Iyengar G Protein Pathways, Part B: G Proteins and Their Regulators

1. Auflage 2001
ISBN: 978-0-08-049692-4
Verlag: Elsevier Science & Techn.
Format: EPUB
Kopierschutz: 6 - ePub Watermark

E-Book, Englisch, Band Volume 344, 813 Seiten

Reihe: Methods in Enzymology

This volume covers topics such as the structure and identification of functional domains of G proteins, and activation of G proteins by receptors or other regulators. The text takes an integrated approach to studying common experimental questions at many different levels related to G proteins. Methods related to G proteins using molecular modeling, systems biology, protein engineering, protein biochemistry, cell biology, and physiology are all accessible in the same volume. The critically acclaimed laboratory standard for more than forty years, Methods in Enzymology is one of the most highly respected publications in the field of biochemistry. Since 1955, each volume has been eagerly awaited, frequently consulted, and praised by researchers and reviewers alike. Now with more than 300 volumes (all of them still in print), the series contains much material still relevant todaytruly an essential publication for researchers in all fields of life sciences.

Autoren/Hrsg.

Iyengar, Ravi

Inhaltsverzeichnis

1;Front Cover;1
2;G Protein Pathways;4
3;Copyright Page;5
4;Table of Contents;6
5;Contributors to Volume 343;10
6;Preface;16
7;Volume in Series;20
8;Section I: G Protein-Coupled Receptors;40
8.1;A. Theoretical Evaluation of Receptor Function;40
8.1.1;Chapter 1. Considerations in the Evaluation of Inverse Agonism and Protean Agonism at GProtein-Coupled Receptors;42
8.1.2;Chapter 2. Theoretical Implications of Receptor Coupling to Multiple G Proteins Based on Analysis of Three-State Model;56
8.2;B. Design and Use of Receptor Ligands;40
8.2.1;Chapter 3. Use of Retinal Analogues for the Study of Visual Pigment Function;68
8.2.2;Chapter 4. Design and Synthesis of Peptide Antagonists and Inverse Agonists for G Protein-Coupled Receptors;88
8.2.3;Chapter 5. Design of Peptide Agonists;112
8.2.4;Chapter 6. Design of Nonpeptides from Peptide Ligands for Peptide Receptors;130
8.2.5;Chapter 7. Strategies for Mapping the Binding Site of the Serotonin 5-HT2A Receptor;162
8.3;C. Structural Characterization of Receptor Proteins;40
8.3.1;Chapter 8. Use of the Substituted Cysteine Accessibility Method to Study the Structure and Function of G Protein-Coupled Receptors;176
8.3.2;Chapter 9. Mass Spectrometric Analysis of GProtein-Coupled Receptors;196
8.3.3;Chapter 10. Probing the Higher Order Sructure of G Protein- Coupled Receptors Using Tethered Cleavage Methods;201
8.3.4;Chapter 11. Use of Fluorescence Spectroscopy to Study Conformational Changes in the ß2-Adrenoceptor;209
8.3.5;Chapter 12. Crystallization of Membrane Proteins in Cubo;222
8.3.6;Chapter 13. N-Linked Carbohydrates on G Protein-Coupled Receptors: Mapping Sites of Attachment and Determining Functional Roles;239
8.3.7;Chapter 14. Magic Angle Spinning Nuclear Magnetic Resonance of Isotopically Labeled Rhodopsin;251
8.3.8;Chapter 15. Use of Nuclear Magnetic Resonance to Study the Three-Dimensional Structure of Rhodopsin;262
8.4;D. Design and Use of Engineered Receptor Proteins;40
8.4.1;Chapter 16. Tools for Dissecting Signaling Pathways in Vivo: Receptors Activated Solely by Synthetic Ligands;271
8.4.2;Chapter 17. Analysis of Structure–Function from Expression of G Protein-Coupled Receptor Fragments;287
8.4.3;Chapter 18. Construction and Analysis of Function of G Protein-Coupled Receptor–G Protein Fusion Proteins;299
8.4.4;Chapter 19. Synthetic Gene Technology: Applications to Ancestral Gene Reconstruction and Structure Function Studies of Receptors;313
8.4.5;Chapter 20. Considerations in the Design and Use of Chimeric G Protein-Coupled Receptors ;334
8.5;E. Molecular Modeling Studies of Receptor Structure and Function;40
8.5.1;Chapter 21. Strategies for Modeling the Interactions of Transmembrane Helices of G Protein-Coupled Receptors by Geometric Complementarity Using the GRAMM Computer Algorithm;352
8.5.2;Chapter 22. Three-Dimensional Representations of G Protein- Coupled Receptor Structures and Mechanisms;368
8.6;F. Analysis of Receptor Protein Coupling;40
8.6.1;Chapter 23. Reconstitution of G Protein-Coupled Receptors with Recombinant GProteinaand ß. Subunits;411
8.6.2;Chapter 24. Cell-Free Membrane Desensitization Assay for G Protein-Coupled Receptors;433
8.6.3;Chapter 25. Methods to Determine the Constitutive Activity of Histamine H2 Receptors;444
8.6.4;Chapter 26. Expression of G Protein-Coupled Receptors and G Proteins in Sf9 Cells: Analysis of Coupling by Radioligand Binding;456
8.6.5;Chapter 27. G Protein-Coupled Receptors and Proliferative Signaling;469
8.7;G. Characterization of Receptor Heterogeneity;40
8.7.1;Chapter 28. Genetic Analysis of G Protein-Coupled Receptor Genes;487
8.7.2;Chapter 29. IdentiIfication of Adrenergic Receptor Polymorphisms;498
8.7.3;Chapter 30. Strategies and Requirements for the Detection of RNA Editing in G Protein Coupled-Receptor RNA;515
8.8;H. The Study of Receptor Trafficking;40
8.8.1;Chapter 31. Fluorescence MicroscopyTechniques for the Study of G Protein-Coupled Receptor Trafficking;531
8.8.2;Chapter 32. Measurement of Receptor Desensitization and Internalization in Intact Cells;545
8.8.3;Chapter 33. Morphological and Biochemical Strategies for Monitoring Trafficking of Epitope-Tagged G Protein-Coupled Receptors in Agonist- Naive and Agonist-Occupied States;569
9;Section II: Regulators of GPCR Function;584
9.1;A. G Protein-Coupled Receptor Kinases (GRKs);584
9.1.1;Chapter 34. Characterization of G Protein-Coupled Receptor Kinases;586
9.1.2;Chapter 35. Regulation of G Protein-Coupled Receptor Kinase 2;598
9.1.3;Chapter 36. Rhodopsin and Its Kinase;617
9.2;B. Arrestins and Novel Proteins;584
9.2.1;Chapter 37. Characterization of Arrestin Expression and Function;639
9.2.2;Chapter 38. Identification of Novel G Protein-Coupled Receptor-Interacting Proteins;650
10;Author Index;662
11;Subject Index;700

Leseproben

[1] Analysis of G Protein Activation in Sf9 and Mammalian Cells by Agonist-Promoted [35S]GTP?S Binding
Rolf T. Windh; David R. Manning Introduction
G-protein-coupled receptors (GPCRs) mediate many of their effects through the activation of heterotrimeric G proteins. The activated receptor accelerates an exchange of GTP for GDP on the G protein a subunit. The corresponding change in conformation of the a subunit results in the release of ß?, and both the GTP-bound a subunit and released ß? dimers interact with a variety of effectors, including adenylyl cyclase, phospholipases, and ion channels. In time, the intrinsic GTP hydrolysis activity of the a subunit converts GTP to GDP, and the GDP-bound a subunit reassociates with ß? to reform a heterotrimer. Traditional analysis of GPCR signaling has relied on changes in the activity of downstream effectors as readouts for receptor and G protein function. Amplification at each step in the transduction pathway makes events distal to receptor activation easy to detect biochemically, and a great deal has been deduced about both signal transduction pathways and receptor–ligand interactions from these studies. It is clear, however, that receptor pharmacology is often system-dependent; that is, the relative efficacies of a variety of receptor ligands differ depending on which of various downstream events is measured.1 Differences in the type of G protein activated or which G protein subunit interacts with the effector, as well as differential regulation of each step of the amplification scheme, can contribute to this system-dependent pharmacology. By measuring the activation of G proteins themselves, the first step in the traditional signaling cascade, some of these issues can be bypassed. Direct assessment of G protein activation by G-protein-coupled receptors is typically accomplished using either of two conserved features of the G protein cycle. One group of assays measures agonist-induced increases in the rate of GTP hydrolysis by the a subunit.2 In these GTPase assays, the release of inorganic phosphate from radiolabeled GTP is determined. These assays can be performed either as single turnover studies, by preloading the G proteins with [32P]GTP, or as steady-state production of inorganic [32P]phosphate release in the continued presence of [32P]GTP. Exchange of GDP for analogs of GTP on the a subunit is the basis for the other major assay of G protein activation by receptor. Hydrolysis-resistant, radiolabeled forms of GTP are used to monitor the exchange. The most widely used GTP analog is guanosine 5'-(?-[35S]thio)triphosphate ([35S]GTP?S). Since GTP?S cannot be hydrolyzed to GDP, GDP–GTP?S exchange assays measure the progression of an irreversible activation rather than steady-state activation/deactivation cycles. As GDP release is the rate-limiting step in the activation of G proteins,3 however, these assays can measure a highly relevant aspect of G protein activation. Furthermore, because the background binding can be controlled more tightly than in GTP hydrolysis studies, the signal strength for GDP–GTP?S exchange assays tends to be greater than for GTPase assays. Whereas direct assay of G protein activity, rather than changes in effector activity, provides a closely linked measure of receptor–ligand interactions, neither GTPase nor GDP–GTP?S exchange assays, when applied to cell membranes, indicate which G proteins the receptor is engaging. Identifiying the G proteins that couple to a given receptor provides a great deal of information about the downstream pathways to be regulated. It has become clear that receptors can couple to multiple G proteins. A receptor often couples to multiple members of a single family of G proteins; a receptor that activates Gi2 will probably also activate other members of the Gi family.4,5 Whereas it is not surprising that structurally related family members will couple to the same receptor, it is not uncommon for a receptor to engage members of two, three, or even all four families of G proteins.6–9 We describe here a GDP–GTP?S exchange assay in which the [35S]GTP?S-bound G protein a subunit is immunoprecipitated with subtype-specific antisera, making it possible to confidently identify which subtypes of G proteins are being activated by receptor in membranes expressing multiple G proteins. In addition to providing a means of identifying which G proteins are activated by a given receptor, the immunoprecipitation method also greatly improves the signal-to-noise ratio. In bypassing the binding of [35S]GTP?S to the filtration membrane or other GTP-binding proteins, the immunoprecipitation method greatly reduces background binding without significantly altering binding induced by ligand. Thus the measured GTP?S binding following activation represents a larger increase over basal, as high as 20-fold in some preparations.10 This increased signal strength allows for more accurate ranking of efficacies for partial agonists and partial inverse agonists as well as a more confident assignment of the G protein activated. We describe these assays using cell expression models that we have employed successfully—Sf9 (Spodoptera frugiperda) cells expressing mammalian receptors and G proteins,9,11,12 and HEK293 (human embryonal kidney) cells12,13 and CHO (Chinese hamster ovary) cells10 in which selected receptors have been introduced by transfection. In some instances, we have been able to measure activation of G proteins in mammalian cells through receptors endogenous to these cells,12 although not in all instances because of issues of sensitivity. Experimental Procedures
Preparation of Protein A
To 0.5 g of protein A immobilized on Sepharose CL-4B (Sigma, St. Louis, MO), add 10 ml of 10 mM HEPES, pH 7.4, and shake gently for 30 min at 4° to allow the beads to swell and to wash away stabilizers added for storage and shipment. Pellet the protein A-Sepharose beads by centrifuging for 5 min at approximately 4000g. Pour off the supernatant, and add 10 ml of ice-cold wash buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 20 mM MgCl2) containing 0.5% (v/v) Nonidet P-40 (NP-40) and 4 mg/ml of bovine serum albumin (BSA) to block nonspecific binding sites on the Sepharose beads. Mix to resuspend the slurry, and shake 30 min at 4°. Pellet the protein A-Sepharose beads as before, and wash the pellet twice by resuspending in 10 ml of cold wash buffer with NP-40 but without BSA. Finally, resuspend the pellet in 10 ml of cold wash buffer containing NP-40 and 0.33% (v/v) aprotinin (Sigma, St. Louis, MO; or 5µg protein/ml), and store at 4°. Preparation of Cell Membranes
HEK293 or CHO cells (or Sf9 cells; see Ref. 14 for growth, infection, and membrane preparation protocols) grown in monolayer and expressing the desired receptors (and G proteins in Sf9 cells) should be washed several times on the plate with cold phosphate-buffered saline and left on ice. Add 0.5 ml/plate of ice-cold HE/PI buffer (20 mM HEPES, pH 8.0, 2 mM EDTA; add protease inhibitors [2 µg/ml aprotinin, 10 µg/ml leupeptin, and 0.1 mM phenylmethylsulfonyl fluoride (PMSF)] immediately before using, scrape cells, and transfer to a microfuge tube. Break the scraped cells by passing gently through a 26-gauge needle 15 times. Pellet the nuclei and unbroken cells by centrifuging for 5 min at 660g, and spin the supernatant for 30–45 min at 20,000g, to pellet the membranes. Resuspend the pellet in HE/PI buffer such that the final protein concentration is 1–3 mg/ml (usually 0.1 or 0.25 ml HE/PI buffer per original confluent 10 cm plate of CHO or HEK293 cells, respectively). Determine protein concentration, aliquot the membranes into lots appropriate for individual assays, freeze in a dry ice/ethanol bath, and store at -70°. Membranes prepared in this manner can be thawed and used only once and keep well for at least 2–3 months. [35S]GTP?S Assay Overview
A standard assay to demonstrate agonist-induced activation of a given G protein requires a minimum of three conditions (Fig. 1). In the first two conditions, the G protein a subunit is immunoprecipitated with a subunit-selective antiserum following incubation of the membranes with [35S]GTP?S in the absence or presence of agonist. The third condition serves as a control for the immunoprecipitation step; membranes that have been incubated with [35S]GTP?S and vehicle are carried through the immunoprecipitation protocol with a preimmune or non-immune serum. Each condition is performed in duplicate, so the most basic assay is six individual assay points. Fig. 1 Typical [35S]GTP?S binding...