Das Glykoprotein M6a beeinflusst Endozytose und Rezyklisierung, sowie die Desensibilisierung des {_m63-Opioidrezeptors [my-Opioidrezeptors] [Elektronische Ressource] / von Daifei Wu

Description

Das Glykoprotein M6a beeinflusst Endozytoseund Rezyklisierung, sowie die Desensibilisierungdes µ-OpioidrezeptorsDissertationzur Erlangung des akademischen Gradesdoctor rerum naturalium (Dr. rer. nat.)genehmigt durchdie Fakultät für Naturwissenschaftender Otto-von-Guericke-Universität Magdeburgvon Magister Daifei Wugeb. am 03. April 1969in Fujian, ChinaGutachter: Prof. Dr. Volker HölltProf. Dr. Eckart D. GundelfingerProf. Dr. Klaus-Armin NaveEingereicht am: 28. November 2005Verteidigung am: 04. Juli 2006Table of contents i1 Introduction............................................................................................................... 11.1 Opioid receptors ......................................................................................................................11.2 Functional activities of opioid receptors...............................................................................31.3 Intracellular trafficking of opioid receptors ........................................................................31.4 Down-regulation of opioid receptors ....................................................................................51.5 Desensitization of opioid receptors........................................................................................61.6 Adenylate cyclase superactivation .............

Sujets

• Biologie

Informations

• Publié par: otto-von-guericke-universitat_magdeburg
• Publié le: 01 janvier 2006
• Nombre de lectures: 29
• Langue: Deutsch
• Poids de l'ouvrage: 47 Mo

Extrait

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 .82NçîÉãÄÉê052 40. Jìäá0 2

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Data analysis ..........................................................................................................................22

Determination of receptor desensitization .........................................................................22

3.15

3.14

3.1

3.2

Primary neuronal cell culture, transfection and immunostaining..................................21

Transferrin trafficking .........................................................................................................21

3.4

3.3

3.6

3.5

3.13

3.7

3.11

3.12

3.9

3.10

Cloning of M6a cDNA by RT-PCR......................................................................................20

3.8

Radioligand binding assay ...................................................................................................20

Quantitative analysis of receptor internalization and recycling by ELISA...................20

Cell culture and generation of stable cell lines ..................................................................19

Calcium-phosphate-mediated transfection of HEK293 cells...........................................19

In situ hybridization..............................................................................................................17

Immunocytochemistry ..........................................................................................................17

BRET assay ............................................................................................................................15

Co-immunoprecipitation ......................................................................................................15

Plasmid sequencing ...............................................................................................................15

Gene subclone ........................................................................................................................15

3

Methods ................................................................................................................... 15

2.9

Buffers and solvents ..............................................................................................................14

2.1

2.2

2.3

2.4

2.5

2.6

Chemicals ...............................................................................................................................12

Bacterium and eukaryotic cell line......................................................................................13

cDNAs and plasmids .............................................................................................................13

Mediums .................................................................................................................................13

Enzymes..................................................................................................................................13

2.7

Antibodies and antibiotics....................................................................................................13

2.8

1.8

1.6

The present research project................................................................................................ 11

1.9

Materials.................................................................................................................. 12

2

Instruments ............................................................................................................................12

Kits ..........................................................................................................................................12

1.4

Down-regulation of opioid receptors ....................................................................................5

1.5

Desensitization of opioid receptors........................................................................................6

Opiate tolerance and dependence .........................................................................................8

Adenylate cyclase superactivation ........................................................................................7

Membrane glycoprotein M6 and its family..........................................................................8

1.7

1 Introduction ............................................................................................................... 1

TaÄäÉ çÑ çåíÉåíë  

1.1

Opioid receptors ......................................................................................................................1

1.2

Functional activities of opioid receptors...............................................................................3

1.3

Intracellular trafficking of opioid receptors ........................................................................3

TaÄäÉ çÑ çåíÉåíë  

Results ..................................................................................................................... 23

4

Co-immunoprecipitation of MOR1 and M6a ....................................................................23

Analysis of protein-protein interaction by BRET .............................................................24

Agonist-mediated subcellular distribution of MOR1-M6a/M6b.....................................25

Analysis of MOR1-M6a interacting domains by BRET...................................................27

BRET-aåalysis of M6a interaction withPC  Rëaêîìëáç......................2...........................9 Formation of a dimer (oligomer) of MOR1 or M6a..........................................................30

Co-expression of MOR1 and M6a in rat brain..................................................................32

M6a does not influence binding and inhibition of AC by MOR1 agonist ......................35

M6a augments µ-opioid receptor trafficking.....................................................................36

4.9

4.6

4.4

4.17

4.7

4.1

Discussion................................................................................................................ 55

4.3

4.2

5.2

5.1

Interaction of the µ-opioid receptor with the membrane glycoprotein M6a .................55

Co-expression of MOR1 with members of the PLP/DM20 family in rat brain ............56

The effect of M6a on signal regulation of the µ-opioid receptor .....................................60

5.4

The role of M6a in the trafficking of opioid receptors .....................................................56

5.3

Expression of M6a mRNA in HEK293 cell ........................................................................41

4.10

Quantitative analysis of µ-opioid receptor trafficking .....................................................40

4.8

M6a increases accumulation of MOR1 and DOR1 in recycling endosomes..................44

4.12

The effect of M6a dominant negative mutants on MOR1 internalization.....................43

4.11

M6a decreases agonist-induced desensitization of the µ-opioid receptor ......................47

4.14

M6a attenuates down-regulation of the µ-opioid receptor...............................................46

4.13

M6a attenuates MOR1 desensitization in primary cultured neurons ............................53

4.16

Role of M6a in MOR1 internalization in primary cultured neurons .............................49

4.15

áá

5

7

References ............................................................................................................... 66

8

List of figures and table ........................................................................................... 78

5.5

Molecular basis of opiate tolerance.....................................................................................62

6

Summary.................................................................................................................. 65

10.3 Acknowledgement .................................................................................................................84

9

11

Zusammenfassung ................................................................................................... 85

10 Appendix.................................................................................................................. 82

Abbreviations ........................................................................................................... 80

10.2 Publications and presentation..............................................................................................83

10.1 Curriculum vitae ...................................................................................................................82

1. Introduction  

1  

Introduction

-1-

1.1 Opioid receptors Oéáìã Üaë ÄÉÉå âåçïå íç êÉäáÉîÉ éaáå aåÇ aäíÉê ãççÇ ëáåÉ íÜÉ aÇîÉåí çÑ êÉçêÇÉÇ Üáëíçêó ( çìêäaó, 002.5 )Tç ÇaíÉ, çéáaíÉë ëìÜ aë ãçêéÜáåÉ aêÉ ëíáää íÜÉ ÄÉëí aåaäÖÉëá ÜçáÉ áå íÜÉ íêÉaíãÉåí çÑ Üêçåá aåÇ ëÉêáçìë éaáå, ëìÜ aë aåÉê éaáå .HçïÉîÉê, íÜÉáê ÉñíÉåëáîÉ aåÇ äçåÖ-íÉêã ìëÉ áë äáãáíÉÇ ÇìÉ íç íÜÉ ÇÉîÉäçéãÉåí çÑ çéáaíÉ íçäÉêaåÉ aåÇ ÇÉéÉåÇÉåÉ. Iå aÇÇáíáçå, ÇêìÖ íêÉaíãÉåí aå äÉaÇ íç ÇêìÖ aÄìëÉ .OéáaíÉ ÇêìÖë ãÉÇáaíÉ íÜÉáê éÜóëáçäçÖáaä ÉÑÑÉíë Äó ÄáåÇáåÖ íç çéáçáÇ êÉÉéíçêë ,ïÜáÜ aêÉ aäëç aíáîaíÉÇ Äó ÉåÇçÖÉåçìëäó éêçÇìÉÇ çéáçáÇ åÉìêçéÉéíáÇÉë (îçå  aëíêçï, . 004)2UåÇÉê íÜÉ íêÉãÉåÇçìë ÉÑÑçêíë çÑ íÜÉ éaëí óÉaêë, ïÉ åçï ìåÇÉêëíaåÇ ãÉÜaåáëãë çÑ çéáçáÇ êÉÉéíçê ëáÖåaä íêaåëãáëëáçå áå çåëáÇÉêaÄäÉ ÇÉíaáä. HçïÉîÉê, a ÇÉÑáåáíáîÉ ãÉÜaåáëã Ñçê çéáçáÇ êÉÉéíçê êÉÖìäaíáçå, ÉëéÉáaääó áå íÜÉ çãéäÉñ éÜóëáçäçÖáaä áêìãëíaåÉ çÑ áåíaí åÉêîçìë ëóëíÉã, áë ëíáää Ñaê Ñêçã äÉaê.

Figure 1.1. Structure of opioid receptors (modified from LaForge et al., 2000).Opioid receptor has a central common core made of seven transmembrane helices connected by three intracellular and three extracellular loops. The differences in N-terminal and C-terminal length for each receptor type are shown.

TÜêÉÉ áåÇÉéÉåÇÉåí êÉéçêíë çå íÜÉ áÇÉåíáÑáaíáçå çÑ çéáçáÇ êÉÉéíçêë áå 3791 (PÉêí aåÇ SåóÇÉê, 19; 73Sáãçå Éí aä. ,9137 ;TÉêÉåáìë, 1973) ãaêâÉÇ íÜÉ aÇîÉåí çÑ a åÉï Éêa çÑ íÜÉ çéáçáÇ êÉëÉaêÜ. 20 óÉaêë äaíÉê, ÖÉåÉë ÉåçÇáåÖ íÜêÉÉ ïÉää-ÇÉÑáåÉÇ çê ÚäaëëáaäÛ íóéÉë çÑ íÜÉ çéáçáÇ êÉÉéíçêë ,  ,-δåa Ç -κK(ÉáÑÑêÉÉ  íäa,. 1992; EîaåëíçÉéÉ êáÇáççé- ÇÉåçä ÉêÉï ,ëê

1. Introduction  

CÜÉå Éí aä., 1993a; Máåaãá Éí aä ,.39 ;91 aëìÇa Éí aä 1.,3;9aåWÉíÖ äa .)391 ,

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Éí aä,. 2; 199 .,3;991 Lá Éí aä ,.3991; MÉåÖ Éí aä.,139 ;Y SÉèìÉåÉ aåaäóëáë çÑ íÜÉëÉ äçåÉÇ çéáçáÇ êÉÉéíçêë êÉîÉaäÉÇ íÜaí íÜÉó ÄÉäçåÖ íç íÜÉ ëìéÉêÑaãáäó çÑ   éêçíÉáå-çìéäÉÇ êÉÉéíçêë ( PCRë )aåÇ íÜÉ ëìÄÑaãáäó çÑ êÜçÇçéëáå êÉÉéíçêë. Aë ëÜçïå áå FáÖìêÉ 1 ,1.íÜÉ   ,-δÇ åa -κÜ îaçíëê Éìé ÉÜíîÉíaíááççé-ÉéÉ êáÇ ëÉîÉå Ççãaáåë çÑ 250-2 ÜóÇêçéÜçÄá êÉëáÇìÉë íÜaí Ñçêã α-ÜÉ Çéëåaí áäÉ ëåaaëãäa éÜÉ ãÉãÄêaåÉ ,aå ÉñíêaÉääìäaê N-íÉêãáåìë, íÜêÉÉ ÉñíêaÉääìäaê äççéë, íÜêÉÉ áåíêaÉääìäaê äççéë aåÇ aå áåíêaÉääìäaê C-íÉêãáåaä íaáä (Eîaåë Éí aä., ; 1992KáÉÑÑÉê Éí aä ,.; 9291CÜÉå Éí aä., 1993a; FìâìÇa Éí aä. ,3991 ;Lá Éí aä .,139;9 MÉåÖ Éí aä ,.91 ;39YaëìÇa Éí aä., 991;3 WaäÇÜçÉê Éí aä .,2. 4)00TÜÉëÉ êÉÉéíçêë aêÉ aÄçìí 06 %áÇÉåíáaä íç ÉaÜ çíÜÉê, ïáíÜ íÜÉ ÖêÉaíÉëí ÜçãçäçÖó ÑçìåÇ áå íÜÉ íêaåëãÉãÄêaåÉ Ççãaáåë 7(3Ô67)% aåÇ áåíêaÉääìäaê äççéë 6(8Ô ).0%10TÜÉ äçïÉëí ÜçãçäçÖó áå aãáåç aáÇ ëÉèìÉåÉ áë ÑçìåÇ áå íÜÉ N-íÉêãáåìë 9(Ô1%0 ,)ÉñíêaÉääìäaê äççéë (14Ô7%2,) aåÇ íÜÉ C-íÉêãáåìë 41(Ô%02) (CÜÉå Éí aä., 3991Ä; Laï Éí aä0Ä 2.,).

Figure 1.2. Different amino acid sequence between rat MOR1 and MOR1B.

NìãÉêçìë éÜaêãaçäçÖáaä ëíìÇáÉë ÜaîÉ ëìÖÖÉëíÉÇ ëìÄíóéÉë çÑ íÜÉ  -çéáçáÇ  êÉÉéíçê (MOR)1 aåÇ ëíìÇáÉë ÜaîÉ êaáëÉÇ íÜÉ éçëëáÄáäáíó íÜaí ëçãÉ çÑ íÜÉëÉ ãaó êÉÑäÉí ëéäáÉ îaêáaåíë çÑ íÜÉ MOR 1ÖÉåÉ (Wçäçòáå aåÇ PaëíÉêåaâ, ; 8119PaëíÉêåaâ,  99;31PaëíÉêåaâ aåÇ SíaåÇáÑÉê, 991. 5)Tïç MOR1 îaêáaåíë ,MOR1A aåÇ MOR1B, ïÉêÉ áÇÉåíáÑáÉÇ ëÜçêíäó aÑíÉê íÜÉ áåáíáaä äçåáåÖ çÑ MOR1 (BaêÉ Éí aä,. 1 99;4 áãéêáÜ Éí aä. ,95). 91MOR1 aåÇ MOR1B ÇáÑÑÉê aí íÜÉ C-íÉêãáåìë aë ëÜçïå áå FáÖìêÉ .21. TÜÉêÉaÑíÉê, aÇÇáíáçåaä MOR 1ëéäáÉ îaêáaåíë ëìÜ aë MOR1C, D ,E aåÇ F ïÉêÉ áÇÉåíáÑáÉÇ (Paå Éí aä. ,91 ,990002). Aää íÜÉëÉ ëéäáÉ îaêáaåíë aêÉ áÇÉåíáaä áå íÜÉ Ñáêëí 3 68aãáåç aáÇë .TÜçìÖÜ MOR1 aåÇ áíë ëéäáÉ îaêáaåíë aêÉ ÇÉêáîÉÇ Ñêçã íÜÉ ëaãÉ ÖÉåÉ ,aåÇ ÉñÜáÄáí ëáãáäaê ÄáåÇáåÖ éêçéÉêíáÉë, íÜÉó ÇáÑÑÉê áå íÜÉáê Ñìåíáçåaä éêçéÉêíáÉë aåÇ êÉÖáçåaä ÇáëíêáÄìíáçåë (SÜìäò Éí aä, .9819; KçÜ Éí aä,. 91 ;89Paå Éí aä., 1999 ,0200 ;AÄÄaÇáÉ Éí aä. ,2 ;000Bçäaå Éí aä., 2004 .)RÉÉåíäó ,ãçêÉ aêÄçñóä-íÉêãáåaä ëéäáÉ îaêáaåíë çÑ íÜÉ  -çéáçáÇ êÉÉéíçê ïÉêÉ çåíáåìaääó áÇÉåíáÑáÉÇ aåÇ ÜaêaíÉêáòÉÇ( PaëíÉêåaâ Éí aä ;0íÉ åaP2 ,äa 42,.05). TÜÉ  -çéáçáÇ êÉÉéíçê (MOR)1   çéáaíÉ ÇêìÖë ,É.Ö .ãçêéÜáåÉ ,aë ïÉää aë  

ãÉÇáaíÉë íÜÉ aíáçå çÑ ãçëí äáåáaääó áãéçêíaåí ÇêìÖë çÑ aÄìëÉ ëìÜ aë ÜÉêçáå (MaííÜÉë Éí aä. ,;9169