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Protein Domains That Interact with
Receptor Tyrosine Kinases:
Structural Aspects
Ming-Ming Zhou, Ph.D.
MSSM Department of Pharmacology Course on
“Cell Signaling Systems”
January 27, 2005
The Role of Protein Domains in Cell Signaling
Growth Factors
RTK
Ras
GDP
Shc
PLC-
P
P
P
P
P
P
Ras
GTP
PTB
P
CH
Sos
Raf
SH2
SH2
PI(3)K
SH3
MEK
SH3
Grb2
MAPK
c-fos, c-myc, c-jun
Protein Domains in Cell Signal Transduction
SH2
PTB
PH
SH3
WW
-pY-X-X-hy-
-hy-X-N-P-X-(p)Y-
Phospholipids
-P-X-X-P-X-
-P-P-X-Y-
EH
14-3-3
PDZ
-N-P-F-
-R-S-X-pS-X-P-
-E-S/T-D/V-COOH-
...
Ligand Recognition of the Shc PTB and SH2 Domains
Protein
pTyr Sites
pTyr
Peptides
Shc SH2
Kd (M)
Shc PTB
Kd (M)
Human TrkA, pY490
HIIENPQpYFSDA
280
0.053
Mouse Polyoma MT, pY250
SLLSNPTpYSVMRS
320
0.140
Human ErbB3, pY1309
SAFDNPDpYWHSRLF
230
0.020
AENPEpYLGLDV
Human ErbB2, pY1248
1290
N.D.
Human EGFR, pY1086
GSVQNPVpYHNQPLN
220
5.30
Human EGFR, pY1148
SLDNPDpYQQDFF
1000
0.200
Human EGFR, pY1114
TAVGNPEpYLNTVQ
290
0.140
Human EGFR, pY992
VADADEpYLIPQQ
130
680
Human EGFR, pY1173
TAENAEpYLRVA
60
770
50
>10000
Human TCR  Chain, pY141
Ref: Zhou et al (1995) JBC 270, 31119-31123.
GHDGLpYQGLSTATK
Phosphotyrosine Binding Domains
 SH2 Domain:
 PTB Domain:
Q:
---pYxx-----xNPxpY---

Ligand Specificity

Structural Versatility

Functional Diversity
3D Structue of the Shc SH2 Domain/TCR Peptide Complex
TCR  chain peptide: GHDGLpYQGLSTATK
R A2
G
A BG7
pY
L D6
A
BG
F
R B5
R B5
N
EF

D
B
E
D’
C
C

T E4
D
pY+3
L D5

I BG4 F

pY
B
V C3
A
C
pY+3
BG
H D4
G
C

BC
Ref: Zhou et al (1995) PNAS 92, 7784-7788.
N
Structures of SH2 Domains
Shc
Lck
D
G
B
BG
BG
pY
A
pY+3
F
A
pY
pY+3
E
B
D’
F
C
N
E
C
D’
DE
DE
PLC-1
Syp
BG
BG
pY+3
F
E
pY+3
F
D’
DE
pY
pY
E
D’
DE
Structures of SH2 Domains
PLC-1
Syp
Lck
Shc
3D NMR Structure of the Shc PTB Domain/TrkA Complex
Ref: Zhou et al (1995) Nature 378, 584-592.
FXNPXpY Recognition by the Shc PTB Domain
PTB Domain versus SH2 Domain
Shc PTB Domain
Shc SH2 Domain
Biological Ligands of PTB Domains
PTB Domain
Binding Partners
Binding Sites
Shc
hTrkA, pY490
mPolyoma MT, pY250
hErbB3, pY1309
hEGFR, pY1148
IRS-1
hIR, pY960
hIL-4R, pY497
hIGFIR, pY950
Numb
LNX
NAK
Peptide Library
GLDNPAYTSSV
GFSNMSFEDFP
YIGPYL
X11, Fe65, Ced-6
APP
GYENPTYKFFE
HIIENPQpYFSDA
SLLSNPTpYSVMRS
SAFDNPDpYWHSRL
ISLDNPDpYQQDFF
LYASSNPEpYLSAS
LVIAGNPApYRSFS
LYASVNPEpYFSAS
3D Structure of the IRS-1 PTB Domain/IL-4R Complex
Ref: Zhou et al (1996) Nature Structural Biology 3, 388-393.
Ligand Recognition of the IRS-1 PTB domain
Distinct Mechanisms for NPXpY Recognition
IRS-1 PTB Domain
Shc PTB Domain
PTB & PH Domains: A Conserved Structural Fold
A
B
C
D
SNT/FRS2 Binding Sites in Neurotrophic Receptors

FGFRs (juxtamembrane region):
KSDFH SQMAV HKLAK SIPLR RQVTS

NGFRs:
•TrkA,pY960
•TrkB
•TrkC

HIIENPQpYFSDA
PVIENPQpYFGIT
PVIENPQpYFRQG
RET, pY1062
TWIENKLpYGRIS
3D Structure of the SNT-1 PTB Domain/FGFR1 Complex
C
C
8
1
’
4
’
8
5
3
FGFR
6
6
FGFR
C
2
N
7
C
N
7
5
1
1
4
N
Ref: Dhalluin et al (2000) Molecular Cell 6, 921-929.
N
2
1
3
SNT-1 versus IRS-1 PTB domains
SNT-1
A
C
R63
R78
8
4
’
5
3
FGFR
6
7
2
N
IRS-1
1
N
1
B
R227
C
C
pY
IL-4R
1
R212
4
5
3
6
pY-3
N
7
C
2
pY-6
1
N pY-8
Sequence Alignment of the SNT & IRS PTB Domains
SNT-1 Truncation Effect on FGFR & TRK Binding
w/FGFR
Yeast Two-Hybrid Binding Assay
1
FGFR
N
C
5
’
6
Vector
Leu-, Trp-
3
4
C
R63
w/TrkB
SNT-1 PTB
Domain
R78
TrkB
1
1
7
pY-5
N
6
5 2
3
4
R63
R78
C
C
pY
Vector
8
SNT-1 PTB
Domain D8
1
N
7
2
His-, Leu-, Trp-
hFGFR1
Leu-, Trp-
His-, Leu-, Trp-
TRKB
Leu-, Trp-
His-, Leu-, Trp-
Unifying Features of PTB Domains
Ref: Yan & Zhou (2002) FEBS Lett. 513, 67-70.
Structure & Function of the SH3 Domain
A
C
B
Ref: Zarrinpar (2003) Sci. STKE 2003(179), re8
RTKs: Activation by Receptor Dimerization
Ref: Hubbard, et al (1998) JBC 273, 11987-11990.
Autoinhibition of Insulin and FGF Receptors
- Cis-inhibition & Trans-activation
IRK
FGFR1K
Unphosphorylated IRK (IRK0P)
Unphosphorylated FGFR1K
Tris-phosphorylated IRK (IRK3P)
Tris-phosphorylated IRK (IRK3P)
AutoRegulation & Activation of Src Tyrosine Kinase
3D Structure of Protein Tyrosine Kinase Hck
Ref: Xu, et al (1997) Nature 385, 595-602; Sicheri, et al (1997) Nature 385, 602-609.
Rational Design of Nonpeptide Inhibitors of Src SH2 Domain
Ref: Sundaramoorthi et al (2003) Biopolymers 71, 717-729.
3D Structure of the Abl Kinase Domain/Drug Complex
Ref: Daub, H. et al (2004) Nature Review Drug Discovery 3, 1001-1010.
Summary
Structure-Function of Protein Domains in RTKs
 Conserved 3D structural fold with divergent sequences
 Conserved structural fold with different functions
 Single protein domain with multiple functions
 Cooperative regulation of RTK activity in cell signaling