UC San Diego UC San Diego Electronic Theses and Dissertations Title Expression Profile and Activation of Components for a Novel Signaling Module Mediating EGFR Transactivation Permalink https://escholarship.org/uc/item/8pn5q6sq Author Lynch, Rebecca Marrie Publication Date 2015 Peer reviewed|Thesis/dissertation eScholarship.org Powered by the California Digital Library University of California UNIVERSITY OF CALIFORNIA, SAN DIEGO Expression Profile and Activation of Components for a Novel Signaling Module Mediating EGFR Transactivation A Thesis submitted in partial satisfaction of the requirements for the degree Master of Science in Biology by Rebecca Marrie Lynch Committee in charge: Paul A. Insel, Chair Julian Schroeder, Co-Chair Aaron B. Coleman 2015 Copyright Rebecca Marrie Lynch, 2015 All rights reserved The thesis of Rebecca Marrie Lynch is approved and it is acceptable in quality and form for publication on microfilm and electronically: _____________________________________________________________________ _____________________________________________________________________ Co-chair _____________________________________________________________________ Chair University of California, San Diego 2015 iii TABLE OF CONTENTS Signature Page…………………………………………………..………………………..iii Table of Contents…………………………………………………………..……………..iv List of Abbreviations.......………………………………………………..………….……vi List of Figures………………………………………………………..………………..…vii List of Tables………………………………………….………………………………….ix Acknowledgements…………………..…………………………………………………...ix Abstract of the Thesis…………………………………………….…………………….…x Chapter 1. Introduction…..……………………..……………………………………...….1 1.1. G protein-coupled receptors (GPCRs)……………………………….….……1 1.2. Matrix Metalloproteases……..………………………….……………….…...2 1.3. Epidermal Growth Factor Receptor….……………..………………………..4 1.4. Membrane-Delimited EGFR Transactivation Mechanism...…………………5 1.5. Bradykinin……………..…….…………………..……………………………7 1.6. NIH 3T3 cells, rat cardiac myocytes, and HeLa cells………………………...7 1.7. Hypothesis and Goals…………...………………………………………...…8 Chapter 2. Materials and Methods…….…………………………………………………10 2.1. Reagents ………………………………………………………….................10 2.2. Cell Culture……………..……………………………………………..….....10 2.3. Isolation of Adult Rat Ventricular Myocytes……………………………..…10 2.3. Membrane Isolation………………………………………………………....11 2.3.1. Crude Membrane Isolation……………………………………..…11 2.3.2. 101 Bio® Plasma Membrane Protein Extraction……………….....12 2.4. Protein Analysis……………………………………………………...……...13 2.4.1. Bradford Protein Assay…………………………………...…….....13 2.4.2. Western Blots……………………………………………………..13 2.5. TaqMan® GPCR Array……………………………………………………..14 Chapter 3. Results: GPCR Expression Profiles for NIH 3T3 and HeLa Cells…………..15 3.1. Quantification of GPCR Expression in NIH 3T3 and HeLa Cells…………15 3.2. GPCR Expression in NIH 3T3 cells………..……………………………….15 3.3. GPCR Expression in HeLa cells………….…………………………...…….21 Chapter 4. Results: Baseline Expression of the Signaling Molecules in the Proposed Mechanism……………………………………………………………….........………....26 Chapter 5. Results: GPCR agonists mediating EGFR transactivation………..………….33 iv Chapter 6. Discussion...……………………………………………………....……...…..39 Chapter 7. Future Directions……………………………………………………………..47 Chapter 8. Appendix…….…………………………………………………………….…49 8.1. GPCR Expression in NIH 3T3 cells………………………………………...49 8.2. GPCR Expression in HeLa cells…………………………………………….54 References…………………………………………….……………..…………………...58 v LIST OF ABBREVIATIONS GPCR = G protein-coupled receptor MMP = matrix metalloprotease MT1-MMP = Membrane Type-1 Matrix Metalloprotease Hb-EGF= Heparin-binding Epidermal Growth Factor EGFR= Epidermal Growth Factor Receptor PBST= 1X Phosphate Buffered Saline Tween-20 UT= untreated EGF= epidermal growth factor BK = bradykinin AG 1478/EGFRinh = inhibitor of EGFR MMP14inh = inhibitor of MT1-MMP vi LIST OF FIGURES Figure 1. GPCR-mediated transactivation of EGFR………………………………....…...5 Figure 2. Proposed Mechanism of GPCR-mediated EGFR transactivation……………...6 Figure 3.1. Total GPCR expression in NIH 3T3 cells…………………………………..17 Figure 3.2. Total GPCR expression in HeLa cells……………..…………..……………22 Figure 3.3. Comparison of GPCR Expression in NIH 3T3 and HeLa cells…………….22 Figure 4.1. Protein Expression of MT1-MMP…………..………………………………28 Figure 4.2. Protein Expression of hb-EGF…………...…………………….……………29 Figure 4.3. Protein Expression of EGFR………………………………………………..30 Figure 4.4. Protein Expression of PEGFR………………………………………………31 Figure 4.5. Protein Expression of ß-Arrestin 1/2 and Src……………………………….32 Figure 5.1. HeLa lysates and membranes treated with recombinant human EGF………35 Figure 5.2. HeLa lysates and membranes treated with the GPCR agonist bradykinin….36 Figure 5.3. EGF Time-course ……………………………………………...………..….37 . Figure 5.4. HeLa membranes treated with EGF, GTPyS, Bradykinin (BK), and the inhibitor AG 1478……………………………………………………………….…….…38 Figure 5.5. Pooled data of HeLa membranes treated with EGF, Bradykinin (BK), AG 1478, and Peptide G……………………………………….…………………………..…38 vii LIST OF TABLES Table 3.1. The Twenty Highest Expressed GPCRs in NIH 3T3 cells……………….…18 Table 3.2. The Five Highest Expressed Orphan Receptors in NIH 3T3 cells…………...18 Table 3.3. The Five Highest Expressed Gi-linked Receptors in NIH 3T3 cells…..…….19 Table 3.4. The Five Highest Expressed Gq-linked Receptors in NIH 3T3 cells …...…..19 Table 3.5. The Five Highest Expressed Gs-linked Receptors in NIH 3T3 cells………...20 Table 3.6. The Five Highest Expressed G12/G13 Receptors in NIH 3T3 cells…….…...20 Table 3.7. The Twenty Highest Expressed GPCRs in HeLa cells………………………23 Table 3.8. The Five Highest Expressed Orphan Receptors in HeLa cells………………23 Table 3.9. The Five Highest Expressed Gi-linked Receptors in HeLa cells……....…….24 Table 3.10. The Five Highest Expressed Gq-linked Receptors in HeLa cells..………....24 Table 3.11. The Five Highest Expressed Gs-linked Receptors in HeLa cells…………..25 Table 3.12. The Five Highest Expressed G12/G13 Receptors in HeLa cells……….......25 viii ACKNOWLEDGEMENTS I would like to acknowledge Paul Insel for his support as the chair of my committee. Throughout the past two years, Paul has given me countless opportunities to participate in novel research and has given me experiences that will be invaluable to me in my professional and personal life. I would also like to acknowledge my advisor and mentor, Aaron Overland, for his unfailing guidance, encouragement, and support throughout the duration of my time in the lab. His advice and insight into the direction of my project was key in allowing me to complete this study. I would like to thank Nakon Aroonsakool and the rest of the Insel Lab for all the help and encouragement they have given me over the past two years. Most importantly, I would like to thank my parents for their love, devotion, and incredible support throughout my years in graduate school. Finally, I would like to acknowledge and thank Julian Schroeder and Aaron Coleman for taking the time to serve on my thesis committee. ix ABSTRACT OF THE THESIS Expression Profile and Activation of Components for a Novel Signaling Module Mediating EGFR Transactivation by Rebecca Marrie Lynch Master of Science in Biology University of California, San Diego, 2015 Paul A. Insel, Chair Julian Schroder, Co-Chair Activation of G protein-coupled receptors (GPCRs) mediates the transactivation of epidermal growth factor receptor (EGFR)-dependent signaling pathways, yet the mechanisms underlying this phenomenon are not fully understood. EGFR transactivation can occur via protease “shedding” of EGFR ligands (e.g., heparin-binding EGF [hb- EGF]) from the cell surface. Work in the Insel laboratory has shown the direct activation x of MT1-MMP (membrane type-1 matrix metalloprotease) via GPCRs and heterotrimeric G proteins and in support of the hypothesis that transactivation of EGFR might occur via components of a novel, membrane-associated signaling module (GPCR/MT1-MMP/HB- EGF/EGFR). In order to identify and quantify GPCRs that might regulate this proposed mechanism, I conducted targeted GPCR arrays using NIH 3T3 and HeLa cells. Next, I assessed the expression of the components in the putative signaling module by using whole cell lysates and isolated membranes from various cell types (NIH 3T3 fibroblasts, HeLa cells, and primary rat cardiac myocytes). I found that these signaling components localize to the plasma membrane and that bradykinin, a GPCR agonist, can activate EGFR in isolated HeLa cell membranes. This response was attenuated by AG1478, an inhibitor of EGFR, and by Peptide G, a selective inhibitor of MT1-MMP. Taken together, these findings provide evidence in support of the hypothesis that components of a plasma membrane-delimited signaling module that mediates EGFR transactivation can be activated by GPCR stimulation. Because over-activation of EGFR has been implicated in a variety of disease states, such as the progression of cancer and cardiovascular disease, further understanding of this mechanism may lead to the discovery of novel therapeutic targets. xi Chapter 1. Introduction: 1.1 G Protein-Coupled Receptors G protein-coupled receptors (GPCRs) are the largest family of membrane signaling receptors, with 900 GPCRs encoded by the human genome (1). These receptors are involved in a large number of physiological responses, from transduction of vision, taste, and smell, to regulation of the immune system, cardiovascular system, and digestive tract. GPCRs have a variety of ligands, including light, odorants, neurotransmitters, hormones, ions, and