3
co ela es wi h i s abili y o inhibi he y osine phospho yla ion
o some in acellula p o ein subs a es.31,32 Once we showed
ha he molecule ecognized by he 143-41 MoAb is a
memb ane p o ein y osine phospha ase, we we e in e es ed o
know whe he his molecule could in luence he signal ansduc-
ion h ough he an igen ecep o . The mos s iking ac o his
analysis was he obse a ion ha he c osslinking o CD148
alone was able o induce a clea inc ease in [Ca21]i. This
phenomenon was no obse ed a e CD45 c osslinking. The
kine ic o he p ocess was simila o he Ca21mobiliza ion
induced a e CD3 c osslinking, al hough he lag ime was
clea ly mo e p olonged in he case o CD148. Ano he di e -
ence was based on he in ensi y o he esponse. The CD148
c osslinking p oduced bo h a lowe le el o [Ca21]imobiliza-
ion and a lowe numbe o esponding cells (39% 69%). In
ac , CD148 c osslinking was able o dec ease he Ca21
mobiliza ion in 35% o he cells esponding o CD3 (69%), a
pe cen age simila o he pe cen age o cells esponding o
CD148 c osslinking (39%).
Conce ning he mechanisms in ol ed in his p ocess, we
conside i plausible ha y osine phospho yla ion could be
in ol ed because he coc osslinking wi h CD45 o he p esence
o genis ein we e able o abolish he esponse induced by
CD148. This assump ion was p o en o be co ec , because a e
CD148 c osslinking a clea pa e n o y osine phospho yla ion
could be obse ed. Especially in e es ing was he obse a ion
ha CD148 c osslinking was able o induce y osine phospho y-
la ion o PLC-g1. Al hough i may seem con adic o y ha a
p o ein y osine phospha ase is able o induce y osine phospho -
yla ion, a simila si ua ion has been desc ibed o he molecule
CD148 INDUCES [CA21]iINCREASE ON LYMPHOCYTES 2807
Fo pe sonal use only. a Ha a d Lib a ies on Augus 27, 2013. bloodjou nal.hema ologylib a y.o gF om