ANALYSIS OF RECEPTOR-G PROTEIN INTERACTIONS IN PERMEABILIZED CELLS

Receptor-induced binding of the stable GTP analogue, guanosine 5'-[gam ma-thio]triphosphate (GTP [gamma S]), to guanine nucleotide-binding re gulatory proteins (G proteins) was measured in various permeabilized c ells. In myeloid differentiated human leukemia (HL-60) cells, permeabi lized digitonin, streptolysin O or Staphylococcus aureus a-toxin, bind ing of GTP[gamma S] induced by three distinct chemoattractant receptor s was observed. The extent of receptor-stimulated GTP[gamma S] binding (maximally about Z-fold) was independent of the type of permeabilizin g agent used. In human erythroleukemia cells permeabilized with digito nin, agonist activation of thrombin and neuropeptide Y receptors incre ased GTP[gamma S] binding by 1.8- and 1.5-fold, respectively. Finally, in adherently grown human embryonic kidney cells permeabilized with d igitonin, activation of the stably expressed human muscarinic m3 recep tor increased GTP[gamma S] binding by about 1.6-fold. In digitonin-per meabilized HL-60 cells, a quantitative analysis of formyl peptide rece ptors and interacting G proteins was performed. About 50,000 formyl pe ptide receptors per cell were detected. Agonist binding to these recep tors was fully sensitive to regulation by guanine nucleotides and pert ussis toxin. The number of high-affinity GTP[gamma S] binding sites, m ost likely representing heterotrimeric G proteins, was calculated to b e about 670,000 per cell. Stimulation of formyl peptide receptors led to the activation of about 130,000 of high-affinity GTP[gamma S] bindi ng sites, indicating a ratio of about three activated G proteins per o ne agonist-activated receptor. Overall, this study indicates that rece ptor-stimulated GTP[gamma S] binding to G proteins in permeabilized ce lls is a sensitive and rapid method for analyzing receptor-G protein i nteractions, which can be applied to a variety of cultured cells and f or various receptor systems.