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Receptor biochemistry: A practical approach
Cell, Vol. 65, 925-926,
June
14, 1991, Copyright
D 1991 by Cell Press
Book Review
The Practical Approach to Receptor Biochemistry: Pure and Functional Receptor Biochemistry: A Practical Approach. Edited by E. C. Hulme. Oxford, England: IRL Press at Oxford University Press, (1990). 352 pp. $83.00.
Purification of pharmacologically important binding sites for structural analysis (e.g., subunit composition, transmembrane topology, and amino acid sequencing) has contributed greatly to our understanding of transmembrane receptors. Partial amino acid sequences or antibodies raised against purified receptors, together with the advent of DNA cloning technology, were necessary for the rapid accumulation of the primary sequence of numerous transmembrane receptors. Analysis of these sequences suggested that essentially all receptors are members of a few large gene families with homologous domains. This finding has subsequently limited the necessity for receptor-specific antibodies or partial primary sequence prior to DNA cloning; many novel receptors are now cloned by low stringency hybridization or utilization of polymerase chain reaction methodology. Hence, the popularity of DNA cloning of receptors for structural analysis has increased more rapidly than the interest in receptor biochemistry. Cloning and other recombinant DNA techniques, however, provide only a partial solution to questions concerning transmembrane receptor structure. Still, the most promising approach for discerning conformational changes upon ligand binding, or comprehending the relationship of primaly sequence to secondary, tertiary, or quaternary structure, is through physical means such as X-ray crystallography and nuclear magnetic resonance spectroscopy. Furthermore, understanding receptor coupling is still best achieved by reconstitution of receptors and other proteins into phospholipid vesicles. The study of receptors by physical methods or reconstitution experiments, however, requires large amounts of purified receptor. Thus, we come full circle again to the art and necessity of receptor purification. A new addition to the Practical Approach series, Receptor Biochemistry is intended to be a useful guide for obtaining purified receptor for protein sequence analysis or for use in structural or functional studies. General strategies for receptor purification and over 150 detailed protocols are presented. Although the primary focus is receptor purification, a detailed chapter on peptide mapping and isolation of peptides for sequence analysis, a short chapter on transfection and receptor expression in cultured cells, and a review of the strategies for deducing secondary structure from primary sequence all contribute to making this book of more general interest. The volume begins with an excellent review of the general
strategies used in solubilization and purification of receptors (by T. Haga, K. Haga, and the editor of this volume, E. C. Hulme). This chapter introduces the three large families of transmembrane receptors: ligand-gated ion channels, receptors with a single transmembrane segment, and G protein-coupled receptors. The authors emphasize that these receptor families have different physical properties and often require different strategies for solubilization and purification. The calculation of yield at each step of purification often requires the binding of radiolabeled ligand to solubilized receptor; hence, the authors also stress the preservation of a receptors ligand-binding activity during solubilization and purification. A review of the different types of chromatography, with special attention to affinity chromatography, is also given. The chapter concludes with a short discussion on molecular characterization of receptors, which includes protein estimation, determination of polypeptide composition, and reconstitution of purified receptors, as well as the basic strategies used for obtaining and expressing the cDNAs encoding receptors. The next eight chapters present protocols for the solubilization and purification of the following receptors: muscarinic acetylcholine receptors(T. Haga, K. Haga, and E. C. Hulme), dopamine (DP) receptors (P. G. Strange and R. A. Williamson), opioid receptors (C. D. Demoliou-Mason and E. A. Barnard), 8-adrenergic receptor (J. L. Benovic), a-adrenergic receptor (J. W. Regan and H. Matsui), muscle-type nicotinic receptors from Torpedo (S. HertlingJaweed, G. Bandini, and F. Hucho), GABAA receptor (F. A. Stephenson), and epidermal growth factor receptor (G. N. Panayotou and M. Gregoriou). This section of the book provides valuable examplesof proven approaches taken in receptor purification. The cross-referencing of these chapters with the first chapter of the text allows the reader to turn to a specific example, and proven protocol, once an appropriate solubilization procedure, ligand-binding assay, or purification strategy has been decided. Much of the “meat and potatoes” of receptor biochemical techniques are contained within a chapter entitled “Peptide mapping and the generation and isolation of sequenceable peptides from receptors” by M. Wheatley. This chapter pre sents many basic techniques, including radioiodination, peptide cleavage, SDS-PAGE, preparing peptides for sequencing, and ashort discussion of HPLC and FPLC. The attention to methodological detail in this chapter is exemplified by subheadings and protocols entitled “pouring and running gels,““autoradiography,““elution of peptidesfrom gel slices,” and many others. Receptors are often expressed only in low abundance. Therefore, overexpression of a receptor in a tissue culture cell line may ease the burden of receptor purification. Thus, Receptor Biochemistry contains a review by C. M. Fraser of the strategies for “Expression of receptor genes in cultured cells.” Although brief, the author presents a useful discussion of factors influencing the choice of recipient cell lines, promoters, and the selection systems used for isolation of stable transfectants. The chapter also contains a practical sum-
Cell 926
mary of, and a protocol for transfection with, calcium phosphate-precipitated DNA. Alternate transfection procedures and the baculovirus expression system are mentioned only briefly, but are well referenced. Other expression systems, such as bacteria and Xenopus oocytes, have been omitted, presumably because only transfected cultured cells can produce large amounts of functional receptor. These omissions leave open the possibility of an additional Practical Approach volume on receptor expression systems. The last chapter contains a review of “Structural deductionsfrom receptor sequences.” Here, N. M. Green presents ageneral strategy for analysisof receptor sequences. Hydrophobicity plots, functional sites in receptors, prediction of secondary structure, and the software for sequence analysis are introduced. The discussion is brief, but is a reasonable review of the general strategies for sequence analysis with a plethora of references. E. C. Hulme completes this volume with an appendix containing a brief outline of receptor binding. This segment of the book contains many of the general principles of binding assays, including the factors influencing the source and preparation of receptors, how the kinetics of ligand binding may determine the assay method, and preliminary analysis and interpretation of binding data. Again, cross-referencing allows the reader to choose an appropriate assay, then turn elsewhere in the text for a specific example. Receptor Biochemistry is not intended as a review encompassing all aspects of receptor biochemical analysis, but is designed to be a guide for obtaining purified receptor for structural and functional studies. Dr. Hulme hassuccessfully met this goal by assembling outlines and protocols for a practical approach to receptor solubilization, purification, and preliminary analysis. The editor cautiously warns the reader in the preface that inevitably there are repetitions and omissions. In reality, the repetitions are few, and many of the omissions are either well referenced or contained in another Practical Approach volume. Two related Practical Approach volumes, also edited by Dr. Hulme, are Receptor-Effector Coupling and Receptor-Ligand Interactions. If these additional volumes are as well organized as ReceptorBiochemistry, then the trilogy would be a “practical” reference guide for any laboratory working with receptors. Paul Blount Department of Anatomy and Neurobiology Washington University School of Medicine St. Louis, Missouri 63110 Books Received Bertini, I., Bolinari, H., and Niccolai, N. (1991). NMR and Biomolecular Structure. VCH Publishers, New York. 209 pp. $85.00. Ciba Foundation (1990). Steroids York. 284 pp. 563.50. Ciba Foundation Liss, New York.
and Neuronal
(1991). Myopia and the Control 256 pp. 563.50.
Ciba Foundation (1991). Sioactive New York. 242 pp. $63.50.
Compounds
Activity.
Wiley-Liss,
of Eye Growth.
New Wiley-
from Plants. Wiley-Liss,
Dennis, E. A. (1991). Methods in Enzymology, Press, San Diego, California. 640 pp. $79.00.
Volume
197. Academic
Edelman, G. M., Gall, W. E., and Cowan, W. M. (1990). Signal and Sense: Local and Global Order in Perceptual Maps. Wiley-Liss, New York. 727 pp. 5149.95. Gribskov, Stockton
M., and Devereux, J. (1991). Sequence Press, New York. 279 pp. 539.95.
Analysis
Primer.
Harrison, F. W., and Bogitsh, B. J. (1990). Microscopic Anatomy Invertebrates, Volume 3. Wiley-Liss, New York. 347 pp. $150.00.
of
Harrison, F. W., and Corliss, J. 0. (1990). Microscopic Anatomy Invertebrates, Volume 1. Wiley-Liss, New York. 493 pp. 5150.00.
of
Harrison, F. W., and Westtall, J. A. (1990). Microscopic Anatomy Invertebrates, Volume 2. Wiley-Liss, New York. 436 pp. 5150.00.
of
Kosower, Princeton
E. M. (1991). Molecular Mechanisms for Sensory University Press, New Jersey. 438 pp. $79.50.
Moody, P. E. C., and Wilkinson, A. J. (1991). Protein Oxford University Press, New York. 85 pp. 513.95. Oppenheim, J. J., and Shevach, ford University Press, New York.
Engineering.
E. M. (1991). Immunophysiology. 424 pp. 555.00.
Rasmussen, H. (1991). Cell Communication in Health W. H. Freeman & Co., New York. 185 pp. 513.95.
Signals.
Ox-
and Disease.
June
14, 1991, Copyright
D 1991 by Cell Press
Book Review
The Practical Approach to Receptor Biochemistry: Pure and Functional Receptor Biochemistry: A Practical Approach. Edited by E. C. Hulme. Oxford, England: IRL Press at Oxford University Press, (1990). 352 pp. $83.00.
Purification of pharmacologically important binding sites for structural analysis (e.g., subunit composition, transmembrane topology, and amino acid sequencing) has contributed greatly to our understanding of transmembrane receptors. Partial amino acid sequences or antibodies raised against purified receptors, together with the advent of DNA cloning technology, were necessary for the rapid accumulation of the primary sequence of numerous transmembrane receptors. Analysis of these sequences suggested that essentially all receptors are members of a few large gene families with homologous domains. This finding has subsequently limited the necessity for receptor-specific antibodies or partial primary sequence prior to DNA cloning; many novel receptors are now cloned by low stringency hybridization or utilization of polymerase chain reaction methodology. Hence, the popularity of DNA cloning of receptors for structural analysis has increased more rapidly than the interest in receptor biochemistry. Cloning and other recombinant DNA techniques, however, provide only a partial solution to questions concerning transmembrane receptor structure. Still, the most promising approach for discerning conformational changes upon ligand binding, or comprehending the relationship of primaly sequence to secondary, tertiary, or quaternary structure, is through physical means such as X-ray crystallography and nuclear magnetic resonance spectroscopy. Furthermore, understanding receptor coupling is still best achieved by reconstitution of receptors and other proteins into phospholipid vesicles. The study of receptors by physical methods or reconstitution experiments, however, requires large amounts of purified receptor. Thus, we come full circle again to the art and necessity of receptor purification. A new addition to the Practical Approach series, Receptor Biochemistry is intended to be a useful guide for obtaining purified receptor for protein sequence analysis or for use in structural or functional studies. General strategies for receptor purification and over 150 detailed protocols are presented. Although the primary focus is receptor purification, a detailed chapter on peptide mapping and isolation of peptides for sequence analysis, a short chapter on transfection and receptor expression in cultured cells, and a review of the strategies for deducing secondary structure from primary sequence all contribute to making this book of more general interest. The volume begins with an excellent review of the general
strategies used in solubilization and purification of receptors (by T. Haga, K. Haga, and the editor of this volume, E. C. Hulme). This chapter introduces the three large families of transmembrane receptors: ligand-gated ion channels, receptors with a single transmembrane segment, and G protein-coupled receptors. The authors emphasize that these receptor families have different physical properties and often require different strategies for solubilization and purification. The calculation of yield at each step of purification often requires the binding of radiolabeled ligand to solubilized receptor; hence, the authors also stress the preservation of a receptors ligand-binding activity during solubilization and purification. A review of the different types of chromatography, with special attention to affinity chromatography, is also given. The chapter concludes with a short discussion on molecular characterization of receptors, which includes protein estimation, determination of polypeptide composition, and reconstitution of purified receptors, as well as the basic strategies used for obtaining and expressing the cDNAs encoding receptors. The next eight chapters present protocols for the solubilization and purification of the following receptors: muscarinic acetylcholine receptors(T. Haga, K. Haga, and E. C. Hulme), dopamine (DP) receptors (P. G. Strange and R. A. Williamson), opioid receptors (C. D. Demoliou-Mason and E. A. Barnard), 8-adrenergic receptor (J. L. Benovic), a-adrenergic receptor (J. W. Regan and H. Matsui), muscle-type nicotinic receptors from Torpedo (S. HertlingJaweed, G. Bandini, and F. Hucho), GABAA receptor (F. A. Stephenson), and epidermal growth factor receptor (G. N. Panayotou and M. Gregoriou). This section of the book provides valuable examplesof proven approaches taken in receptor purification. The cross-referencing of these chapters with the first chapter of the text allows the reader to turn to a specific example, and proven protocol, once an appropriate solubilization procedure, ligand-binding assay, or purification strategy has been decided. Much of the “meat and potatoes” of receptor biochemical techniques are contained within a chapter entitled “Peptide mapping and the generation and isolation of sequenceable peptides from receptors” by M. Wheatley. This chapter pre sents many basic techniques, including radioiodination, peptide cleavage, SDS-PAGE, preparing peptides for sequencing, and ashort discussion of HPLC and FPLC. The attention to methodological detail in this chapter is exemplified by subheadings and protocols entitled “pouring and running gels,““autoradiography,““elution of peptidesfrom gel slices,” and many others. Receptors are often expressed only in low abundance. Therefore, overexpression of a receptor in a tissue culture cell line may ease the burden of receptor purification. Thus, Receptor Biochemistry contains a review by C. M. Fraser of the strategies for “Expression of receptor genes in cultured cells.” Although brief, the author presents a useful discussion of factors influencing the choice of recipient cell lines, promoters, and the selection systems used for isolation of stable transfectants. The chapter also contains a practical sum-
Cell 926
mary of, and a protocol for transfection with, calcium phosphate-precipitated DNA. Alternate transfection procedures and the baculovirus expression system are mentioned only briefly, but are well referenced. Other expression systems, such as bacteria and Xenopus oocytes, have been omitted, presumably because only transfected cultured cells can produce large amounts of functional receptor. These omissions leave open the possibility of an additional Practical Approach volume on receptor expression systems. The last chapter contains a review of “Structural deductionsfrom receptor sequences.” Here, N. M. Green presents ageneral strategy for analysisof receptor sequences. Hydrophobicity plots, functional sites in receptors, prediction of secondary structure, and the software for sequence analysis are introduced. The discussion is brief, but is a reasonable review of the general strategies for sequence analysis with a plethora of references. E. C. Hulme completes this volume with an appendix containing a brief outline of receptor binding. This segment of the book contains many of the general principles of binding assays, including the factors influencing the source and preparation of receptors, how the kinetics of ligand binding may determine the assay method, and preliminary analysis and interpretation of binding data. Again, cross-referencing allows the reader to choose an appropriate assay, then turn elsewhere in the text for a specific example. Receptor Biochemistry is not intended as a review encompassing all aspects of receptor biochemical analysis, but is designed to be a guide for obtaining purified receptor for structural and functional studies. Dr. Hulme hassuccessfully met this goal by assembling outlines and protocols for a practical approach to receptor solubilization, purification, and preliminary analysis. The editor cautiously warns the reader in the preface that inevitably there are repetitions and omissions. In reality, the repetitions are few, and many of the omissions are either well referenced or contained in another Practical Approach volume. Two related Practical Approach volumes, also edited by Dr. Hulme, are Receptor-Effector Coupling and Receptor-Ligand Interactions. If these additional volumes are as well organized as ReceptorBiochemistry, then the trilogy would be a “practical” reference guide for any laboratory working with receptors. Paul Blount Department of Anatomy and Neurobiology Washington University School of Medicine St. Louis, Missouri 63110 Books Received Bertini, I., Bolinari, H., and Niccolai, N. (1991). NMR and Biomolecular Structure. VCH Publishers, New York. 209 pp. $85.00. Ciba Foundation (1990). Steroids York. 284 pp. 563.50. Ciba Foundation Liss, New York.
and Neuronal
(1991). Myopia and the Control 256 pp. 563.50.
Ciba Foundation (1991). Sioactive New York. 242 pp. $63.50.
Compounds
Activity.
Wiley-Liss,
of Eye Growth.
New Wiley-
from Plants. Wiley-Liss,
Dennis, E. A. (1991). Methods in Enzymology, Press, San Diego, California. 640 pp. $79.00.
Volume
197. Academic
Edelman, G. M., Gall, W. E., and Cowan, W. M. (1990). Signal and Sense: Local and Global Order in Perceptual Maps. Wiley-Liss, New York. 727 pp. 5149.95. Gribskov, Stockton
M., and Devereux, J. (1991). Sequence Press, New York. 279 pp. 539.95.
Analysis
Primer.
Harrison, F. W., and Bogitsh, B. J. (1990). Microscopic Anatomy Invertebrates, Volume 3. Wiley-Liss, New York. 347 pp. $150.00.
of
Harrison, F. W., and Corliss, J. 0. (1990). Microscopic Anatomy Invertebrates, Volume 1. Wiley-Liss, New York. 493 pp. 5150.00.
of
Harrison, F. W., and Westtall, J. A. (1990). Microscopic Anatomy Invertebrates, Volume 2. Wiley-Liss, New York. 436 pp. 5150.00.
of
Kosower, Princeton
E. M. (1991). Molecular Mechanisms for Sensory University Press, New Jersey. 438 pp. $79.50.
Moody, P. E. C., and Wilkinson, A. J. (1991). Protein Oxford University Press, New York. 85 pp. 513.95. Oppenheim, J. J., and Shevach, ford University Press, New York.
Engineering.
E. M. (1991). Immunophysiology. 424 pp. 555.00.
Rasmussen, H. (1991). Cell Communication in Health W. H. Freeman & Co., New York. 185 pp. 513.95.
Signals.
Ox-
and Disease.