Agonist‐Stimulated [35S]GTPγS Binding

Rita Raddatz1, Richard H. Alper2

1 Cephalon, Inc., West Chester, Pennsylvania, 2 University of Kansas School of Medicine, Kansas City, Kansas
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 2.6
DOI:  10.1002/0471141755.ph0206s36
Online Posting Date:  March, 2007
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Abstract

This unit presents a convenient method for studying receptor function using a binding assay to assess the initial step in receptor‐mediated G protein activation in membranes prepared from rat brain. While the example procedure employs receptor agonists for serotonin1A receptors in the hippocampus, the method can be employed to study the activity of any substance that activates a receptor coupled to a G protein, particularly Gi. Alternate protocols describe modifications that can be used to simplify and increase the accuracy of the basic protocol, as well the use of the basic protocol to study receptor antagonists.

Keywords: GTPγS; 5‐HT; serotonin; 5‐HT1A; hippocampus; membrane preparation; binding; G protein

     
 
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Table of Contents

  • Basic Protocol 1: Assay of Receptor‐Mediated [35S]GTPγS Binding in Rat Hippocampal Membranes: Receptor Agonists
  • Alternate Protocol 1: Modifications to Simplify and Increase Accuracy and Precision of [35S]GTPγS‐Binding Assay
  • Alternate Protocol 2: Assay of Receptor‐Mediated [35S]GTPγS Binding in Rat Hippocampal Membranes: Receptor Antagonists
  • Alternate Protocol 3: Assay of Receptor‐Mediated [35S]GTPγS Binding Using a Scintillation Proximity Assay Format
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Assay of Receptor‐Mediated [35S]GTPγS Binding in Rat Hippocampal Membranes: Receptor Agonists

  Materials
  • Live rats or frozen rat brain (Pel‐Freez)
  • Isopentane (optional, if using live rats)
  • 50 mM Tris⋅Cl, pH 7.4 ( appendix 2A), 4°C
  • Assay buffer (see recipe), 4°C
  • Test compounds: receptor agonists
  • Unlabeled GTPγS: guanosine 5′‐O‐(3‐thiotriphosphate), tetralithium salt (Sigma), or guanosine 5′‐O‐(3‐thiotriphosphate), dilithium salt (Boehringer Mannheim)
  • Guanosine 5′‐diphosphate (GDP), sodium salt (Sigma)
  • Guanosine 5′‐(γ‐[35S]thio)triphosphate (Perkin‐Elmer)
  • 20 mM (8.93 g/liter) Na 4P 2O 7⋅10 H 2O
  • Scintillation cocktail (Ready Protein, Beckman)
  • Tissue homogenizer (Tekmar or Brinkman) and homogenization tubes
  • Refrigerated centrifuge
  • 12 × 75–mm tubes or microcentrifuge tubes
  • 37°C shaking water bath
  • 500‐ml squirt bottle
  • Glass fiber filters (Whatman GF/B)
  • Cell harvester (Brandel)
  • 7‐ml scintillation vials
  • Software for nonlinear regression analysis (e.g., SigmaPlot and SigmaStat)
  • Additional reagents and equipment for protein assay (Bradford or Lowry; appendix 3A)

Alternate Protocol 1: Modifications to Simplify and Increase Accuracy and Precision of [35S]GTPγS‐Binding Assay

  • Assay buffer containing 0.136 mM DTT: add 13.6 µl of 1 M DTT stock (store in aliquots at −20°C) per 100 ml assay buffer (see recipe) just before use

Alternate Protocol 2: Assay of Receptor‐Mediated [35S]GTPγS Binding in Rat Hippocampal Membranes: Receptor Antagonists

  • Test compounds: receptor agonists and antagonists

Alternate Protocol 3: Assay of Receptor‐Mediated [35S]GTPγS Binding Using a Scintillation Proximity Assay Format

  • Wheat germ agglutinin (WGA)‐coated scintillation proximity beads (SPA beads, Amersham Biosciences)
  • Scintillation counter with bottom‐reading capability (such as Wallac Microbeta)
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Figures

Videos

Literature Cited

   Alper, R.H. and Nelson, D.L. 1998. Characterization of serotonin1A receptor‐mediated [35S]GTPγS binding in rat hippocampal membranes. Eur. J. Pharmacol. 343:303‐312.
   Cheng, Y. and Prusoff, W.H. 1973. Relationship between the inhibition constant (Ki) and the concentration of inhibitor which causes 50% inhibition (I50) of an enzymatic reaction. Biochem. Pharmacol. 22:3099‐3108.
   DeLapp, N.W. 2004. The antibody‐capture [35S]GTPgammaS scintillation proximity assay: a powerful emerging technique for analysis of GPCR pharmacology. Trends Pharmacol Sci. 25:400‐401.
   DeLapp, N.W., McKinzie, J.H., Sawyer, B.D., Vandergriff, A., Falcone, J., McClure, D., and Felder, C.C. 1999. Determination of [35S]Guanosine‐5‐O‐(3‐thio)triphosphate binding mediated by cholinergic muscarinic receptors in membranes from Chinese hamster ovary cells and rat striatum using an anti‐G protein scintillation proximity assay. J. Pharmacol. Exper. Therap. 289:946‐955.
   De Vivo, M. and Maayani, S. 1986. Characterization of the 5‐hydroxytryptamine1A receptor‐mediated inhibition of forskolin‐stimulated adenylate cyclase activity in guinea pig and rat hippocampal membranes. J. Pharmacol. Exp. Ther. 238:248‐253.
   Harrington, M.A., Oksenberg, D., and Peroutka, S.J. 1988. 5‐Hydroxytryptamine1A receptors are linked to a Gi‐adenylate cyclase complex in rat hippocampus. Eur. J. Pharmacol. 154:95‐98.
   Harrison, C. and Traynor, J.R. 2003. The [35S]GTPγS binding assay: Approaches and applications in pharmacology. Life Sci. 74:489‐508.
   Kenakin, T. 1996. The classification of seven transmembrane receptors in recombinant expression systems. Pharmacol. Rev. 48:413‐463.
   Lazareno, S. and Birdsall, N.J. 1993. Estimation of competitive antagonist affinity from functional inhibition curves using the Gaddum, Schild and Cheng‐Prusoff equations. Br. J. Pharmacol. 109:1110‐1119.
   Lazareno, S., Farries, T., and Birdsall, N.J. 1993. Pharmacological characterization of guanine nucleotide exchange reactions in membranes from CHO cells stably transfected with human muscarinic receptors m1‐m4. Life Sci. 52:449‐456.
   Morisset, S., Rouleau, A., Ligneau, X., Gbahou, F., Tardivel‐Lacombe, J., Stark, H., Schunack, W., Ganellin, C.R., Schwartz, J.‐C., and Arrang, J.‐M. 2000. High constitutive activity of native H3 receptors regulates histamine neurons in brain. Nature 408:860‐864.
   Odagaki, Y. and Toyoshima, R. 2006. 5‐HT‐stimulated [35S]guanosine‐5′‐O‐(3‐thio)triphosphate binding as an assay for functional activation of G proteins coupled with 5‐HT1B receptors in rat striatal membranes. Naunyn Schmiedebergs Arch. Pharmacol. 372:335‐345.
   Okuhara, D.Y. and Beck, S.G. 1994. 5‐HT1A receptor linked to inward‐rectifying potassium current in hippocampal CA3 pyramidal cells. J. Neurophysiol. 71:2161‐2167.
   Oleskevich, S. 1995. Gαo1 decapeptide modulates the hippocampal 5‐HT1A potassium current. J. Neurophysiol. 74:2189‐2193.
   Panchalingam, S. and Undie, A.S. 2000. Optimized binding of [35S]GTPγS to Gq‐like proteins stimulated with dopamine D1‐like receptor agonists. Neurochem. Res. 25:759‐767.
   Porter, A.C., Bymaster, F.P., DeLapp, N.W., Yamada, M., Wess, J., Hamilton, S.E., Nathanson, N.M., and Felder, C.C. 2002. M1 muscarinic receptor signaling in mouse hippocampus and cortex. Brain Res. 944:82‐89.
   Schlegel, J.R. and Peroutka, S.J. 1986. Nucleotide interactions with 5‐HT1A binding sites directly labeled by [3H]‐8‐hydroxy‐2‐(di‐n‐propylamino)tetralin ([3H]‐8‐OH‐DPAT). Biochem. Pharmacol. 35:1943‐1949.
   Sim, L.J., Selley, D.E., and Childers, S.R. 1995. In vitro autoradiography of receptor‐activated G proteins in rat brain by agonist‐stimulated guanylyl 5′‐[γ[35S]thio]‐triphosphate binding. Proc. Natl. Acad. Sci. U.S.A. 92:7242‐7246.
   Sim, L.J., Selley, D.E., Dworkin, S.I., and Childers, S.R. 1996. Effect of chronic morphine administration on µ opioid receptor‐stimulated [35S]GTPγS autoradiography in rat brain. J. Neurosci. 16:2684‐2692.
   Zgombick, J.M., Beck, S.G., Mahle, C.D., Craddock‐Royal, B., and Maayani, S. 1989. Pertussis toxin‐sensitive guanine nucleotide‐binding protein(s) couple adenosine A1 and 5‐hydroxytryptamine1A receptors to the same effector systems in rat hippocampus: Biochemical and electrophysiological studies. Mol. Pharmacol. 35:484‐494.
Key Reference
   Harrison and Traynor, 2003. See above.
  Provides references for GTPγS binding assays for many G protein–coupled receptors and describes the effects of changing assay conditions.
   Weiland, T. and Jakobs, K.H. 1994. Measurement of receptor‐stimulated guanosine 5′‐O‐(γ‐thio)triphosphate binding by G proteins. Methods Enzymol. 237:3‐13.
  Describes how the assay should be optimized for any G protein–coupled receptor.
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