Differential parathyroid hormone receptor signaling directed by adaptor proteins:
Gespeichert in:
| 1. Verfasser: | |
|---|---|
| Format: | Abschlussarbeit Buch |
| Sprache: | English |
| Veröffentlicht: |
2012
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| Schlagworte: | |
| Online-Zugang: | Volltext Inhaltsverzeichnis |
| Beschreibung: | Online erschienen: 2013 |
| Beschreibung: | VII, 120, IV Blatt graph. Darst. |
Internformat
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| 245 | 1 | 0 | |a Differential parathyroid hormone receptor signaling directed by adaptor proteins |c von Alexander Emami-Nemini |
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| 264 | 1 | |c 2012 | |
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Datensatz im Suchindex
| _version_ | 1804151522397782016 |
|---|---|
| adam_text | TABLE OF CONTENTS
1. INTRODUCTION 1
1.1. BACKGROUND 1
1.2. G PROTEIN-COUPLED RECEPTORS 1
1.2.1. SIGNAL TRANSDUCTION VIA G PROTEINS 2
1.2.2. P-ARRESTIN 4
1.2.3. G PROTEIN-INDEPENDENT SIGNAL TRANSDUCTION BY GPCRS 5
1.2.4. BIASED AGONISM 5
1.2.5. CLASSIFICATION OF GPCRS 6
1.3. THE PARATHYROID HORMONE RECEPTOR 6
1.3.1. STRUCTURE-FUNCTION RELATIONSHIPS OF THE PTHR 7
1.3.2. PARATHYROID HORMONES 9
1.3.3. PARATHYROID HORMONE RECEPTOR ACTIVATION AND SIGNALING 10
1.3.4. PTH-MEDIATED MITOGEN-ACTIVATED PROTEIN KINASE ACTIVATION 12
1.3.5. ALTERNATIVE DOWNSTREAM EFFECTS OF PTHR SIGNALING 12
1.3.6. PARATHYROID HORMONE-REGULATED BONE METABOLISM 13
1.3.7. PARATHYROID HORMONE RECEPTORS IN PHARMACOLOGICAL TREATMENT 14
1.3.8. BIASED AGONISTS AND BONE FORMATION 15
1.4. SODIUM/HYDROGEN-EXCHANGER REGULATORY FACTOR (NHERF) 16
1.5. RHO FAMILY OF SMALL GTPASES 17
1.6. VAV FAMILY OF GUANINE-NUCLEOTIDE EXCHANGE FACTORS 18
1.6.1. THE GUANINE-NUCLEOTIDE EXCHANGE FACTOR VAV2 20
1.6.2. REGULATION OF RHOA GUANINE-NUCLEOTIDE EXCHANGE FACTORS BY GPCRS
21
1.7. FLUORESCENCE LIGAND BINDING TOOLS FOR PTHR 22
1.8. MOTIVATION 24
2. MATERIALS 26
2.1. EUKARYOTIC CELL CULTURE 26
2.1.1. CELL LINES 26
2.1.2. MEDIUM 26
2.1.3. TRANSFECTION REAGENTS 26
2.2. BACTERIAL CELL CULTURE 27
IV
HTTP://D-NB.INFO/1045320749
2.2.1. BACTERIA STRAINS 27
2.2.2. MEDIA FOR BACTERIA 27
2.3. BUFFER AND SOLUTIONS 27
2.3.1. BUFFER FOR MOLECULAR BIOLOGY 27
2.3.2. BUFFER FOR BIOCHEMISTRY 28
2.3.3. STAINING SOLUTIONS 30
2.4. ANTIBODIES 30
2.5. PLASMIDS 31
2.6. SMALL INTERFERING RNA 31
2.7. ENZYMES 32
2.8. COLUMNS AND RESIN FOR CHROMATOGRAPHY 32
2.9. PARATHYROID HORMONE ANALOGUES AND POLYPEPTIDES 32
2.10. FLUORESCENT PTH ANALOGUES 33
2.11. CHEMICALS 33
3. METHODS 35
3.1. GENETIC ENGINEERING 35
3.1.1. DNA ISOLATION AND PREPARATION 35
3.1.2. POLYMERASE CHAIN REACTION 35
3.1.3. DNA GEL ELECTROPHORESIS 35
3.1.4. RESTRICTION OF DNA 36
3.1.5. LIGATION OF DNA 36
3.1.6. TRANSFORMATION OF E.COII STRAINS 36
3.2. CELL CULTURE 37
3.2.1. BACTERIAL CELL CULTURE 37
3.2.2. EUKARYOTIC CELL CULTURE 37
3.2.3. TRANSFECTIONS 37
3.2.4. GENERATION OF STABLY EXPRESSING CELL LINES 38
3.3. BASIC BIOCHEMICAL TECHNIQUES 39
3.3.1. SODIUM DODECYL SULFATE POLYACRYLAMIDE GEL ELECTROPHORESIS 39
3.3.2. SILVER-STAINING 40
3.3.3. COOMASSIE STAINING 40
3.3.4. WESTERN-BLOTS AND IMMUNOBLOTTING 40
3.4. PROTEOMIC APPROACH 41
V
3.4.1. RECOMBINANT EXPRESSION AND PURIFICATION OF THE PTHR C-TERMINUS 41
3.4.2. PREPARATION OF KIDNEY AND OSTEOSARCOMA CELL LYSATES 42
3.4.3. INTERACTION SCREENING WITH TISSUE AND CELL LYSATES 42
3.4.4. MASS SPECTROMETRY AND DATA ANALYSIS 43
3.5. PROTEIN BIOCHEMISTRY 45
3.5.1. IMMUNOPRECIPITATION OF VAV2 AND PTHR 45
3.5.2. IMMUNOFLUORESCENCE 45
3.5.3. IMMUNOPRECIPITATION AND DETECTION OF PHOSPHO-PROTEINS 46
3.5.4. CO-IMMUNOPRECIPITATION OF G-ALPHA SUBUNITS 47
3.5.5. ACCUMULATION OF IP OR CAMP IN LIVING CELLS 47
3.6. TIME-RESOLVED FLUORESCENCE LIGAND BINDING ASSAYS 49
3.6.1. TIME-RESOLVED FLUORESCENCE SEPARATION ASSAY (TRFS) 49
3.6.2. HOMOGENEOUS TIME-RESOLVED FLUORESCENCE USING SNAP-PTHRS 50
3.6.3. HOMOGENEOUS TIME-RESOLVED FLUORESCENCE WITH ANTIBODIES 51
3.7. MICROSCOPY 51
3.7.1. WIDE-FIELD MICROSCOPY 51
3.7.2. CONFOCAL MICROSCOPY 52
3.8. DATA ANALYSIS AND SOFTWARE 52
4. RESULTS 53
4.1. PTHR INTERACTING PROTEINS - A PROTEOMIC APPROACH 53
4.1.1. EXPRESSION AND PURIFICATION OF THE PTHR C-TERMINAL DOMAIN 55
4.1.2. INTERACTION SCREENINGS WITH TISSUE LYSATES 57
4.1.3. VALIDATION OF CONSTRUCT INTEGRITY AFTER INCUBATION WITH TISSUE
LYSATES 58
4.1.4. MASS SPECTROMETRY ANALYSIS 60
4.2. VAV2 DIRECTS PTHR SIGNALING 63
4.2.1. VAV2 INTERACTS WITH INTRACELLULAR DOMAINS OF PTHR 63
4.2.2. PTH INDUCES PHOSPHORYLATION OF VAV2 68
4.2.3. VAV2 SPECIFICALLY COUPLES TO
GCTQ-
BUT NOT TO
GA
S
-G
PROTEIN SUBUNITS 71
4.2.4. VAV2 AND ACTIVATED G-ALPHA
Q
COMPETE FOR PTHR 72
4.2.5. PTH-INDUCED PLC SIGNALING IS IMPAIRED BY VAV2 75
4.2.6. PLC-PATHWAY INHIBITION BY VAV2 REQUIRES MERM DOMAIN OF NHERF 77
4.3. LIGAND BINDING ASSAYS FOR PARATHYROID HORMONE RECEPTORS 79
4.3.1. TIME-RESOLVED FLUORESCENCE PTH BINDING ASSAYS 79
VI
4.3.2. GENERATION OF FLUORESCENT PTH ANALOGUES 80
4.3.3. EVALUATION OF THE INTEGRITY OF FLUORESCENT PTH ANALOGUES 80
4.3.4. TIME-RESOLVED FLUORESCENCE SEPARATION ASSAY (TRFS) 81
4.3.5. HOMOGENEOUS TIME-RESOLVED FLUORESCENCE BY TAG-LITE TECHNOLOGY 84
4.3.6. ANTIBODY-BASED HOMOGENEOUS TIME-RESOLVED FLUORESCENCE 87
5. DISCUSSION 92
5.1. IDENTIFICATION OF ADAPTOR PROTEINS FOR THE PTHR 92
5.2. VAV2 AFFECTS PTHR SIGNALING 94
5.2.1. VAV2 BINDS TO INTRACELLULAR DOMAINS OF PTHR 94
5.2.2. VAV2 PHOSPHORYLATION IS REGULATED BY PTH-INDUCED PTHR ACTIVATION
95
5.2.3. VAV2 COMPETES WITH
GOIQ
FOR COUPLING TO PTHR 96
5.2.4. VAV2 AFFECTS IP GENERATION DEPENDENT ON NHERF MERM
DOMAINS 97
5.2.5. VAV2 AND BONE METABOLISM 99
5.3. TIME-RESOLVED LIGAND BINDING ASSAYS FOR PTHR 100
5.3.1. TIME-RESOLVED FLUORESCENCE SEPARATION ASSAY (TRFS) 100
5.3.2. HOMOGENEOUS TIME-RESOLVED FLUORESCENCE TAG-LITE TECHNOLOGY 102
5.3.3. ANTIBODY-BASED HOMOGENEOUS TIME-RESOLVED FLUORESCENCE 103
6. CONCLUSIONS 104
7. ABBREVIATIONS 107
8. LITERATURE 110
9. ANNEX 120
VII
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| spelling | Emami-Nemini, Alexander Verfasser (DE-588)1044032251 aut Differential parathyroid hormone receptor signaling directed by adaptor proteins von Alexander Emami-Nemini Steuerung differenzieller Signalgebung des Parathormon Rezeptors durch Adapterproteine 2012 VII, 120, IV Blatt graph. Darst. txt rdacontent n rdamedia nc rdacarrier Online erschienen: 2013 Würzburg, Univ., Diss., 2012 Guaninnucleotid-Austauschfaktoren (DE-588)4796677-4 gnd rswk-swf Parathormon (DE-588)4173328-9 gnd rswk-swf G-Protein gekoppelter Rezeptor (DE-588)7723480-7 gnd rswk-swf (DE-588)4113937-9 Hochschulschrift gnd-content G-Protein gekoppelter Rezeptor (DE-588)7723480-7 s Parathormon (DE-588)4173328-9 s Guaninnucleotid-Austauschfaktoren (DE-588)4796677-4 s DE-604 Erscheint auch als Online-Ausgabe urn:nbn:de:bvb:20-opus-72369 https://nbn-resolving.org/urn:nbn:de:bvb:20-opus-72369 Resolving-System kostenfrei Volltext DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=026858448&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Emami-Nemini, Alexander Differential parathyroid hormone receptor signaling directed by adaptor proteins Guaninnucleotid-Austauschfaktoren (DE-588)4796677-4 gnd Parathormon (DE-588)4173328-9 gnd G-Protein gekoppelter Rezeptor (DE-588)7723480-7 gnd |
| subject_GND | (DE-588)4796677-4 (DE-588)4173328-9 (DE-588)7723480-7 (DE-588)4113937-9 |
| title | Differential parathyroid hormone receptor signaling directed by adaptor proteins |
| title_alt | Steuerung differenzieller Signalgebung des Parathormon Rezeptors durch Adapterproteine |
| title_auth | Differential parathyroid hormone receptor signaling directed by adaptor proteins |
| title_exact_search | Differential parathyroid hormone receptor signaling directed by adaptor proteins |
| title_full | Differential parathyroid hormone receptor signaling directed by adaptor proteins von Alexander Emami-Nemini |
| title_fullStr | Differential parathyroid hormone receptor signaling directed by adaptor proteins von Alexander Emami-Nemini |
| title_full_unstemmed | Differential parathyroid hormone receptor signaling directed by adaptor proteins von Alexander Emami-Nemini |
| title_short | Differential parathyroid hormone receptor signaling directed by adaptor proteins |
| title_sort | differential parathyroid hormone receptor signaling directed by adaptor proteins |
| topic | Guaninnucleotid-Austauschfaktoren (DE-588)4796677-4 gnd Parathormon (DE-588)4173328-9 gnd G-Protein gekoppelter Rezeptor (DE-588)7723480-7 gnd |
| topic_facet | Guaninnucleotid-Austauschfaktoren Parathormon G-Protein gekoppelter Rezeptor Hochschulschrift |
| url | https://nbn-resolving.org/urn:nbn:de:bvb:20-opus-72369 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=026858448&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
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