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RhoGTPases – Function and Regulation RhoGTPases – Function and Regulation
Thomas WielandInstitut für Pharmakologie und Toxikologie
Fakultät für Klinische Medizin Mannheim
Ruprecht-Karls-
Universität
Heidelberg
Physiological function of Rho GTPases
Regulation of the actin cytoskeleton
Actin Vinculin Actin Vinculin
U = TXA2-Analog
Y = Rho-Kinase Inhibitor
C3 = exoenzyme C3 transferase (C3T) from Clostridium botulinumInhibition of RhoA-C
Shape change of platelets
Klages et al. J. Cell Biol. 144 (4) 1999
Keratinocytes
Regulation of cell-cell contacts
C3 = exoenzyme C3 transferase (C3T) from Clostridium botulinumInhibition of RhoA-C
Endothelial cellsThrombin is a strong activator of RhoA
Cadherine
Regulation of proliferation
Neointima formation after vascular injury of a carotid artery
Y27632 = Rho Kinase Inhibitor
Shibata et al. Circulation 103(2) 2001
Cardiac specific overexpression of RhoA in the heart
Posttranslational modification of RhoGTPases
Statins
Beneficial pleiotropic effects of statins
Activation cycle of RhoGTPases
Rho-specific guanine nucleotide exchange factors
DH PH
Catalytic activity~200 aa
Membrane localization ?
Variable specifity for RhoGTPases
Multidomain proteins
Variable size (~ 60 to 800kd)
Variable expression patterns (ubiquitiniously to specific)
Model of PH domain assisted GEF activity
RhoGEFs: The Dbl family
Regulation of RhoGEFs - Autoinhibition
Regulation of RhoGEFs – Protein-Protein-Interaction
Cellular Localization of RhoGEFs
b
Experimental approaches
directpull down assay
Rac1Cdc42 Rho
GEF
GTP GTP GTP
Pak1-GST Rhotekin-GSTp6
3Rho
GEF
cont
rol
p63R
hoG
EF-D
H
Tiam
-1D
bl-D
H
RhoA-GTP
Rac1-GTP
Cdc42-GTP
total RhoA
total Rac1
total Cdc42
Direct detection of RhoGTPase activation
rela
tive
luci
fera
se e
xpre
ssio
n control RhoGEF
0
1
2
3
4
5
+C3T
C3T
indirectluciferase reporter assay
Rac1
pSREfirefly luciferase
renilla luciferase
SRF
const.
Cdc42
Rho
GEF
Detection of RhoGTPase activation II
Detection of RhoGTPase activation IIIFRET
GiPCR GqPCR
Gi Gq
PMG12PCR
G12/13
p63RhoGEFp63RhoGEF LARGLARGPI3K
TIAM-1TIAM-1
RhoARhoARac1Rac1
VEGF-R
NO
cGMP
p164, Grinch, Gef10 p164, Grinch, Gef10
Focus AG Wieland
p63RhoGEF
DH PH
580 aa
H2O
Car
diom
yocy
tes
Non
-Car
diom
yocy
tes
H2Ol
Car
diom
yocy
tes
Non
-Car
diom
yocy
tes
p63RhoGEF Control
Hea
rtB
rain
Place
nta
Lung
Live
rSc.
mus
cle
Kid
ney
Pancr
eas
9,57,5
4,4
2,4
1,3
kb
Northern Blot:mRNA from different human
tissues
RT-PCR:Total RNA from isolated
primary rat cells
Expression of p63RhoGEFExpression of p63RhoGEF
p63RhoGEF induces stress fiber formation
anti-c-myc
20 µm
TRITC-Phalloidin
20 µm
stress fiber
Lutz et al. Naunyn Schmiedebergs Arch Pharmacol. 369:540-6 (2004 )
p63R
hoG
EF
cont
rol
p63R
hoG
EF-D
H
Tiam
-1D
bl-D
H
RhoA-GTP
Rac1-GTP
Cdc42-GTP
total RhoA
total Rac1
total Cdc42
rela
tive
luci
fera
se e
xpre
ssio
n
control p63RhoGEF
0
1
2
3
4
5
+C3T
Specificity of p63RhoGEFSpecificity of p63RhoGEF
Upstream regulatory mechanism (1)Upstream regulatory mechanism (1)
Control M3p63 p63 + M3
0
20
40
60
80
100 Basal
Carbachol
n=8
rela
tive
luc
ifer
ase
exp
res
sio
n
Lutz et al. J Biol Chem. 280:11134-9 (2005 )
Upstream regulatory mechanism (2)Upstream regulatory mechanism (2)re
lati
ve l
uci
fera
seex
pre
ssio
n
0
50
100
150
200
+p63
Basal GqQL G11QL Basal0
50
100
150
+p63
G12QL G13QL
M3-Ach-Receptor
Gq/11 G12/13
Contro
lp6
3
Gq
RCGq
RC+p63
G1
2QL+
p63
G1
2QL
G1
1QL
G1
1QL+
p63
G1
3QL+
p63
G1
3QL
RhoA-GTP
total RhoA
p63
RhoG
EF
p63R
hoG
EF
p63R
hoG
EF
WB:anti-c-myc
WB:anti-Gq/11
anti-Gq/11
IP:anti-c-myc
Lysate
Con
trol
G11QLGqRC
Con
trol
Con
trol
IP:anti-EE
WB:anti-c-myc
WB:anti-Gq/11
p63-
EE-GqQL
Physical interaction of p63RhoGEF with GPhysical interaction of p63RhoGEF with Gq q in the cell in the cell
p63 149-580
p63 149-580
Courtesy of John J. Tresmer
Physical interaction of p63RhoGEF with GPhysical interaction of p63RhoGEF with Gqq in vitroin vitro
DH PH
p63RhoGEF580
RhoA RhoA
GTP GDP
GqPCR
Gq/11
Physiological relevance of Physiological relevance of p63RhoGEF mediated RhoA activationp63RhoGEF mediated RhoA activation
Ad p63RhoGEF+ Endothelin 1
TRITC-Phalloidin
RhoA-GTP
total RhoA
cont
rol
p63R
hoG
EFco
ntro
lp6
3Rho
GEF
+ Endothelin 1
p63RhoGEF
Neonatal Cardiomyocytes
• p63RhoGEF is mainly expressed in heart and brain tissue.
• p63RhoGEF activates RhoA, but not Rac1 or Cdc42
• p63RhoGEF is activated by Gq/11 proteins.
• p63RhoGEF interacts directly with active Gq/11 proteins.
• p63RhoGEF enhances the Endothelin 1 and Phenylephrine induced RhoA activation in cardiomyocytes.
b
p63RhoGEF - Summaryp63RhoGEF - Summary
H1-R M3-R
His
Gq/11
p63RhoGEFLARG
RhoA
G12/13 PLC/PKC
TXA2-R
U-46619
Carb
Signaling cascades involving p63RhoGEF and LARG
Larg
Control
anti-Flag
p63
Control
anti-c-myc
DH PH588
1543
DH PHRGSPDZ
RhoA-GTP
total RhoA
Control
Larg
p63
C
p63
Larg
0
5
10
15
rela
tive
Lu
cife
rase
p63RhoGEF (p63) Dominant expression in brain and heart
LARGWidespread expression
Basal activity of p63RhoGEF and LARG
Control
TXA2-R
TXA2-R+L
ARG
TXA2-R+p
630
25
50
75BasalU-46619 (200nM)
rela
tive
Lu
cife
rase
G12/13PCR Gq/11PCR
Control
M3-
R
M3-
R+LARG
M3-
R+p63
0
25
50
75
100
125BasalCarbachol (1mM)
rela
tive
Lu
cife
rase
GPCR induced RhoA activation mediated by p63RhoGEF and LARG
0 5 10 15 20 250
5
10
15
20
25
30
35Largp63RhoGEF
ng Gq
rela
tive
Lu
cife
rase
Control p63RhoGEF LARG0
25
50
75
100none+GqRC+G12QL
rela
tive
Lu
cife
rase
Conclusion: Larg needs an active state receptor to induce RhoA activation, whereas p63RhoGEF needs only traces of Gq protein for its activation.
The LARG induced Rho activation requires activated GPCRs
Gq/i
1-28aa Gi-3
29-353aa Gq
0
10
20
30
40
50
60
70re
lati
ve L
uci
fera
se
p63 - + - - - + + - - - + + - -
Larg - - + - - - - + + - - - + +
M3-R - - - + - - + - + - - + - +
Gq - - - - + + + + + - - - - -
Gq/i - - - - - - - - - + + + + +
The Larg activation ist dependent on the N-terminal part of Gq proteins
Gq Gq/i
Conclusion: p63RhoGEF and LARG apparently hold divergent binding sites for Gq
RGS3
RGS proteins contribute to the regulation of GRGS proteins contribute to the regulation of G--gated Kgated K+ + -channels-channels
ACh, 1 MControl
500 nA
25 s
Activation of GIRK by M2-AChR in Xenopus oocytes
Doupnik et al. 1997, Proc Natl Acad Sci 94:10461
Regulation by RGS3
+ RGS3
500 nA
25 s
ACh, 1 M
RGS3 - A highly abundant protein in human heartRGS3 - A highly abundant protein in human heart
RGS3 RGS domain
1 394 519509
G binding70kDa
• RGS3 mRNA is upregulated in human HF Owen et al. 2001, Eur Heart J 22:1015
• RGS3 can be induced in NRCM by treatment with bFGF Zhang et al. 1998, J Mol Cell Cardiol 30:269
• RGS3 inhibits G-stimulated PLC activity and PI3K/Akt signaling G scavenger Shi et al. 2001, J Biol Chem 276:24293