Cell Surface Receptors: A Short Course on Theory and Methods

250 Pages·1996·4.833 MB·English

by  Lee E. Limbird (auth.)

CELL SURFACE RECEPTORS:
A SHORT COURSE ON THEORY AND METHODS
SECOND EDITION
CELL SURFACE RECEPTORS:
A SHORT COURSE ON THEORY AND METHODS
SECOND EDITION
LEE E. LIMBIRD
Vanderbilt University
Nashville, Tennessee
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Library of Congress Cataloging-in-Publication Data
A C.I.P. Catalogue record for this book is available
from the Library of Congress.
ISBN-13:978-1-4612-8535-9 e-ISBN-13:978-1-4613-1255-0
DOl: 10.107/978-1-4613-1255-0
Copyright ~ 1996 by Kluwer Academic Publishers. Third Printing 2003.
Softcover reprint of the hard cover 2nd Edition 1996
All rights reserved. No part of this publication may be reproduced, stored in
a retrieval system or transmitted in any form or by any means, mechanical,
photo-copying, recording, or otherwise, without the prior written permission of
the publisher, Kluwer Academic Publishers, 101 Philip Drive, Assinippi Park,
Norwell, Massachusetts 02061
Printed on acid-free paper.
This printing is a digital duplication of the original edition.
Research is essentially 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-Gyorgi, a paraphrase
CONTENTS
Preface xi
Preface to the first edition xiii
Acknow ledgments xvii
1 Historical perspective
The origin of the receptor concept
Mass action law and occupancy theory 7
The concept of efficacy 11
The concept of spare receptors 14
Rate theory 15
Allosteric theory 19
Operational models of pharmacological agonism 21
Summary 23
2 Methods for characterization of receptors based on receptor-mediated
responses in tissue or intact cell preparations 27
Characterization of receptor specificity 27
Generalizations regarding the determination of equilibrium dissociation
constants (K values) for receptor-ligand interactions in intact tissue
D
preparations 36
vii
viii Contents
Determination of KD values for receptor-agonist interactions, KDA 37
Determination of KD values for receptor-partial agonist interactions, KDp 46
Determination of KD values for receptor-antagonist interactions, K DB 50
Summary 58
3 Identification of receptors using direct radioligand binding techniques 61
Methods-data generation 61
Choice of a radioligand 62
The incubation 64
Separation of bound from free radioligand 65
Equilibrium dialysis 66
Centrifugation 68
Vacuum filtration 71
Criteria expected for binding of D to the physiological receptor, R 72
Data obtained to establish the criteria for a physiologically relevant
receptor 73
Determining the saturability of the radioligand binding 75
Plotting the saturation binding data 80
Linear transformations of saturation binding data 82
The Scatchard plot 83
The Rosenthal plot 86
Analysis and interpretation of saturation binding data 87
Interpretation of the linear transformations
for saturation binding 91
Kinetic strategies to resolve the origins of
complex binding phenomena 93
A reminder about assumptions in the development
of linear transformations 95
The Ifill plot 96
Determination of the specificity of radio ligand binding 100
Quantitation of the potency of competing agents 101
Quantitation of the shape of the competition binding curve 104
Computer-assisted analysis of steady state binding data 108
ix
Determination of rate constants for radio ligand association
and dissociation 109
Determination of the association rate constant 110
Determination of the dissociation rate constant 113
Distinguishing negative cooperativity from mUltiple
independent receptor populations using dissociation
strategies 116
Use of changes in the rates of radioligand dissociation to
reveal allosteric modulation of receptor binding properties 117
Summary 119
4 Complex binding phenomena 123
Mathematical descriptions of complex binding phenomena 124
Computer-assisted analysis of complex binding phenomena 132
Analysis of receptor subtypes 140
Independent data consistent with the existence of receptor subtypes 147
Receptor affinity states 148
The ternary complex model (TCM) and expansions of the TCM 152
Thermodynamic parameters of receptor-ligand interactions 158
Summary 163
5 The preparation and study of detergent-solubilized receptors 169
General properties of biological membranes and detergent micelles 170
Choice of a biological detergent 181
Solubilizing receptors from biological membranes 184
Methods for analysis of detergent-solubilized receptors 186
Equilibrium dialysis 186
Hummel-Dreyer chromatographic procedure 187
Gel filtration 189
Precipitation 192
Adsorption to filters 193
Receptor immobilization 194
Summary 196
x Contents
6 The topographical fate of ligand-receptor complexes as
reflected by the properties of ligand binding to intact cells 201
Biochemical approaches for discriminating between cell
surface receptors versus intracellular receptor-ligand complexes 203
Accessibility of impermeant ligands to interaction
with receptors 203
Protease-resistant ligand binding as a measure of
internalized receptor-ligand complexes 209
Rapid dissociation of exofacial ligand-receptor complexes
to resolve surface-bound from internalized ligand 211
Biochemical evidence consistent with recycling of cell
surface receptors 212
Assessment of rate constants for receptor turnover using a
steady state mathematical analysis of intact cell radioligand
binding data 216
The heavy amino acid density-shift technique for quantitating
receptor synthesis and turnover 225
Summary 228
Index 233
PREFACE
In preparing the first edition of this book, I was motivated to pass on
a practical understanding of receptor identification and char
acterization that I had acquired during my postdoctoral training as a
result of the tutelage and shared enthusiasm of Dr. Robert J.
Lefkowitz, my postdoctoral mentor, my colleagues in his laboratory,
especially Andre DeLean, and collaborators, including Pierre De
Meyts. Other aspects of this book deal with concepts I learned from
Joel G. Hardman while teaching a course on receptor theory and
characterization together at Vanderbilt University Medical Center. Not
unsurprisingly, a major source of input into my continued education
concerning receptor theory and characterization has been the inquiries
from motivated, highly inquisitive and persistent graduate students
whom I have experienced at Vanderbilt; I must also acknowledge the
educational "reminders" provided by national colleagues, including
Rick Neubig (University of Michigan), Harvey Motulsky (University
of California-San Diego and GraphPad, Inc.) and Terry Kenakin
(Burroughs Wellcome). The students' commitment, in particular, to
acquiring a strong knowledge base for later application to discovery
of new knowledge is extremely motivating for one's own self-renewal.
The pace at which new knowledge now can be acquired also affirms
another underlying premise of this text, i.e., that receptor
identification might begin with in vivo observations or, alternatively,
following expression cloning of a novel recognition molecule;
xi
xii Preface
nonetheless, the final elucidation of the structure, function, and in vivo
relevance of this receptor ultimately will require its study as a
molecule and as a participant in complex physiological regulation.
Hopefully, the information provided in this primer will be of value in
characterizing receptor function along this continuum.
Lee E. Limbird
Nashville, Tennessee
Leonardo DaVinci's contemplation on "The Lesson of Nature"-
where and when artists find their creative source in other artists
rather than in nature, the state of their art declines.