Agonist-induced [ 35S]GTP γS binding in the membranes of Spodoptera frugiperda insect cells expressing the human D3 dopamine receptor

• Published in: Neuroscience letters Vol. 226; no. 2; pp. 91 - 94
• Main Authors: Pregenzer, Jeffrey F, Alberts, Glen L, Bin Im, Wha
• Format: Journal Article
• Language: English
• Published: Shannon Elsevier Ireland Ltd 25.04.1997; Elsevier
• Subjects: [ 35S]GTP γS binding; Biological and medical sciences; Cell receptors; Cell structures and functions; D 3 dopamine receptor; Fundamental and applied biological sciences. Psychology; G-protein activation; Molecular and cellular biology; Monoamines receptors (catecholamine, serotonine, histamine, acetylcholine); Spodoptera frugiperda; Spodoptera frugiperda insect cells; Spodoptera frugiperda insect cells; D 3 dopamine receptor; [ 35S]GTP γS binding; G-protein activation; Human; Agonist; GTP; Dopamine; Insecta; Spodoptera frugiperda; Gene expression; D3 Dopamine receptor; Cell line; Arthropoda; Lepidoptera; Noctuidae; Invertebrata; G protein
• ISSN: 0304-3940; 1872-7972
• DOI: 10.1016/S0304-3940(97)00251-6

In the membranes of Spodoptera frugiperda (Sf-9) insect cells heterologously expressing the human D3 dopamine receptor, agonists selective for the receptor, but not antagonists, robustly enhanced [ 35S]GTP γS binding. Quinpirole, for instance, dose-dependently enhanced [ 35S]GTP γS binding with a half-maximal concentration of 2.3±0.2 nM. Its action was absent in the cells infected with wild type viruses, and competitively blocked by an antagonist, YM-09151-2. A number of known agonists enhanced [ 35S]GTP γS binding to variable degrees, probably reflecting their differential efficacy to activate target G-proteins via the receptor. This agonist-induced [ 35S]GTP γS binding was abolished by N-ethylmaleimide, a selective blocking agent for Gi/Go proteins, with no appreciable effect on ligand binding. We propose coupling of the cloned D3 receptor to endogenous G-proteins in Sf-9 cells, probably homologs of mammalian Gi/Go proteins. Despite the apparent coupling of the D3 receptor to G-proteins, GTP γS (10 μM) failed to decrease agonist binding ([ 3H]dopamine) to the D3 receptor, probably due to small affinity differences between low and high affinity states for agonists in the D3 receptor, as well as due to high receptor density in Sf-9 cells. We conclude that agonist-induced [ 35S]GTP γS binding for the D3 receptor is suitable for estimating ligand intrinsic efficacy and pharmacological characterizations of ligand-receptor interactions.