Abstract
Receptor-induced binding of the stable GTP analogue, guanosine 5′-[γ-thio]triphosphate (GTP [γS]), to guanine nucleotide-binding regulatory proteins (G proteins) was measured in various permeabilized cells. In myeloid differentiated human leukemia (HL-60) cells, permeabilized with either digitonin, streptolysin O or Staphylococcus aureus α-toxin, binding of GTP [γS] induced by three distinct chemoattractant receptors was observed. The extent of receptor-stimulated GTP [γS] binding (maximally about 2-fold) was independent of the type of permeabilizing agent used. In human erythroleukemia cells permeabilized with digitonin, agonist activation of thrombin and neuropeptide Y receptors increased GTP [γS] binding by 1.8- and 1.5-fold, respectively. Finally, in adherently grown human embryonic kidney cells permeabilized with digitonin, activation of the stably expressed human muscarinic m3 receptor increased GTP[γS] binding by about 1.6-fold. In digitonin-permeabilized HL-60 cells, a quantitative analysis of formyl peptide receptors and interacting G proteins was performed. About 50,000 formyl peptide receptors per cell were detected. Agonist binding to these receptors was fully sensitive to regulation by guanine nucleotides and pertussis toxin. The number of high-affinity GTP [γS] binding sites, most likely representing heterotrimeric G proteins, was calculated to be about 670,000 per cell. Stimulation of formyl peptide receptors led to the activation of about 130,000 of high-affinity GTP [γS] binding sites, indicating a ratio of about three activated G proteins per one agonist-activated receptor.
Overall, this study indicates that receptor-stimulated GTP [γS] binding to G proteins in permeabilized cells is a sensitive and rapid method for analyzing receptor-G protein interactions, which can be applied to a variety of cultured cells and for various receptor systems.
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Wieland, T., Liedel, K., Kaldenberg-Stasch, S. et al. Analysis of receptor-G protein interactions in permeabilized cells. Naunyn-Schmiedeberg's Arch Pharmacol 351, 329–336 (1995). https://doi.org/10.1007/BF00169072
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DOI: https://doi.org/10.1007/BF00169072