Functional characterization of the cytoplasmic domain of LRP2:
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| Format: | Abschlussarbeit Buch |
| Sprache: | English |
| Veröffentlicht: |
Berlin
2016
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| Online-Zugang: | Inhaltsverzeichnis |
| Beschreibung: | VI, 172 Seiten Illustrationen, Diagramme |
Internformat
MARC
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|---|---|---|---|
| 001 | BV044032457 | ||
| 003 | DE-604 | ||
| 005 | 20220915 | ||
| 007 | t | ||
| 008 | 170207s2016 a||| m||| 00||| eng d | ||
| 035 | |a (OCoLC)973044763 | ||
| 035 | |a (DE-599)BVBBV044032457 | ||
| 040 | |a DE-604 |b ger |e rda | ||
| 041 | 0 | |a eng | |
| 049 | |a DE-11 | ||
| 084 | |a WE 5200 |0 (DE-625)148300: |2 rvk | ||
| 100 | 1 | |a Herzog, Katja |d 1986- |e Verfasser |0 (DE-588)1125540990 |4 aut | |
| 245 | 1 | 0 | |a Functional characterization of the cytoplasmic domain of LRP2 |c von M.Sc. Katja Herzog |
| 264 | 1 | |a Berlin |c 2016 | |
| 300 | |a VI, 172 Seiten |b Illustrationen, Diagramme | ||
| 336 | |b txt |2 rdacontent | ||
| 337 | |b n |2 rdamedia | ||
| 338 | |b nc |2 rdacarrier | ||
| 502 | |b Dissertation |c Humboldt-Universität zu Berlin |d 2016 | ||
| 655 | 7 | |0 (DE-588)4113937-9 |a Hochschulschrift |2 gnd-content | |
| 776 | 0 | 8 | |i Reproduziert als |a Herzog, Katja |t Functional characterization of the cytoplasmic domain of LRP2 |d Berlin : MIK-Center GmbH, 2016 |h 2 Mikrofiches |w (DE-604)BV044313994 |
| 856 | 4 | 2 | |m DNB Datenaustausch |q application/pdf |u http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=029439693&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |3 Inhaltsverzeichnis |
| 999 | |a oai:aleph.bib-bvb.de:BVB01-029439693 | ||
Datensatz im Suchindex
| _version_ | 1804177036649955328 |
|---|---|
| adam_text | TABLE OF CONTENT
1
ZUSAMMENFASSUNG..........................................................................................................1
2 SUMMARY
.........................................................................
2
3 INTRODUCTION
.......
.
...........................................................................................................
3
3.1 T
HE
LOW
-
DENSITY
LIPOPROTEIN
(LDL)
RECEPTOR
GENE
FAMILY
........................................3
3.1.1 STRUCTURE OF
LRPS......................................................................................................3
3.1.2 EVOLUTION OF LDL RECEPTOR-RELATED
PROTEINS...............................................................6
3.2
LRP2................................................................................................................................6
3.2.1 STRUCTURE OF
LRP2.....................................................................................................6
3.2.2 LRP2 FUNCTIONS AS A MORPHOGEN RECEPTOR DURING MAMMALIAN
DEVELOPMENT
...........
7
3.2.3 ROLE OF LRP2 IN THE ADULT
ORGANISM........................................................................
10
3.3 I
NTRACELLULAR
FATE
OF
LRP2........................................................................................13
3.3.1 INTRACELLULAR TRAFFICKING AND RECEPTOR LOCALIZATION
.....................................................
13
3.3.2 ROLE FOR LRP2 IN CELL
SIGNALING...............................................................................17
4
AIMS.....................................................................................................................................23
5
RESULTS.............................................................................................................................24
5.1 G
ENERATION
OF
CYTOPLASMIC
TAIL
MUTANTS
OF
LRP2..................................................24
5.1.1 TARGETING STRATEGY TO INTRODUCE KNOCK-IN MUTATIONS IN THE LRP2
LOCUS
.....................
24
5.1.2 IDENTIFYING POSITIVE MURINE EMBRYONIC STEM CELLS CARRYING LRP2
KNOCK-IN ALLELES ....25
5.1.3 ADULT FOUNDERS AND GENOTYPIC
DISTRIBUTIONS.............................................................29
5.1.4 EMBRYONIC LETHALITY OF THE 1ST APDZ EMBRYONIC STEM CELL CLONE
..............................
32
5.1.5 RATIONAL FOR USING COMPOUND HETEROZYGOUS ANIMALS TO STUDY
PHYSIOLOGICAL
PHENOTYPES..............................................................................................................33
5.2 R
OLE
OF
THE
INTRACELLULAR
DOMAINS
OF
LRP2
IN
ADULT
KIDNEY
FUNCTION
..................
36
5.2.1 NORMAL EMBRYONIC DEVELOPMENT OF APDZ, APA AND DPD MUTANTS
........................
36
5.2.2 RENAL RESORPTION REQUIRES INTRACELLULAR INTERACTION AND
PHOSPHORYLATION OF LRP2... 39
5.2.3 INTRACELLULAR DOMAINS OF LRP2 ARE DISPENSABLE FOR NORMAL KIDNEY
MORPHOLOGY
.....
41
5.3 S
IGNAL
TRANSDUCTION
BY
LRP2
REQUIRES
PHOSPHORYLATION
OF
THE
RECEPTOR
.........
43
5.3.1 LRP2 TRANSCRIPTION DEPENDS ON PROPER PHOSPHORYLATION OF THE
CYTOPLASMIC TAIL OF THE
RECEPTOR
......
............................................................................................................
43
5.3.2 DETECTION OF THE LRP2-ICD IN NUCLEAR
EXTRACTS.......................................................45
5.3.3 LRP2 TAIL MODIFICATION DOES NOT IMPACT RECEPTOR
SHEDDING....................................46
5.4 F
UNCTIONAL
RELEVANCE
OF
PDZ
INTERACTIONS
AT
THE
CYTOPLASMIC
TAIL
OF
LRP2
FOR
PROPER RECEPTOR TRAFFICKING IN THE PROXIMAL TUBULE
...............................................48
5.4.1 CYTOPLASMIC MUTANT VARIANTS OF LRP2 ARE EXPRESSED AT NORMAL LEVELS
AND LOCALIZE
PROPERLY TO THE APICAL SURFACE OF PROXIMAL TUBULE CELLS IN THE KIDNEY
.......................
48
5.4.2 SEGMENT-SPECIFIC SUBCELLULAR LOCALIZATION OF APDZ VARIANTS IN THE
KIDNEY
..............
50
5.4.3 ANALYSIS OF MEMBRANE PROTEINS THAT DEPEND ON LRP2-MEDIATED
TRAFFICKING
..........
50
5.4.4 APDZ VARIANTS ARE NOT GLOBALLY ENRICHED IN THE RENAL BRUSH BORDER
........................
53
5.5 L
IGAND
UPTAKE
STUDIES
IN
VIVO
AND
IN
VITRO
.................................................................56
5.5.1 IN VIVO ENDOCYTOSIS IN APDZ
MUTANTS.....................................................................56
5.5.2 LIGAND ENDOCYTOSIS IN PRIMARY PROXIMAL CONVOLUTED TUBULE CELLS
............................
59
5.6 I
NTRACELLULAR
TRAFFICKING
AND
ADAPTORS
.......................................... 62
5.6.1 DAB-2 AND
ENDOCYTOSIS..........................................................................................
62
5.6.2 NHERF1 AND ION TRANSPORTER
REGULATION..................................................................
64
5.6.3 GIPC1 LOCALIZATION TO THE APICAL MEMBRANE DEPENDS ON THE
PDZ-DOMAIN BINDING
SITE IN
LRP2.............................................................................................................
66
6
DISCUSSION........................................................................................................................
69
6.1 E
VOLUTION
OF
TAIL
MOTIFS
IN
THE
LDLR
GENE
FAMILY
.....................................................69
6.2 PDZ INTERACTIONS AT THE CARBOXY TERMINUS AND PHOSPHORYLATION AT THE
PPPSPS
MOTIF OF LRP2 ARE DISPENSABLE FOR EMBRYONIC DEVELOPMENT
...................................
74
6.2.1 A ROLE FOR INTRACELLULAR TAIL MOTIFS IN EMBRYONIC DEVELOPMENT?
.................................
74
6.2.2 FUNCTIONAL COMPENSATION BY OTHER MEMBERS OF THE SHH RECEPTORSOME
................
76
6.3 R
EGULATORY
MOTIFS
IN
THE
CYTOPLASMIC
DOMAIN
OF
LRP2
ARE
REQUIRED
FOR
RENAL
RESORPTION.....................................................................................................................
78
6.3.1 RELEVANCE OF THE INTRACELLULAR DOMAIN OF LRP2 FOR RENAL PHYSIOLOGY
.......................
78
6.3.2 A PHOSPHORYLATION SITE AND THE TERMINAL PDZ MOTIF IN THE TAIL OF
LRP2 ARE NOVEL
ELEMENTS TO CONTROL RENAL
ENDOCYTOSIS.....................................................................
80
6.4 R
OLE
OF
THE
CYTOPLASMIC
REGULATORY
MOTIFS
IN
LRP2
FOR
TRANSCRIPTIONAL
REGULATION.....................................................................................................................
81
6.4.1 PHOSPHORYLATION AT THE PROLINE-RICH REGION IN THE CYTOPLASMIC
DOMAIN OF LRP2
REGULATES ITS OWN
TRANSCRIPTION.................................................................................
81
6.4.2 TRANSCRIPTIONAL REGULATION OF LRP2 EXPRESSION
.............................................
82
6.4.3 RELEASE OF THE INTRACELLULAR DOMAIN OF LRP2 BY RIP AND NUCLEAR
TRANSLOCATION
.....
83
6.4.4 OTHER INTERACTION PARTNERS BINDING TO THE INTRACELLULAR DOMAIN OF
LRP2 AND
POTENTIALLY INVOLVING THE RECEPTOR IN TRANSCRIPTIONAL REGULATION
.................................
85
6.5 R
ENAL
SEGMENT
-
SPECIFIC
REGULATION
OF
LRP2
LOCALIZATION
BY
GIPC1
.....................
85
6.5.1 DIFFERENTIAL SORTING OF LRP2 IN THE S3 SEGMENT OF THE RENAL
PROXIMAL TUBULE
..........
87
6.5.2 PDZ DOMAIN-CONTAINING ADAPTORS IMPLICATED IN LRP2 ACTIVITY IN THE
S3 SEGMENT OF
THE
KIDNEY................................................................................................................
88
6.6 O
UTLOOK
........................................................................................................................
90
7 MATERIALS AND
METHODS..............................................................................................91
7.1 M
ATERIAL
........................................................................................................................
91
7.1.1 GENERAL TECHNICAL
EQUIPMENT..................................................................................
91
7.1.2 REAGENTS &
CHEMICALS...........................................................................................
91
7.1.3 BUFFERS &
SOLUTIONS.................................................................................................
92
7.1.4
KITS..........................................................................................................................
94
7.1.5 MOUSE-SPECIFIC
OLIGONUCLEOTIDES.............................................................................
94
7.1.6
ANTIBODIES...............................................................................................................
96
7.1.7 FLUOROPHORE-COUPLED TOXINS
....................................................................................
99
7.2 M
ICROBIOLOGICAL
METHODS
...........................................................................................99
7.2.1 BACTERIA CULTURE
MEDIUM.........................................................................................
99
7.2.2 TRANSFORMATION OF BACTERIA WITH PLASMID
DNA..........................................................99
7.2.3 BLUE-WHITE SELECTION OF BACTERIA
..............................................................................
99
7.2.4 CRYOPRESERVATION OF
BACTERIA..................................................................................100
7.2.5 PLASMID DNA ISOLATION FROM BACTERIA
....................................................................
100
7.3 M
OLECULAR
BIOLOGY
METHODS
.....................................................................................100
7.3.1 DNA FRAGMENT AMPLIFICATION BY
PCR......................................................................100
7.3.2 AGAROSE GEL ELECTROPHORESIS OF DNA FRAGMENTS AND RNA
....................................
101
7.3.3 DNA PURIFICATION FROM AGAROSE GELS
......................................................................
101
7.3.4 DETERMINATION OF DNA AND RNA CONCENTRATIONS
...................................................
101
7.3.5 ENZYMATIC DIGESTION OF
DNA.................................................................................
102
7.3.6 LIGATION OF DNA
FRAGMENTS....................................................................................
102
7.3.7 DNA
SEQUENCING....................................................................................................103
7.3.8 RNA ANALYSIS BY QUANTITATIVE REAL TIME PCR (QRT PCR)
.......................................
103
7.3.9 PROTEIN ANALYSIS BY WESTERN
BLOT...........................................................................
105
7.4 G
ENERATION
OF
KNOCK
-
IN
MOUSE
MODELS
.................................................................... 110
7.4.1 CLONING AND GENERATION OF TARGETING VECTOR CONSTRUCTS
.........................................
110
7.4.2 EMBRYONIC STEM (ES) CELL
CULTURE...........................................................................117
7.4.3 GENOTYPING OF MUTANT MICE AND EMBRYOS
.............................................................
120
7.5 C
ELL
CULTURE
..............................................................................................................124
7.5.1 CULTURE OF BROWN NORWAY RAT YOLK SAC CARCINOMA CELLS
..........................................
124
7.5.2 ISOLATION AND CULTURE OF PRIMARY PROXIMAL CONVOLUTED TUBULE CELLS
(PCTS)
...........
124
7.6 H
ISTOLOGY
& S
TAININGS
...............................................................................................126
7.6.1 TISSUE CONSERVATION BY
PERFUSIONS.........................................................................
126
7.6.2 PREPARATION OF TISSUE SECTIONS ON GLASS SLIDES
......................................................
127
7.6.3 HEMATOXYLIN AND EOSIN (HE) STAINING
...................................................................
128
7.6.4
IMMUNOHISTOCHEMISTRY..........................................................................................
128
7.6.5 IN SITU HYBRIDIZATIONS
(ISH)....................................................................................
129
7.7 C
ELLULAR
BIOLOGY
&
PHYSIOLOGY
METHODS
................................................................ 131
7.7.1 IN VIVO ENDOCYTOSIS QUANTIFICATION BY FLUORESCENCE UPTAKE MEASURES
.....................
131
7.7.2 ENDOCYTOSIS STUDIES IN
CELLS...................................................................................132
7.7.3 ANALYSIS OF URINE
PARAMETERS.................................................................................132
7.8 M
ICROSCOPY
.....................................................................................................
133
7.8.1 CONFOCAL
MICROSCOPY.............................................................................................
133
7.8.2 BRIGHT-FIELD
IMAGING................................................................................................133
7.8.3 IMAGE ACQUISITION
SOFTWARE...................................................................................
133
7.8.4
STEREOMICROSCOPY..................................................................................................134
7.9 A
NIMAL
EXPERIMENTS
....................................................................................................134
7.9.1 MOUSE
HUSBANDRY..................................................................................................134
7.9.2 ANESTHESIA OF
MICE...........................................................................................................
134
7.10 S
TATISTICS
..................................................................................................................
134
7.11 G
ENERAL
COMMENTS
..................................................................................................
134
8
BIBLIOGRAPHY................................................................................................................
135
9 LIST OF
ABBREVIATIONS...............................................................................................
163
10 LIST OF
FIGURES.............................................................................................................
167
11 LIST OF
TABLES...............................................................................................................
169
12 PUBLICATIONS, POSTERS & PARTICIPATIONS
...........................................................
170
12.1 P
UBLICATIONS
.............................................................................................................
170
12.2 A
CKNOWLEDGED
W
ORK
...............................................................................................
170
12.3 P
OSTER
& P
ARTICIPATIONS
.........................................................................................
170
12.4 S
CIENCE
COMMUNICATION
...........................................................................................
170
13
SELBSTSTAENDIGKEITSERKLAERUNG............................................................................
171
14 CURRICULUM
VITAE........................................................................................................
172
|
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| indexdate | 2024-07-10T07:41:42Z |
| institution | BVB |
| language | English |
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| spelling | Herzog, Katja 1986- Verfasser (DE-588)1125540990 aut Functional characterization of the cytoplasmic domain of LRP2 von M.Sc. Katja Herzog Berlin 2016 VI, 172 Seiten Illustrationen, Diagramme txt rdacontent n rdamedia nc rdacarrier Dissertation Humboldt-Universität zu Berlin 2016 (DE-588)4113937-9 Hochschulschrift gnd-content Reproduziert als Herzog, Katja Functional characterization of the cytoplasmic domain of LRP2 Berlin : MIK-Center GmbH, 2016 2 Mikrofiches (DE-604)BV044313994 DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=029439693&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Herzog, Katja 1986- Functional characterization of the cytoplasmic domain of LRP2 |
| subject_GND | (DE-588)4113937-9 |
| title | Functional characterization of the cytoplasmic domain of LRP2 |
| title_auth | Functional characterization of the cytoplasmic domain of LRP2 |
| title_exact_search | Functional characterization of the cytoplasmic domain of LRP2 |
| title_full | Functional characterization of the cytoplasmic domain of LRP2 von M.Sc. Katja Herzog |
| title_fullStr | Functional characterization of the cytoplasmic domain of LRP2 von M.Sc. Katja Herzog |
| title_full_unstemmed | Functional characterization of the cytoplasmic domain of LRP2 von M.Sc. Katja Herzog |
| title_short | Functional characterization of the cytoplasmic domain of LRP2 |
| title_sort | functional characterization of the cytoplasmic domain of lrp2 |
| topic_facet | Hochschulschrift |
| url | http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=029439693&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
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