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P90 Ribosomal S6 Kinase 2 (Rsk2) Directly Phosphorylates The P90 RIBOSOMAL S6 KINASE 2 (RSK2) DIRECTLY PHOSPHORYLATES THE 5-HT2A SEROTONIN RECEPTOR THEREBY MODULATING SIGNALING by RYAN THOMAS STRACHAN Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Dissertation Advisor: Dr. Bryan L. Roth, M.D., Ph.D. Department of Biochemistry CASE WESTERN RESERVE UNIVERSITY August, 2009 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the dissertation of Ryan Thomas Strachan ______________________________________________________ candidate for the Ph.D. degree *. (signed) William C. Merrick, Ph.D. ______________________________________________ (chair of the committee) Bryan L. Roth, M.D., Ph.D. ______________________________________________ Martin D. Snider, Ph.D. ______________________________________________ Paul R. Ernsberger, Ph.D. ______________________________________________ George R. Dubyak, Ph.D. ______________________________________________ ______________________________________________ June 25th, 2009 (date) ________________________ *We also certify that written approval has been obtained for any proprietary material contained therein. TABLE OF CONTENTS LIST OF TABLES…...…………………………………………………………………..x LIST OF FIGURES...……………………………………………………………….......xi ACKNOWLEDGEMENTS…………………………………………………………...xvi LIST Of ABBREVIATIONS..…………………………………………………….....xviii ABSTRACT.……………………………………………………………………….….xxiv CHAPTER 1: INTRODUCTION 1.1 The Receptor Concept…………………………………………………….1 1.2 G Protein-Coupled Receptors (GPCRs) 1.2.1 Introduction……………………………………………………….2 1.2.2 GPCR Structure……………...…………………………………....3 1.2.3 Mechanism of GPCR Activation………………………………….8 1.2.4 GPCR Signal Transduction: Primary Signaling Pathways……………………………………....9 1.2.5 GPCR Activation and Signal Transduction: Secondary Pathways Involving Arrestins and GRKs….………..11 1.2.6 GPCR Activation and Signal Transduction: Secondary Pathways Involving Transactivation of the ERK/MAPK Cascade…………………………..…………14 1.2.7 GPCR Regulation 1.2.7.1 Canonical GPCR Regulation…………………………..22 i 1.2.7.2 Emerging Evidence for Growth Factor Receptor-Mediated Regulation of GPCRs…….……..26 1.3 5-Hydroxytryptamine Receptors 1.3.1 Introduction……………………………………………………..28 1.3.2 5-Hydroxytryptamine Receptor Activation and Signal Transduction…………………………………………….31 1.3.3 5-Hydroxytryptamine 2A (5-HT2A) Receptors: Identification, Localization, and Relevance for Study…………………………33 1.3.4 5-HT2A Receptor-Mediated Signal Transduction………………36 1.3.5 5-HT2A Receptor Regulation: Rapid Desensitization and Internalization……………………...40 1.3.6 5-HT2A Receptor Regulation: Down-Regulation and Resensitization………………………….42 1.3.7 5-HT2A Receptor Interacting Proteins 1.3.7.1 Overview……………………………………………...45 1.3.7.2 PDZ domain-containing proteins Including PSD-95……………………………………..47 1.3.7.3 Caveolin-1…………………………………………….48 1.3.7.4 p90 Ribosomal S6 Kinase2 (RSK2) 1.3.7.4.1 Identification and Activation of p90 Ribosomal S6 Kinases (RSKs)…………....49 1.3.7.4.2 The Multiple Functions of RSK………….52 ii 1.3.7.4.3 RSK and Human Disease: Role of RSK2 in Coffin-Lowry Syndrome………………………………….55 1.3.8 RSK2 Interacts with the 5-HT2A Receptor and Negatively Regulates Signaling………………………………...56 CHAPTER 2: MATERIALS AND METHODS 2.1 Materials 2.1.1 General Reagents………………………………………………..59 2.1.2 Cell Culture Reagents 2.1.2.1 General………………………………………………..60 2.1.2.2 Serum Dialysis………………………………………..60 2.1.3 cDNA Constructs 2.1.3.1 Insertion of the Rat 5-HT2A Receptor into pBABEpuromycin for Retrovirus Production………………………………..63 2.1.3.2 Insertion of the Rat 5-HT2A-GFP-CT Receptor into FUGW for Lentiviral Production…………………….67 2.1.3.3 Construction of a Tandem Affinity-Tagged 5-HT2A Receptor for Receptor Purification…….........68 2.1.3.4 Construction of a Tandem Affinity-Tagged I3 Loop Peptide Construct for Purification…………..71 2.1.3.5 Construction of GFP-tagged RSK2…………………..75 iii 2.1.4 Antibodies 2.1.4.1 Commercial Antibodies………………………………76 2.1.4.2 Custom Antibodies…………………………………...76 2.2 Methods 2.2.1 Cell Culture 2.2.1.1 General Cell Culture………………………………….80 2.2.1.2 Isolation of Mouse Aortic Vascular Smooth Muscle Cells…………………………………80 2.2.1.3 Primary Cortical Neuron Culture and Lentiviral Infections…………………………………..81 2.2.2 Transfection……………………………………………………...82 2.2.3 Retrovirus Production and 5-HT2A Polyclonal Stable Line Generation……………………………...83 2.2.4 Site-directed Mutagenesis……………………………………....83 2.2.5 Tandem Affinity Purification of 5-HT2A Receptors and I3 Loop Peptides 2.2.5.1 5-HT2A Receptor Purification…………………………84 2.2.5.2 I3 Loop Peptide Purification………………………......85 2.2.6 Competition Radioligand Binding Assays………………….…..86 2.2.7 Immunoprecipitation and Western Blotting…………………….87 2.2.8 In Vitro Kinase Assays………………………………………….89 2.2.9 Mass Spectrometry (MS)……………………………………….90 iv 32 2.2.10 [ Pi] Metabolic Labeling……………………………………….92 2.2.11 Fluorometric Imaging Plate Reader (FLIPRTetra) Analysis of Intracellular Ca2+ Release……………………...…..93 2.2.12 Analysis of Inositol Phosphates 2.2.12.1 Analysis of Inositol 1,4,5 Trisphosphate…………….94 2.2.12.2 Analysis of Inositol Phosphate………………………95 2.2.13 Generation and Validation of High Content Immunofluorescence Assays…………...………………………96 2.2.14 Analysis of Intracellular Ca2+ Release in Primary Cortical Neurons………………………………............99 2.2.15 Mouse Behavioral Experiments 2.2.15.1 Locomotion…………………………………………100 2.2.15.2 Head Twitch Response (HTR)……………………...100 2.2.16 Statistical Analysis of In Vitro Kinase Assays, Second Messenger Assays, High Content ERK1/2 Phosphorylation Assays, and Mouse Behavioral Experiments………………….101 2.2.17 Generation of Statistically Rigorous Rank Orders of Efficacy………………………………………………………..102 CHAPTER 3: RSK2 DIRECTLY PHOSPHORYLATES THE 5-HT2A SEROTONIN RECEPTOR THEREBY MODULATING 5-HT2A SIGNALING 3.1 Introduction and Rationale…………………………………………….103 v 3.2 RSK2 Regulates Both Rapid and Prolonged 5-HT2A Receptor Signaling…………………………………………………….105 3.3 RSK2 Kinase Activity is Essential for Negatively Regulating 5-HT2A Receptor Signaling……………………………......107 3.4 RSK2 Phosphorylates the 5-HT2A Receptor In Vitro………………….……………………………………………..110 3.5 RSK2 Phosphorylates the 5-HT2A Receptor I3 Loop In Vitro…………...................................................................................113 3.6 RSK2 Specifically Phosphorylates the Conserved Residue Ser314 within the 5-HT2A Receptor I3 loop In Vitro……………….....116 3.7 The Phospho-Specific Antibody Against Ser314 Verifies Phosphorylation Within the 5-HT2A Receptor I3 loop……………..….121 3.8 RSK2 Specifically Phosphorylates the Full-Length 5-HT2A Receptor at Ser314……………………………………….…..123 3.9 Validation of Ser314 Phosphorylation in 5-HT2A Receptors In Vitro and in MEFs………………………………………125 3.10 RSK2 Requires Ser314 within the 5-HT2A Receptor I3 Loop to Regulate Agonist Signaling…………………………………..128 3.11 Discussion……………………………………………………………..133 vi CHAPTER 4: RECEPTOR TYROSINE KINASE (RTK)-GPCR CROSS-TALK: EVIDENCE FOR A MECHANISM WHEREBY RTKs ACT VIA RSK2 TO MODULATE GPCR SIGNALING 4.1 Introduction and Rationale…………………………………………….140 4.2 Treatment with Epidermal Growth Factor Time-Dependently Decreases 5-HT-induced Intracellular Ca2+ Release…...………...........142 4.3 Treatment with Epidermal Growth Factor Decreases 5-HT-induced Intracellular Ca2+ Release in a Concentration-Dependent Manner……………………………….……146 4.4 RSK2 is Required for hEGF-Mediated Heterologous Desensitization of 5-HT2A Receptor Signaling…………………………………………148 4.5 RSK2 is Required for TGF-α-Mediated Heterologous Desensitization of 5-HT2A Receptor Signaling…………………………………………151 4.6 RSK2 is Required for hEGF-Mediated Heterologous Desensitization of 5-HT2A Receptors-Supporting Data From a Panel of 5-HT2A Agonists with Different Intrinsic Efficacies………………………...…154 4.7 RSK2 is Required for Platelet-Derived Growth Factor-Mediated Desensitization of Endogenous 5-HT2A Receptors in Primary Cultures of Mouse Vascular Smooth Muscle Cells………….………...158 4.8 Pre-Treatment with Insulin Does Not Lead to Desensitization of 5-HT2A Receptors-Implications for Pathway Specificity……….……...162 vii 4.9 RSK2 is Required for hEGF-Mediated Heterologous Desensitization of Endogenous P2Y Purinergic Receptor Signaling…………………...165 4.10 Discussion……………………………………………………………...168 CHAPTER 5: RSK2-DEPENDENT ALTERATIONS IN THE RELATIVE RANK ORDER EFFICACY OF 5-HT2A SEROTONIN RECEPTOR AGONISTS 5.1 Introduction and Rationale…………………………………………….174 5.2 Validation of a Novel High Content Microscopic Approach for ERK1/2 Phosphorylation…………………………………………..177 5.3 5-HT2A Agonist Responses to a Variety of Agonists are Augmented by Genetic Deletion of RSK2…………...………………..180 5.4 5-HT2A Agonists are Functionally Selective for ERK1/2 Phosphorylation in RSK2+/+ MEFs………….………………………..189 5.5 Genetic Deletion of RSK2 Alters Relative Rank Order of Efficacy at 5-HT2AReceptors…………………………………………..194 5.6 Discussion……………………………………………………………...197 CHAPTER 6: SUMMARY AND IMPLICATIONS FOR FUTURE STUDIES 6.1 Summary……………………………………………………………….203 6.2 Implications for Future Studies………………………………………...206 viii 6.2.1 The C-Terminus of the 5-HT2A Receptor I3 Loop is Important for Regulating G Protein-Mediated Signaling…………………………………………….…………207 6.2.2 RSK2 is Required for the RTK-Mediated Heterologous Desensitization of 5-HT2A Receptor Signaling In Vivo…………………………………….211 6.2.3 Phosphorylation of the 5-HT2A Receptor Differentially Affects Agonist Signaling Thereby Promoting Altered Patterns of Functional Selectivity………………………………………….216 6.2.4 RSK2 Regulates 5-HT2A receptors In Vivo……………………218 6.3 Closing Considerations………………………………………………...222
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