Cell Surface Receptors: A Short Course on Theory and Methods PDF
Preview Cell Surface Receptors: A Short Course on Theory and Methods
CELL SURFACE RECEPTORS: A SHORT COURSE ON THEORY AND METHODS CELL SURFACE RECEPTORS: A SHORT COURSE ON THEORY AND METHODS LEE E. LIMBIRD Vanderbilt University Nashville, Tennessee Springer-Science+Business Media, B.V Copyright 1986© by SpringerScience+BusinessMediaDordrecht Softcoverreprintofthehardcover Istedition1986 OriginallypublishedbyMartinusNijhotfBostonin1986. Allrightsreserved.Nopartofthispublication maybereproduced,storedinaretrievalsystern, ortransmittedinanyform or byany means,mechanical,photocopying,recording,or otherwise, withoutwrittenpermissionofthepublisher,SpringerScience+BusinessMediaDordrecht Library ofCongress Cataloging-in-Publication Data Limbird, Lee E. Cellsurface receptors: A Short Course on Theory and Methods Includes bibliographies and index. 1. Cell receptors. 2. Binding sites (Biochemistry) 1. Tide. [DNLM: 1. Binding Sites. 2. Cdl Membrane. 3. Receptors, Endogenous Substances. QH 603.C43 L733h] QH603.C43L56 1985 574.87'5 85-11423 ISBN978-1-4757-1884-3 ISBN978-1-4757-1882-9(eBook) DOI10.1007/978-1-4757-1882-9 Research isessentially a dialogue with Nature. The important thing is not to wonder about Nature's answer - for she is always honest - but to closely examine your question to her. A. Szent-Györgi, aparaphrase CONTENTS Preface IX Acknowledgments Xlll 1 Historicalperspective 1 Theorigin ofthereceptor concept 1 Massaction law andoccupancy theory 6 Theconceptofefficacy 9 Theconceptofsparereceptors 12 Ratetheory 13 Allosterictheory 17 Summary 19 2 Methodsfor characterizationofreceptorsbased on receptor-mediated responsesin tissue or intactcellpreparations 23 Characterizationofreceptorspecificity 23 Generalizations regarding thedeterrnination ofequilibrium dissociation constants (K values)for receptor-ligand interactionsinintacttissue D preparations 32 DeterminationofKD values for receptor-agonist interactions, KD., 33 DeterminationofK values for receptor-partial agonistinteractions, K 38 D Dp vii viii Contents DeterminationofK valuesfor rcceptor-antagonistinteractions, K 41 D DB Summary 47 3 Identificationofreceptors using directradioligand binding techniques 51 Methods- datageneration 51 Choice ofaradioligand 52 The incubation 53 Separation ofbound from freeradioligand 54 Criteriaexpected for binding ofD to the physiological receptor, R 60 Data obtained to establish thecriteria for aphysiologicallyrelevant receptor 60 Summary 94 4 Complexbindingphenomena 97 Mathematicaldescriptions ofcomplexbinding phenomena 98 Cornputer-assistedanalysisofcomplcxbinding phcnomcna 104 The analysisofreceptorsubtypes 111 Receptoraffinity states 117 Thermodynamicparameters ofreceptor-ligandinteractions 124 Summary 129 5 The preparationandstudy ofdetergent-solubilized receptors 133 General propertiesofbiological membranes and dctergent micelles 134 Thechoiceofabiological detergent 143 Solubilizing receptors from biological membranes 145 Methods for analysisofdetergent-solubilizedreceptors 147 Summary 156 6 Biochemicalcorrelates ofthe topographicalfate ofligand-receptor complexes 159 Lifehistories ofcellsurface receptors and how they aredetcrminedat the light andelectron microscopic level 160 The fateofligand-receptorcomplexes inintact cellsbascdon biochemical, rather than morphological, approaches 167 Summary 190 Index 195 PREFACE The purpose ofthis book is to serve as a primer for the study ofcell surface receptors. The simplified discussion of methods and their underlying prin ciples is intended to remove the usual intimidation caused by the specialized vocabulary or sophisticated mathematics that characterize many of the primary papers in this field. In this way, it is hoped that the basic concepts can be emphasized. This book is meant to be a starting point: a textbook as well as a manual to which the investigator can return for a refresher course, when needed. I feit compelled to write this book for several reasons. The primary philosophical reason was to provide, in one volume, an overall perspective on the study ofcellsurface receptors that describes their characterization from pharmacological studies in a whole organ or tissue bath (chapter 2) to studies ofradioligand binding in isolated membrane and detergent-solubilized pre parations (chapters 3-5) and, back again, to studies in intact cells or tissues using morphological and biochemical techniques (chapter 6). It ismy opinion that the ultimate understanding ofthe molecular basis for hormone or drug receptor interactions, and the physiological consequences of these inter actions, will require an assessment ofcell surface receptor function at all of these experimental levels. In many cases an investigator may not choose to pursue personally each line of investigation considered here. Nonetheless, understanding the principles that underlie a certain methodological approach will certainly assist an investigator in reading and appreciating scientific ix x Preface reports from other laboratories. There were pragmatic considerations that urged me to write this book as well. I have been frustrated in teaching part of a graduate course on drug receptor interactions because there exists no single didactic account for the novice ofhow to investigate these interactions. Inaddition, I am often asked by investigators who arejustbeginning experiments on receptoridentification where to turn for a basic primer. Certainly there are excellent review articles in the literature, but their quality in terms ofthorough informational content does not necessarily make them good starting points for someone who isjust entering the experimental area of receptor characterization. Finally, as a reviewer of many manuscripts, I am reminded that many of us, including myself, would benefit from an occasional refresher course on the basic principles ofligand-receptor interactions. I am indebted to many people for teaching me what Ihave tried to pass on to the readers of this volume. It was my present chairman, Joel Hardman, who introduced me to the concepts and literature that form the basis for the discussion of pharmacological approaches to receptor characterization (chapters 1and 2). Iam additionally grateful to Robert Furchgott for critically reading these chapters and clarifying for me some ofthe concepts with which I was uncomfortable. The personal tutelage of Pierce DeMeyts, a former collaborator, helped me appreciate the relationship between the consequences of mass action law and the choice of an appropriate experimental design (chapter 3). Another former colleague, Andre de Lean, refined my under standing ofradioligand-receptor interactions and introduced me to computer modeling as a tool for objectively analyzing experimental data as well as for selecting the most discriminating experimental approach (chapter 4). The empirical comments regarding methodologies for characterizing membrane bound and detergent-solubilized receptors in chapters 3 and 5 result from my own laboratory experiences as well as those shared with my many colleagues in Bob Lefkowitz's laboratory while I was a postdoctoral fellow at Duke University. I remain grateful, as well, to the tutelage offered by Jacqueline Reynolds and CharlesTanfordat DukeUniversity regarding the hydrophobic effect and the principles governing the actions of biological detergents (chapter 5). Finally, it was Joachim R. Sommer, Department ofPathology, Duke University, who made me keenly aware ofthe unambiguous insights that can be gained when morphological and biochemical techniques are combined to address cellular questions (chapter 6). A less easily quantitated impact on one's education is the contagious excitement about a particular discipline that creates the desire to learn more, experiment more, and, finally, to teach more. I am especially grateful to three people who shared with me, in an important way, their joy ofscience: T.R. Williams (College of Wooster, Wooster, Ohio), Joachim R. Sommer (Duke University), and my mentor during my postdoctoral days, RobertJ. Lefkowitz. The genuine enjoyment which they experience when asking xi questions amenable to scientific inquiry has taught me more than any major tome. I hope that I have been able to pass some of that curiosity and excitement on to the readers ofthe present text. Lee E. Limbird Nashville, Tennessee March 1985
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