title: Use of radiolabeled antagonist assays for assessing agonism at D2 and D3 dopamine receptors: comparison with functional GTPγS assays.
abstract: BACKGROUND
  Cell-based drug screening assays are essential tools for drug discovery and development targeting G protein-coupled receptors, which include dopamine D3 receptors. D3 is notorious for its poor coupling to G protein in most heterologous cell lines, and therefore D3 agonist-stimulated binding of [(35)S]GTPγS to G protein cannot be observed in many "non-functional" D3 expressing cell lines.
  
  
  METHODS
  The present work explores the use of an alternate method for assessing agonist activity, consisting of measuring the difference in agonist competition between [(3)H]spiperone bound to low-affinity states of the receptor and that with radioligand bound to high-affinity states (GTP shift assay).
  
  
  CONCLUSIONS
  The current study describes the determination of GTP shifts in [(3)H]spiperone binding assays for the assessment of agonists' potencies (at D2 and D3) and efficacies (at D3). Compared with GTPγ(35)S binding assays, the new method removes the cumbersome need of functional D3 cell lines and limited project duration due to short half-life of isotope (35)S.
  
  
  CONCLUSIONS
  The new method allows the estimation of potency (D2 and D3) and efficacy (D3) at the level of receptor and G protein activation in a simple fashion from shifts in monophasic-inhibition curves. Moreover, it does not require [(35)S]GTPγS binding assays with functional D3 cells. This method will have wide applicability for D3-selective agonist screening. It may also be useful for other GPCRs circumventing the need for functional assays and offering the ability to detect agonist activity regardless of the particular signaling pathway.
authors.affiliation: Department of Psychiatry, New York University School of Medicine, New York, NY, USA. Electronic address: juan.zhen@nyumc.org.
authors.forename: Juan
authors.initials: J
authors.lastname: Zhen
authors.affiliation: Department of Psychiatry, New York University School of Medicine, New York, NY, USA.
authors.forename: Tamara
authors.initials: T
authors.lastname: Antonio
authors.affiliation: Department of Neuroscience and Physiology, New York University School of Medicine, New York, NY, USA.
authors.forename: Solav
authors.initials: S
authors.lastname: Ali
authors.affiliation: Research Service, Department of Veterans Affairs Medical Center, Portland, OR, USA.
authors.forename: Kim A
authors.initials: KA
authors.lastname: Neve
authors.affiliation: Department of Pharmaceutical Sciences, Wayne State University, Detroit, MI, USA.
authors.forename: Aloke K
authors.initials: AK
authors.lastname: Dutta
authors.affiliation: Department of Psychiatry, New York University School of Medicine, New York, NY, USA; Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, New York, NY, USA.
authors.forename: Maarten E A
authors.initials: ME
authors.lastname: Reith
mesh_terms: D000818:Animals; D016466:CHO Cells; D003412:Cricetulus; D018491:Dopamine Agonists / Q000494:pharmacology*; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology*; D057809:HEK293 Cells; D006801:Humans; D017448:Receptors, Dopamine D2 / Q000819:agonists* / Q000235:genetics; D050637:Receptors, Dopamine D3 / Q000819:agonists* / Q000235:genetics; D013134:Spiperone / Q000494:pharmacology; D013462:Sulfur Radioisotopes; D014162:Transfection; D014316:Tritium
chemical_list: D018491:Dopamine Agonists; D017448:Receptors, Dopamine D2; D050637:Receptors, Dopamine D3; D013462:Sulfur Radioisotopes; D014316:Tritium; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D013134:Spiperone
keywords: Affinity; Binding assay; D(2) receptors; D(3) receptors; Dopamine receptors; Efficacy; Functional assay; GTP shift; [(3)H]spiperone binding; [(35)S] GTP(γ)S binding