Growth Factors and Receptors A Practical Approach

by McKay, Ian A.; Brown, Kenneth D.

• ISBN13: 9780199636464
• ISBN10: 019963646X
• Format: Paperback
• Copyright: 1998-09-17
• Publisher: Oxford University Press

Summary

Growth Factors and Receptors provides comprehensive protocols for studiesof growth factors and their interactions with receptors. It covers a wide rangefrom simple analytical techniques to sophisticated in vivo applicationsincluding: RT-PCR and immunocytochemistry for detection of growth factors andreceptors; production and purification of recombinant growth factors andreceptors; labelling of growth factors for binding studies; in vivo mutagenesis;the yeast two-hybrid assay of protein/protein interactions; phage display offactors; application of factors to wound-healing processes using the gene gun;treatment of cancers with factor/toxin chimeras; and analysis of importantfactor domains using chimeric proteins. This book updates and extends thecurrent literature and describes important novel approaches to the study ofgrowth factors and their receptors, including the use of RNA aptamers asreceptor antagonists, and the development of receptor superantagonists. It willbe of tremendous value to both researchers and teachers, and, through anappendix that lists a large number of growth factors and receptors, will serveas a handy reference text.

Table of Contents

List of contributors	xvii
Abbreviations	xxi
Engineered growth factors and receptors, and their applications	1	(18)
Ian A. McKay
Stephen A. Bustin
Kenneth D. Brown
Introduction	1	(1)
Known growth factors and receptors	1	(1)
Modes of factor-receptor interaction	1	(1)
Applications of engineered growth factors and receptors	2	(1)
Principal methods for engineering growth factors and their receptors	2	(2)
Mutagenesis	2	(1)
Formation of chimeric proteins	3	(1)
Manufacture of alternative ligands and receptors	3	(1)
Design of engineered growth factors and receptors	4	(3)
DNA sequence of factor or receptor to be engineered	4	(1)
Analysis of existing DNA sequence for useful restriction sites	4	(1)
Design of wholly synthetic growth factors and receptors	4	(3)
Expression of DNAs encoding growth factors or receptors	7	(12)
Deciding on an expression system	7	(2)
Microbial expression	9	(1)
In vitro expression	10	(1)
Eukaryotic gene expression	11	(6)
Acknowledgements	17	(1)
References	17	(2)
Engineering of novel neurotrophins	19	(32)
Leopold L. Ilag
Carlos F. Ibanez
Introduction	19	(1)
Structure-function analyses of neurotrophins	20	(1)
Site-directed mutagenesis	21	(6)
Kunkel method	22	(4)
PCR-based mutagenesis	26	(1)
Expression of recombinant protein	27	(4)
COS cell expression	27	(2)
Baculovirus expression	29	(1)
Expression of recombinant proteins in prokaryotic systems (E. coli)	30	(1)
Protein purification	31	(2)
Biochemical assays	33	(6)
Binding assay	33	(3)
Cross-linking assay	36	(1)
Phosphorylation assay	37	(2)
Biological assays	39	(6)
Therapeutic potential of engineered neurotrophins	45	(1)
Conclusion	46	(5)
Acknowledgements	47	(1)
References	47	(4)
Designing new agonists/antagonists of growth factor receptors-the rational design of a superantagonist of the IL-6 receptor	51	(32)
G. Ciliberto
A. Lahm
G. Paonessa
R. Savino
C. Toniatti
Introduction	51	(2)
Molecular modelling of a trimeric IL-6/IL-6Rα/gp130 complex	53	(1)
Model construction	53	(1)
Identification of the putative IL-6 site 1 and site 2 epitopes	53	(1)
IL-6 biological assay	54	(4)
In vitro binding assay of IL-6 to IL-6Rα	58	(2)
IL-6 antagonists mutated in site 2: biochemical and biological evaluation	60	(5)
Generation of IL-6 antagonists	61	(2)
Specificity of IL-6 antagonists	63	(2)
Tag-mediated immunoprecipitation assay (TAMIA)	65	(4)
Selection of amino acid substitutions which increase affinity of IL-6 for IL-6Rα	69	(8)
Generation of IL-6 phage particles	69	(3)
Generation of IL-6 phage libraries	72	(2)
Selection of IL-6 superbinders from a phage library	74	(1)
IL-6 variants with higher affinity for IL-6Rα	75	(2)
Combination of antagonistic and superbinder mutations to generate IL-6 superantagonists	77	(1)
Potential use of IL-6 superantagonists in therapy	78	(5)
Acknowledgements	79	(1)
References	79	(4)
A chimeric approach for studying receptor binding domains in EGF-like molecules	83	(26)
E. J. J. van Zoelen
A. E. G. Lenferink
M. J. H. van Vugt
M. L. M. van de Poll
Introduction	83	(1)
Chimeric growth factors	84	(1)
EGF-related factors and their receptors	85	(5)
EGF-related factors	85	(1)
Receptors for the EGF-related factors	86	(1)
Structure-function relationship of EGF and TGFα	87	(2)
Chimeric proteins of the EGF family	89	(1)
Preparation and isolation of recombinant EGF-like growth factors	90	(8)
Introduction	90	(3)
Construction and expression of EGF/TGFα chimeras	93	(5)
Characterization of EGF/TGFα domain-exchange mutants	98	(11)
Introduction	98	(7)
Functional studies on EGF/TGFα chimeras	105	(1)
Acknowledgements	105	(1)
References	105	(4)
Structure-activity relationships of chemokines	109	(16)
Ian Clark-Lewis
Jennifer Anderson
Philip Owen
Luan Vo
Jiang-Hong Gong
Introduction	109	(3)
Chemokines	110	(1)
Primary structure: practical considerations	110	(1)
Importance of the 3D structure	111	(1)
Design: testing an hypothesis	112	(1)
Single substitutions	112	(1)
Multiple substitutions	112	(1)
Hybrids/chimeras	112	(1)
Purification, folding, and analysis	113	(6)
Sample preparation and RP-HPLC analysis	113	(2)
Folding	115	(1)
Purification	116	(2)
Verification of covalent structure	118	(1)
Handling and storage	118	(1)
Functional assays	119	(1)
General considerations	119	(1)
Cell migration	119	(1)
Receptor binding	120	(5)
Iodination of chemokines	120	(2)
Receptor binding assays	122	(1)
Bindability and kinetics	123	(1)
References	123	(2)
Generation and selection of RNA ligands that inhibit the interaction of vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) with its receptors	125	(24)
Louis S. Green
Nebojsa Janjic
Introduction to the SELEX process	125	(1)
Application of the SELEX process to identifying RNA ligands to VPF/VEGF	126	(11)
Random RNA libraries	126	(3)
Selection conditions	129	(2)
Consensus sequence and secondary structure identification	131	(2)
Minimal sequence determination	133	(1)
Selection of VPF/VEGF anatgonists: inhibition of receptor binding	134	(3)
Methods for making RNA ligands nuclease resistant	137	(6)
Clinical applications of VPF/VEGF antagonists	143	(6)
References	146	(3)
Immunolocalization and RT-PCR for the detection and quantification of growth factor and receptor gene expression	149	(26)
Simon R. Myers
Harshad A. Navsaria
Introduction	149	(2)
Assays of protein expression in tissues	149	(1)
Assays of mRNA expression in tissues	150	(1)
Immunolocalization of growth factor and receptor protein	151	(6)
Introduction	151	(1)
Tissue preparation	151	(1)
Storage and dilution of antibodies	152	(1)
Detection methods	152	(1)
Controls	153	(2)
Immunohistochemistry	155	(2)
Semi-quantification of immunohistochemistry by computer image analysis	157	(2)
Introduction	157	(1)
Average OD measurement	158	(1)
Interpretation of immunohistochemistry results	159	(2)
Semi-quantification of mRNA levels using non-competitive RT-PCR	161	(7)
Introduction	161	(1)
Extraction of total RNA from small tissue specimens	162	(1)
Reverse transcription of mRNA to cDNA	163	(1)
PCR amplification of mRNA-derived cDNA with specific oligonucleotide primers	164	(2)
Detection of RT-PCR product and quantification	166	(2)
Competitive RT-PCR quantification of specific mRNA levels	168	(3)
Introduction	168	(1)
Mutant templates	168	(2)
Use of mutant templates in tissue analysis	170	(1)
Interpretation of RT-PCR amplification analyses of gene expression	171	(4)
References	173	(2)
Defining growth factor function through tissue-specific expression of dominant-negative receptor mutants	175	(24)
Sabine Werner
Introduction	175	(3)
The concept of dominant-negative growth factor receptors	176	(1)
Examples of efficient use of dominant-negative growth factor receptors in transgenic mice	177	(1)
Design and synthesis of a gene encoding a dominant-negative receptor mutant	178	(1)
Identification of transgenic mice	179	(2)
Analysis of transgene expression	181	(7)
Advantages and disadvantages of various RNA detection methods	181	(1)
RNase protection assay	182	(6)
In situ detection of transgene mRNA and protein	188	(1)
Characterization of the skin of transgenic mice	188	(11)
Histological analysis	188	(2)
Analysis of differentiation-specific proteins in the epidermis	190	(3)
Labelling of proliferating cells with 5-bromo-2'-deoxyuridine (BrdU)	193	(2)
Acknowledgements	195	(1)
References	196	(3)
Growth factor-toxin chimeras and their applications	199	(28)
Pamela A. Davol
A. Raymond Frackelton, Jr.
Paul Calabresi
Introduction	199	(1)
Detecting chimeric function	200	(14)
Evaluating cytotoxicity as a function of toxin activity	200	(3)
Evaluating cytotoxicity as a function of receptor specificity	203	(5)
Quantitating cytotoxic activity on various cell lines	208	(2)
Assessing antitumour effects in animals	210	(4)
Clinical applications of growth factor-toxin chimeras	214	(2)
Cancer therapy	214	(1)
Other applications	215	(1)
Potential problems in using biologically active chimeras	216	(7)
Pitfalls associated with the toxin moiety	217	(1)
Adverse effects associated with growth factor-toxin chimeras	217	(2)
Mechanisms of resistance	219	(4)
Conclusions	223	(4)
References	223	(4)
Gene therapy applications of growth factors	227	(38)
Tor Svensjo
Feng Yao
Bohdan Pomahac
Elof Eriksson
Introduction to gene therapy applications of growth factors	227	(7)
Gene transfer methods	228	(4)
Gene therapy with growth factors	232	(2)
Growth factor applications to enhance cutaneous wound healing	234	(1)
Growth factor expression in skin	235	(8)
Detecting growth factor expression in target organs	243	(4)
Determining the effects of growth factors on wound healing	247	(1)
Summary	248	(5)
References	248	(5)
Appendices
A1 Growth factors	253	(6)
A2 Amino acid abbreviations	259	(2)
A3 Names and addresses of suppliers	261	(4)
Index	265