Talin/ Radixin, a FERM Family Protein

that Binds Intergrin β2 Cytotail and

Regulates Its Function?

Vascular Biology

Pingtao Tang

• Introduction

• Analysis of protein in lymphocytes

using 2D-electrophoresis

• Integrin β 2 cytoplasmic tail binding protein

1) Talin and Talin F3/ F3S

Interaction between β 2

and Talin

F3

2) ERM family: Radixin

N/C-terminal radixin and β 2

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

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.

“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

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)

AL-500 N-500

pH3 pH10 pH3 pH10

Analysis of Two-Dimensional (2-D) Electrophoresis

of Protein in the Patient’s Lymphocyte Line

(Comsblue)

Empty: Matched, Solid: Unmatched

Blue: AL Red : N ; 4-20% PAGE gel

AL-N-comsblue-Matched Map

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

• 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

Sequence Alignment of FERM Domains

342

173

Talin F3 Subdomain Adopts

A PTB-like Fold

phosphotyrosine-binding, PTB

(DA. Calderwood 2002)

• 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)

8d4 Talin

139 433 1071 2541

210 297 583

Band4.1 ERM

NR CR

Radixin

FERM Domain

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)

68.8

40.0

28.4

(kDa)

Western bolt SDS-PAGE MW (Anti-His Antibody) (Protein Staning)

Purification of His-talin F3S

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

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

His-Talin F3S Subdomain Binds Intergrin β2 Cytotail

0.3 1.2 0.6 1.2 (μg)

0

0.1

0.2

0.3GST-beta2 cytotail (0.31 ug)

GST-alpha M cytotail (0.31 ug)

GST (0.35 ug)

Talin F3S (No. 1) Talin F3 (No. 2)

OD

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

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

A

A

C

C

B

Add DDT

0

200

400

600

800

1000

1200

1400

1600

IgM PAC-1 PAC-

1+DDT

Co

un

tes o

f C

ell B

ind

ing

PA

C-1

0

0.2

0.4

0.6

0.8

1 GFP

NM

B

Expression of the N-terminal radixin promotes integrin

aggregation activity

(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)

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)

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

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

Radixin recognizes the membrane-proximal region

of the integrin b2 cytoplasmic tail

sb2CTD31:

Bo

un

d r

ad

ixin

N-E

RM

AD

(A

550)

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

Bo

un

d r

ad

ixin

N-E

RM

AD

(A

550)

0.0

0.5

1.0

1.5

2.0

DMSO

b2CTD31

GST

sb2CTD31

CWKALIHLSDLREYRRFEKEKLKSQWNNDNPLFKSATTTV M N PKFAES WT b2CT:

CWKALIHLSDLREYRRFEKEKLKSQWNND b2CTD19:

CLYRLEWFHAILRSRKD CWKALIHLSDLREYRRF b2CTD31:

723

|

750

|

738

| I II III

A

B C

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)

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

Bo

un

d r

ad

ixin

fra

gm

en

ts (

A550

)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

Effect of C-radixin on interaction of

N-radixin with integrin tail

Bo

un

d r

ad

ixin

N-E

RM

AD

(A

550

)

0.0

0.1

0.2

0.3

0.4

0.5

0.6

C/N Ratio: 0:1 2:1 4:1

the b2 peptide

the M

peptide

F

N-ERMAD (1-297) C-ERMAD (281-583)

FERM -helix CTD F

Radixin N-ERMAD interacts with the integrin b cytoplasmic tail.

Expression of the N-terminal radixin promotes integrin

adhesive activity

Ce

ll a

dh

es

ion

(A

57

0)

0

1

2

3

4

5

GFP N-CRMAD

clone 6

N-CRMAD

pool

C-CRMAD

pool

N-CRMAD

pool +EDTA

A B

Expression

of the N-

terminal

radixin

promotes

integrin

adhesive

activity

Cytohesin-1 and radixin N-ERMAD recognize overlapping

regions of the b2 cytoplasmic tail

Peptide added (M)

0 20 40 60 80 100

Bo

un

d c

yto

hesin

-1 (

A550)

0.0

0.5

1.0

1.5

2.0

2.5DMSO

b2CTD31

GST

sb2CTD31

SW63

Cytohesin-1 (M)

0 2 4 6 8 10 12

Bo

un

d r

ad

ixin

N-E

RM

AD

(A

550)

0.0

0.1

0.2

0.3

0.4

0.5

0.6

A B

The integrin β tail plays a central role in

the activation process, by undergoing

regulated interations with cytoplasmic

proteins

“Inside-out”

Signaling Conformational Clusterin

changes

Cytohesin-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

ACKNOWLEDGEMENT

Li Zhang

Yumei Xiong

Min Xu

Driss Ehirchiou

Ying Xiong

Yasuo Miura