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The Functional Significance of G Protein -Coupled Receptor Dimerization

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The Functional Significance of G Protein -Coupled Receptor Dimerization THE FUNCTIONAL SIGNIFICANCE OF G PROTEIN-COUPLED RECEPTOR DIMERIZATION by Matthew R. Whorton A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Pharmacology) in The University of Michigan 2008 Doctoral Committee: Assistant Professor Roger K. Sunahara, Chair Assistant Professor Jorge A. Iñiguez-Lluhí Professor Jennifer J. Linderman Professor Richard R. Neubig Professor William B. Pratt Associate Professor John J. G. Tesmer To my parents and Jen for their love, support, and encouragement. ii ACKNOWLEDGEMENTS I would like to thank my advisor Roger Sunahara for his guidance and support. I would also like to extend my gratitude to other members of the Sunahara lab, both past and present, as well as members of the Neubig and Tesmer labs for thoughtful discussions and comments during lab meetings. I would also like to extend a great deal of gratitude to several collaborators who have provided their expertise to various aspects of my research. At Stanford University, Mike Bokoch and Soren Rasmussen in Brian Kobilka’s lab and Bo Huang in Richard Zare’s lab were instrumental in fluorescently labeling β2AR and then imaging it with their powerful TIRF single molecule setup. Beata Jastrzebska and Paul Park in Krzysztof Palczewski’s lab at Case Western University were also invaluable in providing purified rhodopsin as well as performing transducin activation assays. And finally, I would like to thank Dimitrios Fotiadis in Andrea Engel’s lab for using their various microscopy techniques to image rhodopsin-HDL complexes. I would also like to thank my friends and roommates through out the years who have helped to enrich the graduate school experience. I am also indebted to my fiancée Jen and my family for their love and support. iii TABLE OF CONTENTS DEDICATION.................................................................................................................... ii ACKNOWLEDGEMENTS............................................................................................... iii LIST OF FIGURES ........................................................................................................... vi LIST OF ABBREVIATIONS.......................................................................................... viii ABSTRACT....................................................................................................................... xi CHAPTER 1. INTRODUCTION G protein-coupled receptor biology.........................................................................1 GPCR Oligomerization............................................................................................7 High Density Lipoproteins as a Means for Studying Membrane Proteins ............11 CHAPTER 2. A MONOMERIC G PROTEIN COUPLED RECEPTOR EFFICIENTLY COUPLES TO ITS G PROTEIN Introduction............................................................................................................15 Results....................................................................................................................16 Reconstitution of β2AR into HDL ................................................................16 β2AR in rHDL is monomeric by single molecule spectroscopy...................18 Monomeric β2AR in rHDL functionally couples to G proteins....................25 Discussion..............................................................................................................28 Materials and Methods...........................................................................................30 iv CHAPTER 3. EFFICIENT COUPLING OF TRANSDUCIN TO MONOMERIC RHODOPSIN IN A PHOSPHOLIPID BILAYER Introduction............................................................................................................44 Results....................................................................................................................46 Reconstitution of rhodopsin in HDL.............................................................46 Rhodopsin:rHDL stoichiometry....................................................................48 Functional comparison of monomeric and oligomeric rhodopsin ................49 Discussion..............................................................................................................53 Materials and Methods...........................................................................................57 CHAPTER 4. CONCLUSIONS Summary................................................................................................................62 Is There a Functional Role for Dimerization? .......................................................64 Dimerization in Question.......................................................................................66 General Applications of HDL Technology............................................................67 REFERENCES ..................................................................................................................71 v LIST OF FIGURES Figure 1-1 The GPCR signaling pathway.........................................................................2 1-2 Atomic force microscopy of rod outer segment discs from mouse retina ...............................................................................................................5 1-3 Illustration of a typical FRET setup................................................................9 1-4 High density lipoproteins (HDL)..................................................................13 2-1 Depiction of rHDL particles .........................................................................17 2-2 Functional reconstitution of β2AR into rHDL ..............................................19 2-3 Labeling efficiency of β2AR estimated by TIRF imaging and photobleach-step counting. ...........................................................................20 2-4 Purified β2ΑR in DDM micelles is monomeric before reconstitution in vesicles..........................................................................................................21 2-5 TIRF of Cy3- and Cy5-labeled β2AR in rHDL reveals that the vast majority of β2AR are monomeric .................................................................23 2-6 FRET measurements confirm monomeric β2AR in rHDL ...........................24 2-7 Monomeric β2AR incorporated in rHDL particles couples efficiently to G proteins..................................................................................................26 3-1 Illustration of reconstituted HDL particles ...................................................46 3-2 Rhodopsin incorporated in rHDL and resolved by size exclusion chromatography is photoactivatable .............................................................47 3-3 Rhodopsin·rHDL particles were resolved on a size exclusion column and then purified on a ConA-Sepharose column..........................................48 3-4 Rhodopsin exists as a monomer in rHDL particles ......................................50 3-5 Meta II formation and decay.........................................................................51 vi 3-6 Activation of transducin by monomeric rhodopsin in rHDL........................52 3-7 Only one receptor in a receptor dimer is necessary to activate G proteins..........................................................................................................56 4-1 Visualization of rhodopsin in HDL using electron microscopy ...................64 4-2 Molecular model of an arrestin-rhodopsin dimer complex...........................66 4-3 Cryo-EM class averages of HDL..................................................................69 vii LIST OF ABBREVIATIONS AC Adenylyl Cyclase AFM Atomic Force Microscopy apoAI Apolipoprotein A-I β2AR β2 Adrenergic Receptor Bis(NHS)PEO5 bis N-succinimidyl[pentaethylene glycol] ester BBmax Maximum Receptor Binding BRET Bioluminescence Resonance Energy Transfer BS3 Bis(sulfosuccinimidyl) Suberate cAMP Cyclic Adenosine Monophosphate CFP Cyan Fluorescent Protein cGMP Cyclic Guanosine Monophosphate CHAPS 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate CMC Critical Micelle Concentration CNG Cyclic Nucleotide Gated Channel Con A Concanavalin A CREB cAMP Response Element Binding Protein DDM Dodecyl Maltoside DHAP Dihydroalprenolol DOPC Dioleoyl Phosphocholine EDTA Ethylenediaminetetraacetic Acid EM Electron Microscopy ERK Extracellular Signal-Regulated Kinase viii FRET Fluorescence Resonance Energy Transfer G protein GTP-binding protein GABA Gamma-aminobutyric Acid GDP Guanosine Diphosphate GIRK G Protein-Coupled Inwardly-Rectifying Potassium Channel GPCR G Protein-Coupled Receptor GRK G Protein-Coupled Receptor Kinase GTP Guanosine Triphosphate GTPγS Guanosine 5'-O-(3-thiotriphosphate) HDL High Density Lipoprotein HEK Human Embryonic Kidney IP Immunoprecipitate ISO Isoproterenol Khigh High Affinity Ki Ki Inhibitory Constant Klow Low Affinity Ki LtB4 Leukotriene B4 MAP Mitogen-Activated Protein META Metarhodopsin mGluR Metabotropic Glutamate Receptor MLCK Myosin Light Chain Kinase MWCO Molecular Weight Cutoff PBS Phosphate Buffered Saline PDE Phosphodiesterase PI Protease inhibitors PKA Protein Kinase A PLC Phospholipase C ix POPC Palmitoyl-oleoyl Phosphocholine POPG Palmitoyl-oleoyl Phosphoglycerol RET Resonance Energy Transfer RGS Regulators of G Protein Signaling rHDL Reconstituted HDL ROS Rod Outer Segment RT Room Temperature SDS-PAGE Sodium Dodecyl Sulfate - Polyacrylamide Gel Electrophoresis SEC Size Exclusion
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