Dissertation Approved by / Major Advisor r?<-^ Dean A PHARMACOLOGICAL STUDY OF CALCITONIN GENE-RELATED PEPTIDE RECEPTOR SUBTYPES By Boyd R. Rorabaugh A DISSERTATION Submitted to the faculty of the Graduate School of Creighton University in partial tulEllment of the requirements for the degree of Doctor of Philosophy in the Department of Pharmacology Omaha, Nebraska, May 2002 ABSTRACT Calcitonin gene-related peptide (CGRP) receptors are divided into CGRP] and CGRP2 receptor subtypes based on their affinity for CGRPg.37 in functional assays. CGRP] receptors are blocked with high affinity (Kg < 100 nM) by CGRPg.37, and CGRP2 receptors are blocked with low affinity (Kg > 100 nM) by this antagonist. The CGRP] receptor has been cloned and well characterized. In contrast, the CGRP2 receptor has not been well characterized and has only been identified by its low affinity for CGRPs.37 in functional assays. Furthermore, previous investigators reported that radioligand binding assays using [*^I]CGRP do not support the existence of the CGRP2 receptor. The goals of my work were to further characterize the CGRP2 receptor and to determine why this receptor cannot be identified by radioligand binding. Porcine coronary arteries were used to study CGRPi receptors because CGRPgg? blocks CGRP-induced relaxation of this tissue with low affinity. I used RT-PCR, radioligand binding, and affinity values from previously reported isolated tissue studies to compare the CGRP receptor in coronary arteries to the cloned CGRP] receptor I identified mRNA encoding calcitonin receptor-like receptor (CRLR) and receptor activity modifying protein (RAMP) 1 in porcine coronary arteries. In addition, the ligand binding characteristics of CGRP receptors in coronary arteries and in CGRP] receptor-transfected HER 293 cells were similar. There was also a high correlation between antagonist affinities determined by radioligand binding and functional relaxation assays. My data support the conclusion that CGRPg.37 blocks CGRP] receptors with low affinity in functional relaxation assays using porcine coronary arteries. iii SV40LT-SMC cells were also used to study CGRP2 receptors. In contrast to coronary arteries, the putative CGRP? receptor in this cell line was identified as an adrenomedullin receptor that has low affinity for CGRPg.37. The inability of ['^1]CGRP to label adrenomedullin receptors is consistent with the fact that the CGRP2 receptor has not been previously identified by radioligand binding. In summary, my data do not support the widely accepted view that CGRP- induced responses are mediated by two different CGRP receptor subtypes. Rather these data support the conclusions that the putative CGRP2 receptor is an adrenomedullin receptor and that CGRPg.37 blocks CGRP] receptor mediated responses with low affinity in some isolated tissues. I anticipate that this work will have a significant impact on the currently accepted classification of CGRP receptors. iv PREFACE Portions of the work described in this dissertation have been published in: Rorabaugh BR, Abel PW, Smith DD, Scofield MA (2002) Putative CGRP2 Receptors are Identified as Adrenomedullin Receptors in SV40LT-SMC Cells. E/f-SEE Joarna/ 16:A576. Rorabaugh BR. Scofield MA, Smith DD, Jeffries WB, Abel PW (2001) Functional Calcitonin Gene-Related Peptide Subtype 2 Receptors are Identified as Calcitonin Gene-Related Peptide 1 Receptors by Radioligand Binding and Reverse Transcription-Polymerase Chain Reaction. Joaraa/ o/*P/zarTMaco/ogy azzJ Eyperzwe/ztn/ P/zerapeatzc^. 299:1086-1094. Rorabaugh BR, Abel PW, Smith DD, Scofield MA (2001) CGRP Receptor Signaling Pathways in SK-N-MC and HER 293 Cells. ETfEEP Joarrza/ 15:A931. Rorabaugh BR, Abel PW, Smith DD, Scofield MA (2000) Evidence for the CGRP] Receptor in Porcine Coronary Artery. P4EEP Joaraaf 14:A1404. v D eJicoteJ to /?;y /oon'/y. vi ACKNOWLEDGMENTS Many people have contributed to the successful completion of this work. This project could not have been completed without the encouragement and support of my mentor, Dr. Margaret Scofield. I also gratefully acknowledge the members of my research committee, Dr. Peter Abel, Dr. Frank Dowd, Dr. Joe Knezetic, and Dr. David Smith for their insight and guidance throughout this project. I also thank Dr. William Jeffries, Dr. Michael Bradley, and Dr. Charles Bockman for allowing me to use their laboratory equipment and for their contributions to my professional development. Joe Haun of J& J Quality Meats (Elkhom, Nebraska) also contributed significantly to my research. Joe went out of his way to provide the pig tissues that 1 needed and always made me feel welcome at the slaughterhouse. I enjoyed the mornings that I spent at his business, and I learned some things about butchering too. 1 am also thankful for the support of my friends at Omaha Central Church of the Nazarene. They helped keep me focused on the things that are really important in life. This work could not have been completed without the support of my family, i am particularly grateful for the encouragement of my parents, grandparents, my sister, and my in-laws. These people were constant reminders that there is life after graduate school. I especially appreciate the moral support of Tom and Karen, f have enjoyed our weekends together in Kansas City, and we have become good friends. Finally, I am most deeply indebted to my wife for tolerating my late nights and weekends in the lab, for her constant encouragement, and for making all the cookies that helped keep my presence welcome at the slaughterhouse. I could not have done it without you! vii TABLE OF CONTENTS ABSTRACT..........................................................................................................................iii PREFACE..............................................................................................................................v DEDICATION......................................................................................................................vi ACKNOWLEDGEMENTS.................................................................................................vii LIST OF TABLES................................................................................................................xi LIST OF FIGURES.............................................................................................................xii ABBREVIATIONS.................................................... xiv CHAPTER 1. BACKGROUND AND OBJECTIVES......................................................... 1 Intercellular Communication.................................................................................... 1 Calcitonin Gene-Related Peptide.............................................................................. 1 CGRP Receptors and Accessory Proteins.................................................................. 6 Objectives................................................................... 12 CHAPTER 2. FUNCTIONAL CGRPi RECEPTORS IN PORCINE CORONARY ARTERIES ARE IDENTIFIED AS CGRP, RECEPTORS BY RADIOLIGAND BINDING AND REVERSE TRANSCRIPTION-POLYMERASE CHAIN REACTION..........................................................................................................................13 Abstract.....................................................................................................................13 Introduction.............................................................................................................. 14 Materials and Methods............................................................................................. 16 Chemicals and Reagents.............................................................................. 16 Peptide Synthesis......................................................................................... 16 RNA Isolation.............................................................................................. 17 RT-PCR........................................................................................................ 17 Cell Culture..................................................................................................20 Membrane Preparations...............................................................................20 ['*^I]ha.-CGRP Binding Kinetics.................................................................21 Competition Binding Assay............................................ 22 Measurement of Intracellular cAMP...........................................................22 Data Analysis............................................................................................... 23 Results...................................................................................................................... 24 Identification of CRLR and RAMP 1 mRNA in Porcine Coronary Arteries......................................................................................................... 24 Kinetics of ['^I]ha-CGRP Binding to CGRP Receptors........................... 26 Binding of CGRP Receptor Ligands to CGRP, Receptors in HEK 293 Cells ,29 viii Binding of CGRP Receptor Ligands to CGRP Receptors in Porcine Coronary Arteries........................................................... 33 Comparison of Ligand Affinities for CGRP Receptors in Porcine Coronary Arteries and HER 293 Cells.................................... 34 Activation of CGRP] Receptors by a Putative CGRPi Receptor Selective Agonist........................................................................................