(--THIS SECTION DOES NOT PRINT--) Nassr Nama1,2,3 ... · PDF filetitles, subtitles, text, ......
1
RESEARCH POSTER PRESENTATION DESIGN © 2012 www.PosterPresentations.com CXCR4, a chemokine receptor involved in metastasis, signals through two major successive pathways: Gα/i and β-arrestin. β-arrestin terminates G- protein signaling and targets the receptor to endocytosis. Constitutively active GPCR mutants (CAMs) spontaneously activate G- protein. Constitutively inactive mutants (CIMs) that spontaneously recruit β- arrestin are devoid of G-protein signaling. We tested a set of CXCR4 mutants on their signaling capacity of G α/i and ß- Arrestin2 pathways. R134A was found a CIM that was inactive on the G α/i signaling, but constitutively recruited arrestin. N119S was found as a CAM, constitutively active in G-protein signaling as well as β-arrestin recruitment. Imaging of these two mutants showed their constitutive internalization and their different localization in the cell. These results show the presence of varying β-arrestin recruitment, independent of the G-protein signaling. Further studies remain to conclude whether the difference of β-arrestin recruitment is directed by the GRKs or the ubiquitination of the receptor. ABSTRACT WT D133N R134A Y135A N119D N119S N119K INTRODUCTION Recruitment of β-arrestins and its modalities are mainly regulated by the GPCR kinases (GRKs). Four of these regulate the CXCR4, GRK 2,3,5 and 6. The divers modalities of β-arrestins recruitment induce different signaling pathways such as internalization or ERK signaling. MATERIAL & METHODS Gα/i activation measured as cAMP reduction by CXCR4 using EPAC. • The method does not permit showing the constitutive activity of N119S as previously demonstrated (Zhang et al.). • N119K and R134A are unable to activated Gα/i. RESULTS REFERENCES CONCLUSIONS I would like to thank Dr. Michel Bouvier and Guillaume Sylvain-Drolet for the plasmids, Nicolas Montpas for his training and advice, and François Guité-Vinet for being a constant source of inspiration. Metastasis is the major mortality cause in cancer. Detection of cancer patients early before the metastatic process increase the chances of treatment by chemotherapy or surgery. Thus it is primordial to understand the process of metastatic spread in order to be able to predict its targets, and to develop specific drugs to obstruct it. CXCR4, a G-protein coupled receptor (GPCR), binds one chemokine ligand, CXCL12 (SDF-1α). Breast cancer cells that express the CXCR4 tend to migrate to organs with a high level of CXCL12, such as lung, liver, and bone marrow. 1. Department of Biochemistry, Université de Montréal. 2. Department of Microbiology and Immunology, Université de Montréal. 3. CHU Ste-JusFne Children Hospital. 4. Department of Pathology and Cellular Biology, Université de Montréal. Nassr Nama 1,2,3 , Stéphanie Gravel 1,3 , Amel Kechad 3,4 , Gilles Hickson 3,4 , Nikolaus Heveker 1,3 Diverse ModaliHes Of Β-ArresHn2 Recruitment To CXCR4, Independently Of G-Protein AcHvaHon -∞ -12 -10 -8 -6 0 20 40 60 80 100 [CXCL12] % Forskolin Inhibition CXCR4-wt -∞ -12 -10 -8 -6 0 20 40 60 80 100 [CXCL12] % Forskolin Inhibition CXCR4-N119D CXCR4-wt [CXCL12] % Forskolin Inhibition -∞ 0 20 40 60 80 100 -12 -10 -8 -6 CXCR4-D133N CXCR4-wt -∞ -12 -10 -8 -6 0 20 40 60 80 100 [CXCL12] % Forskolin Inhibition CXCR4-N119S CXCR4-wt [CXCL12] % Forskolin Inhibition -∞ 0 20 40 60 80 100 -12 -10 -8 -6 CXCR4-R134A CXCR4-wt [CXCL12] % Forskolin Inhibition -∞ 0 20 40 60 80 100 -12 -10 -8 -6 CXCR4-Y135A CXCR4-wt -∞ 0 20 40 60 80 100 -12 -10 -8 -6 % Forskolin Inhibition [CXCL12] CXCR4-wt CXCR4 N119K -10 -8 -6 0.00 0.02 0.04 0.06 0.08 0.10 0.12 [CXCL12] BRET 2 NET CXCR4 wt -10 -8 -6 0.00 0.02 0.04 0.06 0.08 0.10 0.12 [CXCL12] BRET 2 NET CXCR4 wt CXCR4 N119D -10 -8 -6 0.00 0.02 0.04 0.06 0.08 0.10 0.12 [CXCL12] BRET 2 NET CXCR4 wt CXCR4 D133N -10 -8 -6 0.00 0.02 0.04 0.06 0.08 0.10 0.12 [CXCL12] BRET 2 NET CXCR4 wt CXCR4 N119S -10 -8 -6 0.00 0.02 0.04 0.06 0.08 0.10 0.12 [CXCL12] BRET 2 NET CXCR4 wt CXCR4 R134A -10 -8 -6 0.00 0.02 0.04 0.06 0.08 0.10 0.12 [CXCL12] BRET 2 NET CXCR4 wt CXCR4 N119K -10 -8 -6 0.00 0.02 0.04 0.06 0.08 0.10 0.12 [CXCL12] BRET 2 NET CXCR4 wt CXCR4 Y135A CXCR4 Wt 0 200 400 600 800 1000 0.00 0.03 0.06 0.09 0.12 0.15 GFP10/RLuc3 BRET 2 NET SDF + 200 nM SDF- CXCR4 R134A 0 200 400 600 800 1000 0.00 0.03 0.06 0.09 0.12 0.15 GFP10/RLuc3 BRET 2 NET SDF- SDF + 200 nM CXCR4 N119S 0 500 1000 1500 0.00 0.03 0.06 0.09 0.12 0.15 GFP10/RLuc3 BRET 2 NET SDF- SDF + 200 nM Forskolin CXCL12 AMD3100 TC14012 Forskolin CXCL12 AMD3100 CXCL12 + AMD3100 TC14012 Forskolin CXCL12 AMD3100 TC14012 Forskolin CXCL12 AMD3100 TC14012 0 50 100 150 % Forskolin Inhibition ** **** **** **** pcDNA3 WT R134A N119S N.S CXCL12 AMD3100 CXCL12 + AMD3100 TC14012 N.S CXCL12 AMD3100 TC14012 N.S CXCL12 AMD3100 TC14012 0.00 0.02 0.04 0.06 0.08 BRET 2 NET **** **** **** *** * **** **** **** ** WT R134A N119S -∞ -10 -8 -6 0.00 0.02 0.04 0.06 0.08 0.10 [TC14012] BRET 2 NET CXCR4 N119S CXCR4 WT 0 300 600 900 1200 1500 0.00 0.02 0.04 0.06 0.08 GFP10/RLuc3 BRET 2 NET N.S TC14012 N.S Forskolin CXCL12 0.12 0.14 0.16 0.18 0.20 BRET 2 NET N.S CXCL12 AMD3100 CXCL12 + AMD3100 TC14012 N.S CXCL12 AMD3100 CXCL12 + AMD3100 TC14012 N.S CXCL12 AMD3100 CXCL12 + AMD3100 TC14012 0.00 0.03 0.06 0.09 0.12 BRET 2 NET *** ** ** WT R134A N119S 0 300 600 900 1200 1500 0.00 0.02 0.04 0.06 0.08 GFP10/RLuc3 BRET 2 NET CXCR4 R134A CXCR4 WT CXCR4 N119S CXCR4 signals through two major pathways: G α/i and β-arrestins. G α/i inhibits adenylyl cyclase, responsible of generating cAMP. CXCR4 recruits also the β-arrestin proteins (1 and 2), and they shut off the signal transduction via G α/i , by desensitization. β-arrestins target the desensitized receptor to endocytosis. β-arrestins are able to induce signaling effects too, by activating the ERK pathway leading to enhanced chemotaxis and cellular survival. While the previously believe function of endocytosis is to desensitize the stimulated receptor, increasing evidence support the signaling of GPCRs on the endocytic vesicles. The trafficking patterns of receptor-β-Arrestin complexes may play an important role in the assembly of this signalosomes and their activation β-Arresstin 2 recruitment to CXCR4 mutants by BRET 2 . BRET 2 assay, in which CXCR4 is tagged with RLuc3 and β-arrestin2, is tagged with GFP 10 . RLuc3, when stimulated with a substrate, luminesces. Upon recruitment, the two tags come to proximity and give a fluorescent BRET 2 signal. β-Arresstin 2 recruitment to CXCR4 mutants by BRET 2 . BRET 2 assay, in which CXCR4 is tagged with RLuc3 and β-arrestin2, is tagged with GFP 10 . RLuc3, when stimulated with a substrate, luminesces. Upon recruitment, the two tags come to proximity and give a fluorescent BRET 2 signal. OBJECTIVES • Evaluate the relation between the Gα/i and β-arrestin pathways with CXCR4. • Determine the divers modalities of β-arrestin recruitment and their roles. • Assay the effect of TC14012 and AMD3100 on β-arrestin recruitment. • Illustrate the different pathways of CXCR4 trafficking. β-arrestin2 recruitment by CXCR4 mutants using BRET2. • N119S and R134A show high basal BRET signals. • N119K is unable to recruit β-arrestin2. • β-arrestin2 recruitment is independent of Gα/i activation (R134A). Titrations of β-arrestin2 to evaluate the constitutive recruitment. • N119S and R134A constitutively recruit β-arrestin2 while still showing variation after stimulation with CXCL12, in either the affinity (N119S) or the conformation (R134A). Test the effect TC14012 and AMD3100 on β-arrestin2 recruitment. • TC14012 is a biased inverse agonist showing only an effect on Gα/i. Evaluating the effect of TC14012 on β-arrestin2 recruitment by N119S. Inhibition of Gα/i using Pertussis Toxin (PTX). • β-arrestin2 recruitment to CXCR4 is independent of Gα/i activation. Basal titrations of the β-arrestin2 constitutive mutants R134A and N119S. • Different modalities of β-arrestin2 recruitment by the two mutants. ACKNOWLEDGEMENTS Live cell imaging of HEK293E cells transfected with CXCR4-YFP mutants. • β-arrestin2 recruitment to CXCR4 is independent of Gα/i activation. • TC14012 is a biased CXCR4 inverse agonist with only an effect on Gα/i. • CXCR4 shows different modalities of β-arrestin2 recruitment associated with divers localization. 1. P. M. Murphy, N. Engl. J. Med. 345, 833–835 (2001). 2. R. Zhang, X. Xie, Acta Pharmacol. Sin. 33, 372–384 (2012). 3. W.-B. Zhang et al., J. Biol. Chem. 277, 24515–24521 (2002). 4. B. Ponsioen et al., EMBO Rep. 5, 1176–1180 (2004). 5. Y. A. Berchiche et al., J. Biol. Chem. 282, 5111–5115 (2007). 6. L. M. Luttrell, R. J. Lefkowitz, J. Cell. Sci. 115, 455–465 (2002). 7. E. Reiter et al., Annu. Rev. Pharmacol. Toxicol. 52, 179–197 (2012). PERSPECTIVES 1. Assay the phosphorylation of CXCR4 associated with the different β- arrestin2 modalities. 2. Evaluate the basal level and the stimulated phosphorylation of ERK with the mutants. 3. Identify the localization of the different mutants. 4. Evaluate their chemotactic activity. 5. Test the effect of mutants and compounds on cell survival.
Transcript of (--THIS SECTION DOES NOT PRINT--) Nassr Nama1,2,3 ... · PDF filetitles, subtitles, text, ......
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CXCR4, a chemokine receptor involved in metastasis, signals through two major successive pathways: Gα/i and β-arrestin. β-arrestin terminates G-protein signaling and targets the receptor to endocytosis. Constitutively active GPCR mutants (CAMs) spontaneously activate G-protein. Constitutively inactive mutants (CIMs) that spontaneously recruit β-arrestin are devoid of G-protein signaling. We tested a set of CXCR4 mutants on their signaling capacity of Gα/i and ß-Arrestin2 pathways. R134A was found a CIM that was inactive on the Gα/i signaling, but constitutively recruited arrestin. N119S was found as a CAM, constitutively active in G-protein signaling as well as β-arrestin recruitment. Imaging of these two mutants showed their constitutive internalization and their different localization in the cell. These results show the presence of varying β-arrestin recruitment, independent of the G-protein signaling. Further studies remain to conclude whether the difference of β-arrestin recruitment is directed by the GRKs or the ubiquitination of the receptor.
ABSTRACT
WT D133N R134A Y135A
N119D N119S N119K
INTRODUCTION
Recruitment of β-arrestins and its modalities are mainly regulated by the GPCR kinases (GRKs). Four of these regulate the CXCR4, GRK 2,3,5 and 6. The divers modalities of β-arrestins recruitment induce different signaling pathways such as internalization or ERK signaling.
MATERIAL&METHODS
Gα/i activation measured as cAMP reduction by CXCR4 using EPAC. • The method does not permit showing the constitutive activity of N119S as
previously demonstrated (Zhang et al.). • N119K and R134A are unable to activated Gα/i.
RESULTS
REFERENCES
CONCLUSIONS
I would like to thank Dr. Michel Bouvier and Guillaume Sylvain-Drolet for the plasmids, Nicolas Montpas for his training and advice, and François Guité-Vinet for being a constant source of inspiration.
Metastasis is the major mortality cause in cancer. Detection of cancer patients early before the metastatic process increase the chances of treatment by chemotherapy or surgery. Thus it is primordial to understand the process of metastatic spread in order to be able to predict its targets, and to develop specific drugs to obstruct it.
CXCR4, a G-protein coupled receptor (GPCR), binds one chemokine ligand, CXCL12 (SDF-1α). Breast cancer cells that express the CXCR4 tend to migrate to organs with a high level of CXCL12, such as lung, liver, and bone marrow.
1.DepartmentofBiochemistry,UniversitédeMontréal.2.DepartmentofMicrobiologyandImmunology,UniversitédeMontréal.3.CHUSte-JusFneChildrenHospital.4.DepartmentofPathologyandCellularBiology,UniversitédeMontréal.
NassrNama1,2,3,StéphanieGravel1,3,AmelKechad3,4,GillesHickson3,4,NikolausHeveker1,3
DiverseModaliHesOfΒ-ArresHn2RecruitmentToCXCR4,IndependentlyOfG-ProteinAcHvaHon
-∞ -12 -10 -8 -60
20
40
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[CXCL12]
% F
orsk
olin
Inhi
bitio
n CXCR4-wt
-∞ -12 -10 -8 -60
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100
[CXCL12]
% F
orsk
olin
Inhi
bitio
n
CXCR4-N119DCXCR4-wt
[CXCL12]
% F
orsk
olin
Inhi
bitio
n
-∞0
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-12 -10 -8 -6
CXCR4-D133NCXCR4-wt
-∞ -12 -10 -8 -60
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orsk
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[CXCL12]
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orsk
olin
Inhi
bitio
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-12 -10 -8 -6
CXCR4-R134ACXCR4-wt
[CXCL12]
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orsk
olin
Inhi
bitio
n
-∞0
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-12 -10 -8 -6
CXCR4-Y135ACXCR4-wt
-∞0
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-12 -10 -8 -6
% F
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olin
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bitio
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[CXCL12]
CXCR4-wtCXCR4 N119K
-10 -8 -60.00
0.02
0.04
0.06
0.08
0.10
0.12
[CXCL12]
BR
ET2
NE
T
CXCR4 wt
-10 -8 -60.00
0.02
0.04
0.06
0.08
0.10
0.12
[CXCL12]
BR
ET2
NE
T
CXCR4 wtCXCR4 N119D
-10 -8 -60.00
0.02
0.04
0.06
0.08
0.10
0.12
[CXCL12]
BR
ET2
NE
T
CXCR4 wtCXCR4 D133N
-10 -8 -60.00
0.02
0.04
0.06
0.08
0.10
0.12
[CXCL12]
BR
ET2
NE
T
CXCR4 wtCXCR4 N119S
-10 -8 -60.00
0.02
0.04
0.06
0.08
0.10
0.12
[CXCL12]
BR
ET2
NE
T
CXCR4 wtCXCR4 R134A
-10 -8 -60.00
0.02
0.04
0.06
0.08
0.10
0.12
[CXCL12]
BR
ET2
NE
T
CXCR4 wtCXCR4 N119K
-10 -8 -60.00
0.02
0.04
0.06
0.08
0.10
0.12
[CXCL12]
BR
ET2
NE
T
CXCR4 wtCXCR4 Y135A
CXCR4 Wt
0 200 400 600 800 10000.00
0.03
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0.09
0.12
0.15
GFP10/RLuc3
BR
ET2 N
ET
SDF + 200 nMSDF-
CXCR4 R134A
0 200 400 600 800 10000.00
0.03
0.06
0.09
0.12
0.15
GFP10/RLuc3
BR
ET2 N
ET
SDF-SDF + 200 nM
CXCR4 N119S
0 500 1000 15000.00
0.03
0.06
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GFP10/RLuc3
BR
ET2 N
ET
SDF-SDF + 200 nM
Forsko
lin
CXCL12
AMD3100
TC1401
2
Forsko
lin
CXCL12
AMD3100
CXCL12 +
AMD3100
TC1401
2
Forsko
lin
CXCL12
AMD3100
TC1401
2
Forsko
lin
CXCL12
AMD3100
TC1401
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orsk
olin
Inhi
bitio
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**
**** ****
****
pcDNA3 WT R134A N119S
N.S
CXCL12
AMD3100
CXCL12 +
AMD3100
TC1401
2N.S
CXCL12
AMD3100
TC1401
2N.S
CXCL12
AMD3100
TC1401
20.00
0.02
0.04
0.06
0.08
BR
ET2 N
ET
****
****
*******
*****
****
******
WT R134A N119S
-∞ -10 -8 -60.00
0.02
0.04
0.06
0.08
0.10
[TC14012]
BR
ET2 N
ET
CXCR4 N119SCXCR4 WT
0 300 600 900 1200 15000.00
0.02
0.04
0.06
0.08
GFP10/RLuc3
BR
ET2 N
ET
N.STC14012
N.S
Forsko
lin
CXCL120.12
0.14
0.16
0.18
0.20
BR
ET2
NE
T
N.S
CXCL12
AMD3100
CXCL12 +
AMD3100
TC1401
2N.S
CXCL12
AMD3100
CXCL12 +
AMD3100
TC1401
2N.S
CXCL12
AMD3100
CXCL12 +
AMD3100
TC1401
20.00
0.03
0.06
0.09
0.12
BR
ET2 N
ET
***
****
WT R134A N119S
0 300 600 900 1200 15000.00
0.02
0.04
0.06
0.08
GFP10/RLuc3
BR
ET2 N
ET
CXCR4 R134A
CXCR4 WT
CXCR4 N119S
CXCR4 signals through two major pathways: Gα/i and β-arrestins. Gα/i inhibits adenylyl cyclase, responsible of generating cAMP. CXCR4 recruits also the β-arrestin proteins (1 and 2), and they shut off the signal transduction via Gα/i, by desensitization. β-arrestins target the desensitized receptor to endocytosis. β-arrestins are able to induce signaling effects too, by activating the ERK pathway leading to enhanced chemotaxis and cellular survival.
While the previously believe function of endocytosis is to desensitize the stimulated receptor, increasing evidence support the signaling of GPCRs on the endocytic vesicles. The trafficking patterns of receptor-β-Arrestin complexes may play an important role in the assembly of this signalosomes and their activation
β-Arresstin 2 recruitment to CXCR4 mutants by BRET2. BRET2 assay, in which CXCR4 is tagged with RLuc3 and β-arrestin2, is tagged with GFP10. RLuc3, when stimulated with a substrate, luminesces. Upon recruitment, the two tags come to proximity and give a fluorescent BRET2 signal.
β-Arresstin 2 recruitment to CXCR4 mutants by BRET2. BRET2 assay, in which CXCR4 is tagged with RLuc3 and β-arrestin2, is tagged with GFP10. RLuc3, when stimulated with a substrate, luminesces. Upon recruitment, the two tags come to proximity and give a fluorescent BRET2 signal.
OBJECTIVES• Evaluate the relation between the Gα/i and β-arrestin pathways with
CXCR4. • Determine the divers modalities of β-arrestin recruitment and their roles. • Assay the effect of TC14012 and AMD3100 on β-arrestin recruitment. • Illustrate the different pathways of CXCR4 trafficking.
β-arrestin2 recruitment by CXCR4 mutants using BRET2. • N119S and R134A show high basal BRET signals. • N119K is unable to recruit β-arrestin2. • β-arrestin2 recruitment is independent of Gα/i activation (R134A).
Titrations of β-arrestin2 to evaluate the constitutive recruitment. • N119S and R134A constitutively recruit β-arrestin2 while still showing
variation after stimulation with CXCL12, in either the affinity (N119S) or the conformation (R134A).
Test the effect TC14012 and AMD3100 on β-arrestin2 recruitment. • TC14012 is a biased inverse agonist showing only an effect on Gα/i.
Evaluating the effect of TC14012 on β-arrestin2 recruitment by N119S.
Inhibition of Gα/i using Pertussis Toxin (PTX). • β-arrestin2 recruitment to CXCR4 is independent of Gα/i activation.
Basal titrations of the β-arrestin2 constitutive mutants R134A and N119S. • Different modalities of β-arrestin2 recruitment by the two mutants.
ACKNOWLEDGEMENTS
Live cell imaging of HEK293E cells transfected with CXCR4-YFP mutants.
• β-arrestin2 recruitment to CXCR4 is independent of Gα/i activation. • TC14012 is a biased CXCR4 inverse agonist with only an effect on Gα/i. • CXCR4 shows different modalities of β-arrestin2 recruitment associated
with divers localization.
1. P. M. Murphy, N. Engl. J. Med. 345, 833–835 (2001). 2. R. Zhang, X. Xie, Acta Pharmacol. Sin. 33, 372–384 (2012). 3. W.-B. Zhang et al., J. Biol. Chem. 277, 24515–24521 (2002). 4. B. Ponsioen et al., EMBO Rep. 5, 1176–1180 (2004). 5. Y. A. Berchiche et al., J. Biol. Chem. 282, 5111–5115 (2007). 6. L. M. Luttrell, R. J. Lefkowitz, J. Cell. Sci. 115, 455–465 (2002). 7. E. Reiter et al., Annu. Rev. Pharmacol. Toxicol. 52, 179–197 (2012).
PERSPECTIVES1. Assay the phosphorylation of CXCR4 associated with the different β-
arrestin2 modalities. 2. Evaluate the basal level and the stimulated phosphorylation of ERK with
the mutants. 3. Identify the localization of the different mutants. 4. Evaluate their chemotactic activity. 5. Test the effect of mutants and compounds on cell survival.
