Modulatory signalling in immune cells:SLAM family receptors and
SAP-related adaptors
André Veillette414A
November 2004
TCR(T-cells)
Ig/Ig
BCR(B-cells)
FcRIII(NK cells)
FcRI(mast cells)
= ITAM
DAP-12
NCRs NKG2D
(NK cells)
Immunoreceptors
TCR signalling
Lck FynTSH2
SH2
Zap-70
CD4 TCR
SH2
SH2
Lck
Zap-70
CD4 TCR
FynT
P
PP
P
PP PP P
PP
Ag/MHC
MHC
protein tyrosine phosphorylation
PH
Itk
PH
Itk
PAG(ITIMs)
Csk-PEPSHP-1c-Cbl
TCR-CD4/CD8
Src kinasesZap-70
Btk kinases
T-cell signalling
Positivesignalling
Inhibitorysignalling
Cytokineproduction
(TH1 vs TH2)
AnergyMemoryMaturation Apoptosis
SAP
Modulatorysignalling
CD28
PI 3’ kinase
ICOS
?
SLAM
?
? ? ? ? ? ? ?
SH2 SAP(T-cells, NK cells, NK-T-cells, ?some B-cells)
SH2 EAT-2 (NK cells, macrophages, DCs, ?some B-cells)
SAP family of adaptors
X-linked lymphoproliferative disease (XLP)- caused by inactivating point mutations or deletions in the sap gene;- characterized by abnormal response to EBV
infection, resulting in fatal infectious mononucleosis, as well as a high frequencyof malignant lymphomas;
- SAP-deficient mice exhibit abnormal CD4+ T-cell help (decreased TH2 functions; +/- increased TH1 functions), decreased Ig production and memory B-cell generation, altered anti-viral responses and absent NK-T-cells.
CRACC
V
C2
CD84SLAM 2B4 NTB-A
Y
V
C2
Y
Y
Y
Y
V
Y
Y
Y
V
C2
Y
Ly-9
V
C2
YY
C2
V
YY
Y
YY
Y
Ligands
V
C2
Y
Y
CD84SLAMmeasles
CD48 NTB-A Ly-9 CRACC
Y
C2
Y : TIYxxV/I motif
Y : TVYxxV/I motif YY
Y
Y
Y
SAP family adaptors associatewith SLAM-related receptors
Expression patterns of SLAM-related receptors
SAP
SLAMCD84
2B4
NTB-A
Ly-9
TH2functionsNK cell
cytotoxicity
NK cellcytotoxicity
?
?
XLP is likely caused by multipleSLAM-related receptor dysfunctions
Ig productionmemory B-cells
?
How does SAP regulate SLAM-related receptors?
SLAM (CD150)- expressed on activated T-cells, B-cells,
macrophages and dendritic cells;- self-ligand;- SLAM is the lymphoid-specific receptor
for measles virus in humans;- anti-SLAM stimulation modulates IFN- secretion
by activated T-cells;- defective TCR-induced IL-4 production in
slam-/- mice.
SAP is required for SLAM-induced protein tyrosine phosphorylation
SH2
SH2
Lck
Zap-70
CD4 TCR
P
PFynTPP
PP P P P
PP
MHC
SH2
kinase
PP
SLAM
SLAM
SAP
SHIP-1DokRas-GAP
?Ras ? ?Itk?Akt
- -
PH
Itk
IL-4 production
Possible mechanism(s) by which SAP induces protein tyrosine phosphorylation:1) displacement of a protein tyrosine phosphatase;2) recruitment of a protein tyrosine kinase;3) both.
SH
2
SLAM
SAP
B + SAP
SH
2P
P
SAP
ligand
+ ligand
tyrosine phosphorylation
SH2
SLAM
SHP-2
A - SAP
SH2
SHP-2
ligand
+ ligand
tyrosine phosphorylation
SH2
SH2
SAP: a natural blocker of SH2 domain-mediated interactions?
Recruitment of FynT during SLAM-SAP signalling
SH2
SH2
Lck
Zap-70
CD4 TCR
P
PFynTPP
PP P P P
PP
MHC
SH2
FynT
PP
SLAM
SLAM
SAP PH
Itk
SH3SH2
How does SAP promote recruitment of FynT?
pY
P
SH3
FynT
SAP
SH2
SLAM
SH3
PP
P
SAP is a bifunctional adaptor that links SLAM to FynT
1
2
A B C D E
F G
**** ***
SH2 domain
tail
The FynT SH3 domain binds a novel arginine-basedmotif at the surface of the SAP SH2 domain
1 30 SAP(m)MDAVTVYHGKISRETGEKLLLATGLDGSYLLRDSESVPGVYCLCVLYQGYIYTYRVSQTETGSWSAE SAP(h)MDAVAVYHGKISRETGEKLLLATGLDGSYLLRDSESVPGVYCLCVLYHGYIYTYRVSQTETGSWSAE EAT-2(m)MD.LPYYHGCLTKRECEALLLKGGVDGNFLIRDSESVPGALCLCVSFKKLVYSYRIFREKHGYYRIE EAT-2(h)MD.LPYYHGRLTKQDCETLLLKEGVDGNFLLRDSESIPGVLCLCVSFKNIVYTYRIFREKHGYYRIQ
68 126 SAP(m)TAPGVHKRFFRKVKNLISAFQKPDQGIVTPLQYPVE.KSSGRGPQAPTG.RRDSDICLNAP SAP(h)TAPGVHKRYFRKIKNLISAFQKPDQGIVIPLQYPVEKKSSARSTQGTTGIREDPDVCLKAP EAT-2(m)TDAHTPRTIFPNLQELVSKYGKPGQGLVVHLSNPIMRNNLC...QRGRRMELELNVYENTDEEYVDVLP EAT-2(h)TAEGSPKQVFPSLKELISKFEKPNQGMVVHLLKPIKRTSPS...LRWRGLKLELETFVNSNSDYVDVLP
Chan et al., 2003
Crystal structure SAP SH2-FynT SH3 complex
Arginine 78 is required for SAP-mediated signalling
FynT
SLAM
SLAM
SAP
SH2Y
YYP
P
SH3
P
FynT
SAP
SH2 SH3
P
2B4
CD48
YP
YP
YP
YP
Modulation of cytotoxicityModulation of IFN-
Modulation of IL-4
SHIP-1Dok-1/2
Ras-GAP
Vav-1SHIP-1c-Cbl
A common mechanism for signalling by SLAM-related receptors
Role of FynT in SAP-dependent signalling in vivo?
fyn-/- T-cells exhibit TH2 and, to a lesser extent, TH1 cytokine defects in response to TCR stimulation
exon 1 exon 2 exon 3 exon 4
ATG TGA
SH2 domain
cgggcg
loxP
floxed sapR78A allele
Creation of a sapR78A “knock-in” mouse
sapR78A
R78A
**
IFN-
0
500
1000
1500
2000
2500
sap+
sapR78Asap-
IL-4
0
50
100
150
200
250
300
350
400
IL-13
0
50
100
150
200
250
300 Proliferation
0
50000
100000
150000
200000
250000
300000
Anti-CD3:Anti-CD28:
00
0.30
3.00
0.31
3.01
P+I Anti-CD3:Anti-CD28:
00
0.30
3.00
0.31
3.01
P+I
Anti-CD3:Anti-CD28:
00
0.30
3.00
0.31
3.01
P+IAnti-CD3:Anti-CD28:
00
0.30
3.00
0.31
3.01
P+I
IL-4
(p
g.m
l-1)
IFN
- (
pg
.ml-1
)IL
-13
(p
g.m
l-1)
cp
m
sap+
sapR78Asap-
Cytokine production defect in T-cells from sapR78A mice
0
2
4
6
8
10
0 14 28 35 42 49 70
time (days)
IgE
(g
.ml-1
) sap+
sap-
sapR78A
0123456789
IgE
(g
.ml-1
)
day 0day 14
sap+ sap- sapR78A
05
10152025303540
IgE
(g
.ml-1
)
sap- fyn-/-sapR78Acontrol
0
5
10
15
20
25
30
35
IgG
1 (
g.m
l-1)
sap- fyn-/-sapR78Acontrol
A B
C D
day 0day 14
Defective IgE production in sapR78A mice
Regulation of TH2 cytokine production by SAP-FynT pathway
DC
T-cell
MHC ?
TCR
SAP
FynT FynT
?
IL-4IL-13
Induction of SLAM expression during T-cell activation
Defective TH2 cytokine production inslam-/- T-cells
DC
T-cell
MHC SLAM
TCR
SAPFynT FynT
MHC SLAM
SLAMTCR
SAP
FynT FynT
IFN- IL-4IL-13
P
GATA-3
Regulation of TH2 cytokine production by the SLAM-SAP-FynT pathway
Conclusions1) SAP, a small SH2 domain-containing adaptor
mutated in XLP, is necessary for modulatorysignalling through SLAM-related receptors;
2) SAP functions by recruiting the Src-related PTK FynT, rather than by displacing a PTP;
3) SAP links SLAM-related receptors to FynT through binding of a unique surface in the SAP SH2 domain to the FynT SH3 domain;
4) Defects in SAP-mediated recruitment of FynTto SLAM receptors are likely to underlie at least part of the pathophysiology of XLP.
Sylvain Latour
Riyan ChenRomain RoncagalliOliver UttingJames TaylorLaurent DoucetMing-Chao ZhongMario-Ernesto Cruz-MunozMarceline Côté
Xiaochu ShiShaohua Zhang
Dominique Davidson
Collaborators:Pam SchwartzbergRusung TanYusuke YanagiLuo Yin