Thèse realisée en co tutelle avec l'Université de Bâle

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Niveau: Supérieur, Doctorat, Bac+8
Thèse realisée en co-tutelle avec l'Université de Bâle Présentée pour obtenir le grade de Docteur de l'Université Louis Pasteur Strasbourg I Discipline: Sciences du vivant Spécialité: Aspects moléculaires et cellulaires de la biologie par Sophie Schlumberger Caractérisation des récepteurs de l'hormone de mélano-concentration (MCH) Soutenue le 26 juillet 2002 à 14 heures, devant le jury composé de: Directeur de Thèse: M. A.N. EBERLE, Professeur, Université de Bâle Co-Directeur de Thèse: Mme M-J. FREUND-MERCIER, Professeur, ULP Rapporteur Interne: M. P. PEVET, Directeur de Recherche, ULP Rapporteur Externe: Mme T.J. RESINK, Professeur, Université de Bâle Rapporteur Externe: M. H. VAUDRY, Professeur, Université de Rouen Faculté des sciences de la vie

  • thèse realisée en co-tutelle

  • putative mch

  • binding assay

  • faculté des sciences de la vie

  • mch

  • mch-r1

  • directeur de la thèse


Publié le : lundi 1 juillet 2002
Lecture(s) : 64
Source : scd-theses.u-strasbg.fr
Nombre de pages : 124
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Faculté des sciences de la vie
Thèse realisée en co-tutelle avec l’Université de Bâle
Présentée pour obtenir le grade de Docteur de l’Université Louis Pasteur Strasbourg I
Discipline: Sciences du vivant Spécialité: Aspects moléculaires et cellulaires de la biologie
par Sophie Schlumberger
Caractérisation des récepteurs de l’hormone de mélano-concentration (MCH)
Soutenue le 26 juillet 2002 à 14 heures, devant le jury composé de:
Directeur de Thèse: Bâle Co-Directeur de Thèse:
Rapporteur Interne:
Rapporteur Externe:
Rapporteur Externe:
M. A.N. EBERLE, Professeur, Université de
Mme M-J. FREUND-MERCIER, Professeur, ULP
M. P. PEVET, Directeur de Recherche, ULP Mme T.J. RESINK, Professeur, Université de Bâle M. H. VAUDRY, Professeur, Université de Rouen
Binding characteristics of MCH to MCH-R1_________________ 16
MCH-R1knockout mouse model__________________________ 19
MCH-R ________________________________________ 20 2
MCH /α-MSH antagonism at MCH-R1_____________________19
_
_
MCH transgenic mouse model____________________________ 13
a
e
b.
1.2.5.
1.2.2.
1.1.4.
1.2.3.
a
1.
c.
b.
a.
1.1.2.
1.1.1.
Introduction________________________________ 8
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_
Table of contents _____________________________ 1
In teleost fish____________________________________________ 12
1.2.7.
1.2.6.
1.3.
Page
Table of Contents
.
MCH-R ________________________________________14 1
MCH knockout mouse model_____________________________ 13
In cellular models of MCH-R1ectopic expression_____________17
MCH-R1signalling pathway_______________________________17
s
1.1.3.
1.2.4.
r
t
b
A
r
A
b
b
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_
1.1.
1.2.
1.2.1.
MCH-R1cloning_________________________________________ 14
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_
Structure and localisation___________________________________8
In mammals_____________________________________________12
MCH /α-MSH / NEI antagonism__________________________13
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_
Implication of MCH-R1in a human autoimmune disease______19
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c
_
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_
i
v
o
t
i
s
a
t
n
Table of contents
Melanin-concentrating hormone (MCH)_____________ 8
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7
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6
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1
In cells endogenously expressing MCH-R1__________________ 18
MCH-R1mRNA expression_______________________________ 15
Functions_______________________________________________ 12
Table of Contents
1.4.
1.5.
Putative MCH receptors on melanophores melanoma cells___________________________________22
Aims
of
the
and
thesis____________________________23
2
Table of Contents
2.
Methods___________________________________24
2.1.
2.1.1.
2.1.2.
2.2.
2.3.
2.3.1.
a.
b
.
c.
2.3.2.
a.
b.
2.3.3.
a.
b.
2.4.
2.4.1.
2.5.
2.6.
Cell culture______________________________________24
Cell lines used during this thesis work______________________ 24
Cell culture conditions and media__________________________ 25
Peptides used during this thesis work______________26
Binding assay___________________________________ 27
Binding assay with cell lines_______________________________ 27
Preparation of radioligand________________________________ 27
Receptor binding assay___________________________________ 28
Stability of radioligand after incubation_____________________ 29
Binding assay with porcine brain membranes________________29
Membrane preparation___________________________________ 29
Binding
assay____________________________________________29
Binding assay with embryonic rat neurons__________________ 30
Isolation and culture of the rat embryonic hypothalamic cells__ 30
Binding
assay____________________________________________30
125 Cross-linking of [ I]-MCH_______________________ 31
Cross-linking assay prior to 2-D electrophoresis______________31
Two-dimensional gel electrophoresis_______________ 32
Autoradiographic localisation of MCH binding sites in the central nervous system of the rat_______________33
2.7. Semi-quantitative RT-PCR_________________________34
2.8.
Immunofluorescence
studies_______________________35
3
Table of Contents
2.8.1.
2.8.2.
2.9.
2.10.
External
Internal
staining_________________________________________
35
staining__________________________________________35
Western blotting_________________________________35
FLIPR
assay__________________________________36
4
3.1.2.
Binding assays with porcine brain membranes_______________ 40
primary
3.Results____________________________________37
embryonic
Autoradiographic localisation of MCH binding sites in the central
unlabeled
Can the 50 kDa cross-linked band be isolated by two-
cell
hypothalamic
with
Flag-rat MCH-R1mRNA production________________________52
radioligand
neuroblastoma
Verification
3.1.3.
3.1.4.
3.1.5.
3.1.6.
3.1.7.
a.
3.3.1.
b.
Establishment
3.2.1.
3.2.
3.3.
transgene
proteins________49
Structure-activity
of
of
electrophoresis
with
the
technique
by
overexpressing rat MCH-R1______________________52
on
derivatives
Expression and localisation of Flag-rat MCH-R1______________ 53
Detection of MCH binding sites____________________37
125 13 18 A well established model: I-[DPhe , Tyr ]-MCH binding to the
RT-PCR
and
immunofluorescence______________________________________52
5
cells
?______________________
neurons_________________________________________________ 46
nervous system of the rat__________________________________47
expression
3.2.2.
3.1.
3.1.1.
49
Binding assays with neuroblastoma cell lines_________________38
to
of
rat
MCH
G4F-Cl1 melanoma cells___________________________________37
Binding
of
assays
Binding assay with human primary melanocytes_____________ 45
7
lines_____________________________________________________4
Cross-linking
Table of Contents
2-D electrophoresis of radioligand cross-linked sample________ 50
MCH
dimensional
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6
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0
_
54
6
os
s
-
3
.
3
blotting
assay____________________________________61
3.3.5.
Western
l
3.3.2.
Binding
assays___________________________________________
_
_
4
.
C
.
r
3.3.3.
_
Table of Contents
_
n
i
_
_
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_
_
s
y
a
a
FLIPR assays_____________________________________________56
g
s
i
n
s
k
endogenously
Table of Contents
4.3.
4.3.2.
cell
4.7
obesity
Choice of the radioligand to find novel MCH binding
sites____________________________________________68
Advantages of FLAGtag_______________________72
4.1.
FLIPR assay_____________________________________________ 66
overexpressing cells______________________________69
Binding site_____________________________________________ 71
line
MCH-R1immunofluorescence staining______________________63
for
The case of G4F-Cl1 mouse melanoma binding site___71
125 Competition assay with [ I]-MCH_________________________65
a
anorexia?_______________________________________75
line____________________________________________ 74
loss in humans___________________________________________75
7
and/or
neuroblastoma
Western blotting_________________________________________ 64
3.4.6.
3.4.
3.4.1.
RT-PCR for MCH, MCH-R1and -R2_________________________61
MCH:
3.4.5.
3.4.4.
3.4.3.
3.4.2.
expresses MCH-R1_______________________________ 61
Presence of MCH-R1in Kelly human neuroblastoma cell
MCH-R1regulation by MCH or leptin______________73
Kelly
target
drug
Isolation of the50 kDa cross-linked band__________________71
Example of a neuropeptide administration that induced body fat
Cross-linking
The
6
8
promising
_
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_
4.7.1.
4
.
D
i
s
c
4.2.
4.3.1.
_
4.6.
4.5.
4.4.
_
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_
_
assay_______________________________________66
of
MCH
_
_
_
_
_
_
_
_
on
MCH-R1
derivatives
_
n
o
i
s
s
u
Structure/activity
Table of Contents
4
.
7
.
2
.
Tools to study MCH-MCH-R1axis in humans________________76
8
Table of Contents
5.
6.
7.
Conclusion_________________________________78
5.1.
5.2.
5.2.1.
5.2.2.
a.
b
.
c.
d.
5.2.3.
Conclusion______________________________________78
Perspectives (proposal for future experiments)______ 79
With the Kelly human neuroblastoma cell line_______________ 79
With MCH-R1and -R2coding sequences____________________ 79
Intracellular localisation of MCH-R1and –R2_________________ 79
Study of intracellular dynamics of MCH-R1and –R2___________80
Study of MCH-R1and –R2homo- or heterodimerisation_______80
125 Mapping of the [ I]-MCH contact point on MCH-R1and R2___ 81
With the G4F-Cl1 mouse melanoma cells____________________ 81
References_________________________________82
Appendix__________________________________89
7.1.
7.2.
7.2.1.
7.2.2.
7.2.3.
7.3.
Acknowledgements______________________________89
Curriculum vitae_________________________________90
Personal information_____________________________________90
Education_______________________________________________90
Lectures attended during the Ph.D. fellowship_______________ 91
Original publications_____________________________ 92
9
Abbreviations
Abbreviations
α-MSH bp BSA CHAPS
CHO cpm DTT ECL EDTA FLIPR Fluo-3
GPCR HEK HEPES HPLC IP kDa MCH MCH-R1 MCH-R2 NP-40 PBS PCR PMSF Ponceau S
PP Probenecid RACE RT SDS-PAGE SEM TEMED TFA
α-melanocyte-stimulating hormone basepair (s) bovine serum albumin 3-[(3-cholamidopropyl)dimethyl-ammonio]-1-propane-sulfonate Chinese hamster ovary count (s) per minute dithiothreitol enhanced chemoluminescence ethylenediamine tetraacetic acid fluorimetric imaging plate reader 1-[2-Amino-5-(2,7-dichloro-6-hydroxy-3-oxy-9-xanthenyl)-phenoxy]-2-[2-amino-5-methylphenoxy]ethane-N,N,N’,N’-tetraacetic acid G protein-coupled receptor human embryonic kidney N-(2-hydroxyethyl-1-piperazine-N’-ethane)-sulfonic acid high pressure liquid chromatography intra peritoneal kilodalton (s) melanin-concentrating hormone melanin-concentrating hormone receptor 1 melanin-concentrating hormone receptor 2 nonidet P-40 phosphate-buffered saline polymerase chain reaction phenylmethylsulfonylfluoride 3-hydroxy-4-(2-sulfo-4-[4-sulfophenylazo]-phenylazo)-2,7-naphthalenedisulfonic acid polypropylene p-[Dipropylsulfamoyl]benzoic acid rapid amplification of cDNA ends reverse transcriptase sodium dodecyl sulfate poly acrylamide gel electrophoresis standard error of the mean N,N,N’,N’-tetramethylethylenediamine trifluoroacetic acid
1
0
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