Practical Aspects of Radioligand Binding

Michael McKinney1, Rita Raddatz2

1 Mayo Clinic Jacksonville, Jacksonville, Florida, 2 Cephalon, Inc., West Chester, Pennsylvania
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 1.3
DOI:  10.1002/0471141755.ph0103s33
Online Posting Date:  July, 2006
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Radioligand binding has been used for many years to identify new binding sites, characterize receptors, and identify novel ligands. Although various techniques have been developed to improve the efficiency of preparing the biological source of the receptors and for detecting bound radioligand, the principles of the assays remain the same. This unit reviews theory and provides examples of the parameters that can be calculated from radioligand binding data to characterize ligandÔÇÉreceptor interactions. The important aspects of assay development and validation that allow meaningful interpretation are discussed. The selection of a radioligand, buffer and other assay components is critical to developing a useful binding assay. The nature of the binding interaction can also be probed by varying assay conditions.

Keywords: radioligand binding; filtration; membrane preparation; affinity; saturation binding; displacement

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Fundamentals of Radioligand Binding Assays
  • Guidelines for Establishing a Radioligand Binding Assay
  • Analysis of Binding Data
  • Troubleshooting
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

   Abramson, S.N., McGonigle, P., and Molinoff, P.B. 1987. Evaluation of models for analysis of radioligand binding data. Mol. Pharmacol. 31:103‐111.
   Ator, M.A. and Williams, M. 2005. Safety Pharmacology III—Target profiling. (S.J. Enna and D.B. Bylund, eds.) xPharm, Elsevier, New York. http://www.xpharm.com/
   Beck, J.S. and Goren, H.J. 1983. Simulation of association curves and ‘Scatchard’ plots of binding reactions where ligand and receptor are degraded or internalized. J. Recept. Res. 3:561‐577.
   Bennett, J.P. Jr. and Yamamura, H. 1986. Neurotransmitter, hormone, or drug receptor binding methods. In Neurotransmitter Receptor Binding, 2nd ed. (H.I. Yamamura, S.J. Enna, and M.J. Kuhar, eds.) pp. 61‐90. Raven Press, New York.
   Berry, J. and Price‐Jones, M. 2005. Measurement of radioligand binding by scintillation proximity assay. Methods Mol. Biol. 306:121‐138.
   Borea, P.A., Dalpiaz, A., Varani, K., Gessi, S., and Gilli, G. 1996. Binding thermodynamics at A1 and A2A adenosine receptors. Life Sci. 59:1373‐1388.
   Burgisser, E. 1984. Radioligand‐receptor binding studies: What's wrong with the Scatchard analysis? Trends Pharmcol. Sci. 5:142‐145.
   Cheng, H.C. 2004. The influence of cooperativity on the determination of dissociation constants: Examination of the Cheng‐Prusoff equation, the Scatchard analysis, the Schild analysis and related power equations. Pharmacol. Res 50:21‐40.
   Cheng, Y.‐C. and Prusoff, W.H. 1973. Relation ship between the inhibition constant (KI) and the concentration of inhibitor which causes 50 percent inhibition (I50) of an enzymatic reaction. Biochem. Pharmacol. 22:3099‐3103.
   Childers, S.R. and Snyder, S.H. 1980. Differential regulation by guanine nucleotides or opiate agonist and antagonist receptor interactions. J. Neurochem. 34:583‐593.
   Christopoulos, A. and Kenakin, T. 2002. G‐protein‐coupled receptor allosterism and complexing. Pharmacol. Rev 54:323‐374.
   Contreras, M.L., Wolfe, B.B., and Molinoff, P.B. 1986. Thermodynamic properties of agonist interactions with the beta adrenergic receptor‐coupled adenylate cyclase system. I. High‐ and low‐affinity states of agonist binding to membrane‐bound beta adrenergic receptors. J. Pharmacol. Exp. Ther. 237:154‐164.
   Cuatrecasas, P. and Hollenberg, M.D. 1976. Membrane receptors and hormone action. Adv. Protein Chem. 30:251‐451.
   DeLean, A., Munson, P.J., and Rodbard, D. 1978. Simultaneous analysis of families of sigmoidal curves: Application to bioassay, radioligand assay, and physiological dose‐response curves. Am. J. Physiol. 235:E97‐E102.
   DeLean, A., Stadel, J.M., and Lefkowitz, R.J. 1980. A ternary complex model explains the agonist‐specific binding properties of the adenylate cyclase‐coupled β‐adrenergic receptor. J. Biol. Chem. 255:7108‐7117.
   DeLean, A., Hancock, A.A., and Lefkowitz, R.J. 1982. Validation and statistical analysis of a computer modeling method for quantitative analysis of radioligand binding data for mixtures of pharmacological receptor subtypes. Mol. Pharmacol. 21:5‐16.
   Ehlert, F.J., Roeske, W.R., and Yamamura, H.I. 1981. Mathematical analysis of the kinetics of competitive inhibition in neurotransmitter receptor binding assays. Mol. Pharmacol. 19:367‐371.
   Feldman, H.A. 1972. Mathematical theory of complex ligand‐binding systems at equilibrium: Some methods for parameter fitting. Anal. Biochem. 48:317‐338.
   Feldman, H.A. 1983. Statistical limits in Scatchard analysis. J. Biol. Chem. 258:12865‐12867.
   Filer, C., Hurl, S., and Wan, Y.‐P. 1989. Radioligands: Synthesis and handling. In Receptor Binding. (M. Williams, P.B.M.W.M. Timmermans, and R.A. Glennon, eds.) pp. 105‐135. Marcel Dekker, New York.
   Fleming, W.W., Westfall, D.P., De La Lande, I.S., and Jellett, L.B. 1972. Log‐normal distribution of equieffective doses of norepinephrine and acetylcholine in several tissues. J. Pharmacol. Exp. Ther. 181:339‐345.
   Gaddum, J.H. 1945. Log normal distributions. Nature 156:463‐466.
   Hill, A.W. 1910. The possible effects of the aggregation of the molecules of hemoglobin on its dissociation curves. J. Physiol. (Lond.) 40:iv‐vii.
   Jacobs, S., Chang, K.‐J., and Cuatrecasas, P. 1975. Estimation of hormone receptor affinity by competitive displacement of labeled ligand: Effect of concentration of receptor and of labeled ligand. Biochem. Biophys. Res. Commun. 66:687‐692.
   Jarv, J., Hedlund, B., and Bartfai, T. 1979. Isomerization of the muscarinic receptor‐antagonist complex. J. Biol. Chem. 254:5595‐5598.
   Karlsson, M.O. and Neil, A. 1989. Estimation of ligand binding parameters by simultaneous fitting of association and dissociation data: A Monte Carlo simulation study. Mol. Pharmacol. 35:59‐66.
   Kenakin, T. 1993. Radioligand binding experiments. In Pharmacologic Analysis of Drug‐Receptor Interaction, 2nd ed., pp. 385‐410. Raven Press, New York.
   Kenakin, T. 1996. The classification of seven transmembrane receptors in recombinant expression systems. Pharmacol Rev. 48:413‐63.
   Ketelslegers, J.‐M., Pirens, G., Maghuin‐Rogister, G., Hennen, G., and Frere, J.‐M. 1984. The choice of erroneous models of hormone‐receptor interactions: A consequence of illegitimate utilization of Scatchard graphs. Biochem. Pharmacol. 33:707‐710.
   Koshland, D.E., Nemthy, G., and Filmer, D. 1966. Comparison of experimental binding data and theoretical models in proteins containing subunits. Biochemistry 5:365‐385.
   Lam, C.F. and Cross, A.P. 1979. Comparative study of parameter estimation procedures in enzymic kinetics. Comput. Biol. Med. 9:145‐153.
   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.
   Limbird, L. 1996. Cell Surface Receptors: A Short Course in Theory and Methods, 2nd ed. Kluwer Academic Publishers, Boston.
   Linden, J. 1982. Calculating the dissociation constant of an unlabeled compound from the concentration required to displace radiolabel binding by 50%. J. Cyclic Nucleotide Res. 8:163‐172.
   Maksay, G. 1994. Thermodynamics of gamma‐aminobutyric acid type A receptor binding differentiate agonists from antagonists. Mol. Pharmacol. 46:386‐390.
   Marquardt, D.W. 1963. An algorithm for least‐squares estimation of nonlinear parameters. J. Soc. Indust. Appl. Math. 11:431‐441.
   McKinney, M., Stenstrom, S., and Richelson, E. 1985. Muscarinic responses and binding in a murine neuroblastoma clone (N1E‐115). Mediation of separate responses by high affinity and low affinity agonist‐receptor conformations. Mol. Pharmacol. 27:223‐235.
   Metcalf, M.A., McGuffin, R.W., and Hamblin, M.W. 1992. Conversion of the human 5‐HT1Db serotonin receptor to the rat 5‐HT1B ligand‐binding phenotype by Thr355Asn site directed mutagenesis. Biochem. Pharmacol. 44:1917‐1920.
   Meyer, J.H., Wilson, A.A., Sagrati, S., Hussey, D., Carella, A., Potter, W.Z., Ginovart, N., Spencer, E.P., Cheok, A., and Houle, S. 2004. Serotonin transporter occupancy of five selective serotonin reuptake inhibitors at different doses: An [11C]DASB positron emission tomography study. Am. J. Psychiatry 161:826‐835.
   Monod, J., Wyman, J., and Changeux, J.‐P. 1965. On the nature of allosteric transitions: A plausible model. J. Mol. Biol. 12:88‐118.
   Motulsky, H. and Neubig, R. 1997. Analyzing radioligand binding data. In Current Protocols in Neuroscience (J.N., Crawley, C.R., Gerfen, R., McKay, M.A., Rogawski, D.R., Sibley, and P., Skolnick, eds.) pp. 7.5.1‐7.5.55. John Wiley & Sons, New York.
   Motulsky, H.J., Mahan, L.C., and Insel, P.A. 1985. Radioligand, agonists and membrane receptors on intact cells: Data analysis in a bind. Trends Pharmacol. Sci. 6:317‐319.
   Munson P.J. and Rodbard, D. 1980. LIGAND: A versatile computerized approach for characterization of ligand‐binding systems. Anal. Biochem. 107:220‐239.
   Murphy, D.E., Schnieder, J., Boehm, C., Lehmann, J., and Williams, M. 1987. Binding of [3H]3‐(2‐carboxypiperazin‐4‐yl)propyl‐1‐phosphonic acid to rat brain membranes: A selective, high‐affinity ligand for N‐methyl‐D‐aspartate receptors. J. Pharmacol. Exp. Ther. 240:778‐784.
   Murphy, D.E., Hutchison, A.J., Hurt, S.D., Williams, M., and Sills, M.A. 1988. Characterization of the binding of [3H]CGS 19755: A novel N‐methyl‐D‐aspartate antagonist with nanomolar affinity in rat brain. Br. J. Pharmacol. 95:932‐938.
   Nelder, J.A. and Mead, R. 1965. A simplex method for function minimization. Computer J. 7:308‐313.
   Nelson, N. 1987. A novel method for the detection of receptors and membrane proteins by scintillation proximity radioassay. Analytical Biochemistry 165:287‐293.
   Pellicciari, R., Marinozzi, M., Natalini, B., Costantino, G., Luneia, R., Giorgi, G., Moroni, F., and Thomsen, C. 1996. Synthesis and pharmacological characterization of all sixteen stereoisomers of 2‐(2′‐carboxy‐3′‐phenylcyclopropyl)glycine. Focus on (2S,1′S,2′S,3′R)‐2‐(2′‐carboxy‐3′‐phenylcyclopropyl)glycine, a novel and selective group II metabotropic glutamate receptor antagonist. J. Med. Chem. 39:2259‐2269.
   Rabow, L.E., Russek, S.J., and Farb, D.H. 1995. From ion currents to genomic analysis: Recent advances in GABA‐A receptor research. Synapse 21:189‐274.
   Rodbard, D. 1973. Mathematics of hormone‐receptor interaction. I. Basic principles. Adv. Exp. Med. Biol. 36:289‐329.
   Rodbard, D., Munson, P.J., and Thakur, A.K. 1980. Quantitative characterization of hormone receptors. Cancer 46:2907‐2918.
   Rosenthal, H.E. 1967. Graphic method for the determination and presentation of binding parameters in a complex system. Anal. Biochem. 20:525‐532.
   Scatchard, G. 1949. The attractions of proteins for small molecules and ions. Ann. N.Y. Acad. Sci. 51:660‐672.
   Schreiber, G., Henis, Y.I., and Sokolovsky, M. 1985. Rate constants of agonist binding to muscarinic receptors in rat brain medulla. Evaluation by competition kinetics. J. Biol. Chem. 260:8795‐8802.
   Sullivan, J.P., Decker, M.W., Brioni, J., Donnelly‐Roberts, D., Anderson, D.A., Bannon, A.W., Kang, C.‐H., Adams, P., Piattoni‐Kaplan, M., Buckley, M.J., Gopalakrishnan, M., Williams, M., and Arneric, S.P. 1994. (+)‐Epibatidine elicits a diversity of in vitro and in vivo effects mediated by nicotinic acetylcholine receptors. J. Pharmacol. Exp. Ther. 271:624‐631.
   Taylor, S.J., Michel, A.D., and Kilpatrick, G.J., 1992. In vivo occupancy of histamine H3 receptors by thioperamide and RAMHA measured using histamine turnover and an ex vivo labeling technique. Biochem. Pharmacol. 44:1261–1267.
   Tolkovsky, A.M. and Levitzki, A. 1981. Theories and predictions of models describing sequential interactions between the receptor, the GTP regulatory unit, and the catalytic unit of hormone dependent adenylate cyclases. J. Cyclic Nucleotide Res. 6:139‐150.
   Waelbroeck, M., Tastenoy, M., Camus, J., and Christophe, J. 1990. Binding of selective antagonists to four muscarinic receptors (m1 to m4) in rat forebrain. Mol. Pharmacol. 38:267‐273.
   Waelbroeck, M., Camus, J., Tastenoy, M., Lambrecht, G., Mutschler, E., Kropfgans, M., Sperlich, J., Wiesenberger, F., Tacke, R., and Christophe, J. 1993. Thermodynamics of antagonist binding to rat muscarinic M2 receptors: Antimuscarinics of the pridinol, sila‐pridinol, diphenidol and sila‐diphenidol type. Br. J. Pharmacol. 109:360‐370.
   Weiland, G.A., Minneman, K.P., and Molinoff, P.B. 1979. Fundamental difference between the molecular interactions of agonists and antagonists with the β‐adrenergic receptor. Nature 281:114‐117.
   Wild, K.D., Porreca, F., Yamamura, H.I., and Raffa, R.B. 1994. Differentiation of receptor subtypes by thermodynamic analysis: Application to opioid receptors. Proc. Natl. Acad. Sci. U.S.A. 91:12018‐12021.
   Williams, M., Mehlin, C., Raddatz, R., and Triggle, D.J. 2005. Receptor targets in drug discovery. In Encyclopedia of Molecular Cell Biology and Molecular Medicine (R.A. Meyers, ed.) John Wiley and Sons, New York.
   Wreggett, K.A. and DeLean, A. 1984. The ternary complex model. Its properties and application to ligand interactions with the D2‐dopamine receptor of the anterior pituitary gland. Mol. Pharmacol. 26:214‐227.
   Yasuda, R.P., Ciesia, W., Flores, L.R., Wall, S.J., Li, M., Satkus, S.A., Weisstein, J.S., Spagnola, B.V., and Wolfe, B.B. 1993. Development of antisera selective for m4 and m5 muscarinic cholinergic receptors: Distribution of m4 and m5 muscarinic receptors in rat brain. Mol. Pharmacol. 43:149‐157.
   Zuck, P., Lao, Z., Skwish, S., Glickman, J.F., Yang, K., Burbaum, J., and Inglese, J. 1999. Ligand‐receptor binding measured by laser‐scanning imaging. Proc. Natl. Acad. Sci. U.S.A. 96:11122‐11127.
Key References
   Kenakin, 2004. A pharmacology primer: Theory, application and methods. Elsevier, Inc., London, UK.
  A complete treatise for the advanced student.
   Weiland, G.A. and Molinoff, P.B. 1981. Quantitative analysis of drug‐receptor interactions: I. Determination of kinetic and equilibrium properties. Life Sci. 29:313‐330.
  Useful for explaining common plotting methods.
   Yamamura, H.I., Enna, S.J., and Kuhar, M.J. 1985. Neurotransmitter Receptor Binding, 2nd ed. Raven Press, New York.
  This volume has been a standard in the field for many years and is especially useful for beginners.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library