HUMAN PROGESTIN ADIPOQ RECEPTORS, IDENTIFICATION OF PAQR3 AS A NEW POSSIBLE ADIPONECTIN RECEPTOR By IBON GARITAONANDIA A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2008 1 © 2008 Ibon Garitaonandia 2 To my loving family 3 ACKNOWLEDGMENTS This project was funded by the University of Florida and the NIH Grant R21 DK074812- 01. I would also like to thank the Department of Chemistry for the financial support. I am grateful to my research advisor Dr. Tom Lyons and my doctoral dissertation committee, Dr. Gail Fanucci, Dr. Nicole Horenstein, Dr. William Dolbier and Dr. David Silverman, for their continuous support and guidance throughout all these years. I thank former and current members of the Lyons’ group, Jessica, Brian, Lisa, Nancy, Lidia, for their friendship and guidance throughout these years. Special thanks go to Jessica, whose unconditional friendship, help and useful discussions over the years made the laboratory such a positive place for work. Finally I thank my parents, Pedro and Maria, my sister Alaitz and my grandmother Herminia for being my number one support system. 4 TABLE OF CONTENTS page ACKNOWLEDGMENTS ...............................................................................................................4 LIST OF TABLES...........................................................................................................................7 LIST OF FIGURES .........................................................................................................................8 ABSTRACT...................................................................................................................................12 CHAPTER 1 INTRODUCTION ..................................................................................................................14 Type 2 Diabetes ......................................................................................................................14 Adiponectin.............................................................................................................................14 Adiponectin Receptors............................................................................................................19 PAQR Receptors.....................................................................................................................23 Human Class I PAQRs ....................................................................................................23 Human Class II PAQRs...................................................................................................24 Human Class III PAQRs .................................................................................................25 Ceramidase Activity ........................................................................................................25 2 EXPRESSION AND CHARACTERIZATION OF THE HUMAN CLASS I PAQR PROTEINS IN ESCHERICHIA COLI ...................................................................................38 Introduction.............................................................................................................................38 Materials and Methods ...........................................................................................................38 Cloning of Human PAQRs..............................................................................................38 Growth Conditions ..........................................................................................................39 Detection of Expression ..................................................................................................40 Coomassie staining...................................................................................................40 His-tag in-gel staining ..............................................................................................40 Western blot .............................................................................................................40 Purification ......................................................................................................................41 Ceramidase Assay ...........................................................................................................42 Results and Discussion ...........................................................................................................43 Expression of PAQRs in Escherichia coli.......................................................................43 Ceramidase Assay ...........................................................................................................44 3 EXPRESSION AND CHARACTERIZATION OF THE HUMAN CLASS I PAQR PROTEINS IN SACCHAROMYCES CEREVISAE ................................................................51 Introduction.............................................................................................................................51 Materials and Methods ...........................................................................................................52 5 Yeast Strains and Media..................................................................................................52 Cloning of Human PAQR ...............................................................................................52 Growth Conditions ..........................................................................................................52 Membrane Protein Isolation ............................................................................................53 Western Blot....................................................................................................................53 β-galactosidase Reporter Assays .....................................................................................54 GFP Fluorescence............................................................................................................55 Adiponectin Binding .......................................................................................................55 Results and Discussion ...........................................................................................................56 Repression of FET3 by PAQRs.......................................................................................56 Thaumatin as a Ligand for Izh2p.....................................................................................57 Human Adiponectin Receptors........................................................................................58 Inhibitors and Activators of Human Adiponectin Receptors ..........................................59 PAQR3 and PAQR4 ........................................................................................................60 Involvement of PKA and AMPK ....................................................................................62 Ceramidase Activity ........................................................................................................63 Fluorescence Studies .......................................................................................................63 Adiponectin Binding .......................................................................................................66 4 EXPRESSION AND CHARACTERIZATION OF THE HUMAN CLASS I PAQR PROTEINS IN SF9 INSECT CELLS ....................................................................................94 Introduction.............................................................................................................................94 Materials and Methods ...........................................................................................................95 Cloning of Human Class I PAQR Proteins .....................................................................95 Growth of Sf9 Insect Cells...............................................................................................95 Transfection of Sf9 Insect Cells.......................................................................................96 Cell Extract Preparation and Western Blot .....................................................................96 Adiponectin Binding .......................................................................................................96 Results and Discussion ...........................................................................................................97 Expression of PAQRs in Sf9 cells ...................................................................................97 Adiponectin binding ........................................................................................................97 5 CONCLUSIONS ..................................................................................................................100 APPENDIX A YEAST STRAINS................................................................................................................103 B MEDIA COMPOSITION.....................................................................................................104 C PRIMERS USED FOR CLONING......................................................................................105 LIST OF REFERENCES.............................................................................................................109 BIOGRAPHICAL SKETCH .......................................................................................................118