1. Talin/ Radixin, a FERM Family Protein
that Binds Intergrin β2 Cytotail and β3
Regulates their Function?
Vascular Biology
Pingtao Tang Ph D MD

2. • Introduction
• Analysis of protein in lymphocytes
using 2D-electrophoresis
• Integrinβ
2
cytoplasmic tail binding protein
1) Talin andTalin F3/ F3S
Interaction between β
2
andTalin F3
2) ERM family: Radixin
N/C-terminal radixinandβ
2

3. Intergrin
 > 20 human intergrins ( αβ
heterodimers)
 Activation of intergrins is important in
many biological processes: cell
migration, hemostasis, extracellular
matrix assemble, tumor metastasis,
and immuune response
 The integrin β tail plays a central role
in the activation process, by
undergoing regulated interations with
cytoplasmic proteins
Fb--fibrinogen
FX--factor X
ICAM-1, 2, 3--
intercellular adhesion
molecule-1, 2, 3

4.  The patient: extensive mucosal bleeding,
intercranial hemmorrage, recurrent infection.
 Deficiencies: Glanzmann’s Thrombasthenia (GT): failure of Fg
binding to plateles.
Leukocyte Adhesion Deficiency (LAD)
 αMβ2 ( neutrophils): NIF binding, but defective adhesion to Fg (PAM)
implicating a defect in the “inside-out” signaling
 Lymphocytes: defective adhesion to ICAM-1(αLβ2) and Fn (α4β1).
In contrast, lymphocytes from normal control
adhered to both ligands, and adhesion could be
further enhanced by addition of PAM (a PKC agonist)
 Stable cell lines: the Epstein Bar virostransformed lymphocytes.

5. “Inside-out”
Signaling
Conformational Clusterin
changes
The “inside-out” signaling:
Upon activation, intergrins
change their conformation
and/or cluster on the cell
surface. These processes
leads to increase
in ligand affinity
and avidity, and
hence enhanced
ligand binding
and cell adhesion.
Cytohesin-1, a PIP3-regulated
adaptor molecule for LFA-1
(CD11a/CD18, αLβ2) activation
Cytoskeleton (αIIbβ3)
PAK1 Rhok Cytohesin-1 Talin F3
?
Lipid (PIP2,PIP3)
rho rac cdc42
PI-3K MAPK
PKC
G-protein
Growth factors CD19
Cytokines

6. PKC-driven wound
closure response in 2C4
fibrosarcoma cells stably
transfected with GFP–
PKC.
• Wound closure response was
recorded by time-lapse
microscopy
• GFP–PKC-2C4 cells were
wounded after a 30 min
incubation in media
containing LY294002 (10
µM) (+LY294002) PDBu (1
µM) was added to the media
after wounding and the
wound closure response was
monitored.
(EMBO J2001, 20(11): 2723-2741)

7. AL-500 N-500
pH3 pH10 pH3 pH10
Analysis of Two-Dimensional (2-D) Electrophoresis
of Protein in the Patient’s Lymphocyte Line
(Comsblue)

8. Empty: Matched, Solid: Unmatched
Blue: AL Red : N ; 4-20% PAGE gel
AL-N-comsblue-Matched Map

9. pH10 pH3 pH3.0 pH10
AL N
Phosphorylation of 2-D Electrophoresis of Protein
in the Lymphocyte Line
(Western blot with anti- phosphotyrosine antibody)
4 sec
10 sec

10. • band four-point-one (band4.1)/ezrin/radixin/moesin homology
domain, FERM (173-342aa)
• mediate their interactions with the cytoplasmic domain of
transmembrane protein
F3 , a sandwich of two orthogonal antiparallel b sheets and a helix,
including phosphotyrosine-binding (PTB) and plechstrin homology (PH)
domain.
PTB like F3 subdomain leads to integrin activation.
A Schematic Representation of Talin
Head F1-3 Rod domain
50 -kDa CalpainII 250-kDa
8d4
139 433 1071

11. Sequence Alignment of FERM Domains
342
173

12. Talin F3 Subdomain Adopts
A PTB-like Fold
phosphotyrosine-binding, PTB
(DA. Calderwood 2002)

13. • GST fusion expression system: pGEX-4T-1
• Histin fusion expression system: pET-32a-c(+), pQE30/pQE31
• Mammalian cell expression: MSCV-IRGFP, pcDNA3.1/myc-His
Head Rod domain
(FERM, F1-3)
8d4Talin
139 433 1071 2541
210 297 583
Band4.1 ERM
NR CR
Radixin
FERM Domain

14. Assay of (Functional) Interaction for integrin/talin/radixin:
Focal adhesion plaques in cell line
Disruption of normal phosphoinositide metablism at the plaques interferes with
cell adhesion
Expression level, shape…location.
Migration, Aggregation, adhesion inhibition assay, antibody recognition of
conformational changes of receptor, etc.
GST pull down
Using protein purified or cell lyses, A pull down B, and B pull down A
Co-IP
Over express with HA/myc/flag or cell lyses with higher expression
IP: using the antibody from rabbit/ mice with same original IgG for control,
IB: using mice/rabbit
Biacore:
Monitoring the progress of biomolecular interactions in real time

15. Hygromycin 300
Hygromycin 300
Hygromycin 300
Hygromycin 300
Hygromycin 300
Hygromycin 300
Hygromycin 300
Neomycin 1,200PRSVF3SMSCV-IRGFPCHOmac-1
Neomycin 1,200PRSVF3MSCV-IRGFPCHOmac-1
Neomycin 1,200PRSVTalin 116-435MSCV-IRGFPCHOmac-1
Neomycin 1,200PRSVGFPMSCV-IRGFPCHOmac-1
Neomycin 1,200PRSVC-RadixinMSCV-IRGFPCHOmac-1
Neomycin 1,200PRSVN-RadixinMSCV-IRGFPCHOmac-1
Neomycin 1,200PRSVRadixinMSCV-IRGFPCHOmac-1
Drug and
concentration.(µg/ml)
Selection
vector
Express
molecularVector nameCell lineIntegrin
Establishment of stable cell lines
PMSCV hygF3SMSCV-IRGFPCHOIIb3
PMSCV hygF3MSCV-IRGFPCHO
PMSCV hygTalin 116-435MSCV-IRGFPCHO
PMSCV hygGFPMSCV-IRGFPCHOIIb3
PMSCV hygC-RadixinMSCV-IRGFPCHOIIb3
PMSCV hygN-RadixinMSCV-IRGFPCHOIIb3
PMSCV hygRadixinMSCV-IRGFPCHOIIb3
IIb3
IIb3

16. Purification of talin F3
Eluted Throbin Talin F3 GST
M 1 2 3 Sup Resin 5 6 11 15 16 17 18 19 20
Ni –NTA Mono HR 5/5
F3
GST
GST-F3
61.3
36.4
24.7
19.2
13.1
9.3
(kDa)

17. 68.8
40.0
28.4
(kDa)
Western bolt SDS-PAGE
MW (Anti-His Antibody) (Protein Staning)
Purification of His-talin F3S

18. Purification of GST-Radixin / Talin L
NR M CR Talin L
68.8
52.5
40.0
(kDa)
(Glutathione Sepharose 4B)
GSH (mM) 5 20 5 20 20

19. F
N-ERMAD (1-297) C-ERMAD (281-583)
FERM -helix CTD F
Radixin N-ERMAD interacts with the integrin b cytoplasmic tail.

20. ELISA Assay of Talin/Talin F3
Binding on β2 Cytotail
0
0.4
0.8
1.2 Alpha M
beta2
3 6 3 6 (nM)
Talin Talin F3

21. His-Talin F3S Subdomain Binds Intergrin β2 Cytotail
0.3 1.2 0.6 1.2 (μg)
0
0.1
0.2
0.3
GST-beta2 cytotail (0.31 ug)
GST-alpha Mcytotail (0.31 ug)
GST (0.35 ug)
Talin F3S (No. 1) Talin F3 (No. 2)
OD

22. His-Talin F3S Subdomain Binds
Intergrin β2 Tail (Repeated)
0
0.2
0.4
0.6 GST-beta2 cytotail
GST-alpha M cytotail
GST
0.3 0.6 (μg)
Tail F3S
OD

23. ELISA Assay of Talin / Talin F3
Binding on Intergrin β2 Tail
OD
Concentration of Talin or Talin F3 (nM)
•Coat β2 or
αM cytotail
peptid 21.2
mMol/L
•10% milk and
1% BSA block
•GST-talin/F3
0
0.2
0.4
0.6
0.8
0 2 4 6 8
Talin L+ beta 2
F3 + beta2
Talin + Alpha M
F3 + Alpha M

24. (a) Organization of the
ERM family.
Share a ~300 aa domain
involved both in the
morphogenesis of membrance
structure on which they are
concentrated and in cell adhesion
Ezrin, substrate of EGF receptor
tyrosine kinase
Radixin, F-actin barbed end
capping protein
(b) Mapping of binding sites
for ERM-interaction partners.
(Trends in Cell Biology 2003)

25. Ezrin localization
in human adenocarcinoma
A431cells.
A full-length ezrin–GFP
fusion protein expressed
transiently, appears yellow
in cells that were fixed,
permeabilized and stained
with antibody against ezrin
(red). Ezrin–GFP as well
as endogenous ezrin
localize in actin-rich cell-
surface membrane
structures.
Moesin and radixin
localize similarly, ERM
bind to the plasma
membrane through the
FERM domain
(Paul M, et al. 2003)

26. Possible conformations of ERM
Single
phosphorylation
of a conserved C-
terminal Thr
residue
Dimers and
oligomers are
physiological
forms of ERM
molecules found
associated at the
membrane level
The activation
ERM resulting in
unfolding and
subsequent N–C
ERMAD
association

27. A fold cytosolic
ERM is unable
to associate
with membrane
and
microfilaments
C-terminal Thr- P
Activated forms of
ERM might contol
assemble of actin
microfilaments

28. Radixin recognizes the membrane-proximal region
of the integrin b2 cytoplasmic tail
s2CTD31:
BoundradixinN-ERMAD(A550)
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
WT CTD19 CTD sCTD BSA
Peptide added (M)
0 100 200 300 400
BoundradixinN-ERMAD(A550)
0.0
0.5
1.0
1.5
2.0
DMSO
2CTD31
GST
s2CTD31
CWKALIHLSDLREYRRFEKEKLKSQWNNDNPLFKSATTTV M N PKFAESWT 2CT:
CWKALIHLSDLREYRRFEKEKLKSQWNND2CTD19:
CLYRLEWFHAILRSRKD
CWKALIHLSDLREYRRF2CTD31:
723
|
750
|
738
|I II III
A
B C

29. Radixin Binding on Integrin β2 Cytotail
0
0.2
0.4
0.6
0.8
1
1.2 NR + beta 2 tail
CR + beta 2 tail
NR + alpha M tail
CR + alpha M tail
21 11 5 2.5
Concentration of β2/αM cytotail (nM)

30. Interaction of N, C-terminal Radixin with
Integrin β2 Cytotail
2
Radixin(nM) vs NR(aMtail) Kd=1.4*10
Radixin(nM) vs 5CR(aMtail) Kd=5*10
Radixin(nM) vs NR(b2tail) Kd=3.2*10
Radixin(nM) vs CR(b2tail) Kd=3.2*10
2
6
Radixin fragment added (nM)
0 50 100 150 200 250
Boundradixinfragments(A550)
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4

31. Effect of C-radixin on interaction of
N-radixin with integrin tail
BoundradixinN-ERMAD(A550)
0.0
0.1
0.2
0.3
0.4
0.5
0.6
C/N Ratio: 0:1 2:1 4:1
the 2 peptide
the M peptide

32. Binding of intergrin β2 to
immobilized GST-talin,GST-F3 and GST-NR
Mac-1 standard + +
79 18#
GST-4B + +
GST-F3 +
GST-Talin +
GST-NRadixin +
293 mac-1 lyses + + + + +
*080903A anti- β2 1:1,500
β2

33. Expression of the N-terminal radixin promotes integrin
adhesive activity
Celladhesion(A570)
0
1
2
3
4
5
GFP N-CRMAD
clone 6
N-CRMAD
pool
C-CRMAD
pool
N-CRMAD
pool +EDTA
A B

34. Expression
of the N-
terminal
radixin
promotes
integrin
adhesive
activity

35. Cytohesin-1 and radixin N-ERMAD recognize overlapping
regions of the b2 cytoplasmic tail
Peptide added (M)
0 20 40 60 80 100
Boundcytohesin-1(A550)
0.0
0.5
1.0
1.5
2.0
2.5
DMSO
2CTD31
GST
s2CTD31
SW63
Cytohesin-1 (M)
0 2 4 6 8 10 12
BoundradixinN-ERMAD(A550)
0.0
0.1
0.2
0.3
0.4
0.5
0.6
A B

36.  The integrin β tail plays a central role in
the activation process, by undergoing
regulated interations with cytoplasmic
proteins

37. How about intergrin β3 ?

38. Binding of intergrin β3 to immobilized GST-F3
GST-F3 ++ + - + + (4B)
CHO αIIbβ3 + + + + + + +
GST - - + - -
β3

39. Binding of intergrin β3 to immobilized GST-F3
(Western blot with anti-β3 antibody)
GST + - - - + - -
CHO αIIbβ3 + + + + + + +
(Cells Lysis)
GST-F3 - + - + - ++ +++
(4B ) S (Resin)
β3

40. Binding of intergrin β3 to immobilized GST-F3
(Anti-GST Western blot )
GST-F3 + ++ + -
GST-Talin + +
CHO αIIbβ3 + + + + + +
GST - - +
β3

41. Binding of intergrin β3 to
immobilized GST-talin and GST-F3
GST-4B +
GST-F3 + +
(0804) (0620)
GST-Talin +
CHO αIIbβ3 + + + + + +
β3

42. A
A
C
C
B
Add DDT
0
200
400
600
800
1000
1200
1400
1600
IgM PAC-1 PAC-
1+DDT
CountesofCellBindingPAC-1
0
0.2
0.4
0.6
0.8
1 GFP
NM
B
Expression of the N-terminal radixin promotes integrin β3
aggregation activity

43. “Inside-out”
Signaling
Conformational Clusterin
changesCytohesin-1, a PIP3-
regulated adaptor molecule
for LFA-1 (CD11a/CD18, αLβ2)
activation
Cytoskeleton (αIIbβ3)
PAK1 Rhok Cytohesin-1 Talin F3
?
GTP (active) Lipid (PIP2,PIP3)
rho rac cdc42 GDP
PI-3K MAPK
PKC
G-protein
Growth factors CD19
Cytokines
N-Radixin
Rho GDI

44. ACKNOWLEDGEMENT
Li Zhang
Yumei Xiong
Min Xu
Driss Ehirchiou
Ying Xiong
Yasuo Miura