RELATIONSHIP BETWEEN AGONIST BINDING, PHOSPHORYLATION AND IMMUNOPRECIPITATION OF THE M(3)-MUSCARINIC RECEPTOR, AND 2ND MESSENGER RESPONSES

1 Phosphoinositidase C-Linked m(3)-muscarinic receptors expressed in C hinese hamster ovary cells (CHO-m(3) cells) are phosphorylated on seri ne following agonist stimulation. 2 m(3)-Muscarinic receptor phosphory lation is concentration-dependent requiring a carbachol concentration of 13.2 mu M for half maximal stimulation. 3 The phosphorylation conce ntration-response curve lies to the left of the curve for carbachol bi nding to muscarinic receptors (K-D=100 mu M) in membranes from CHO-m(3 ) cells. In contrast, receptor phosphorylation closely correlates with receptor-mediated phosphoinositidase C activation (EC(50) for inosito l 1,4,5 trisphosphate accumulation during the peak and plateau phases were 7.14 mu M and 5.92 mu M respectively) but not with rapid agonist- mediated calcium elevation (EC(50) = 0.32 mu M) measured in fura-2-AM loaded cells. 4 These data suggest a dissociation of receptor phosphor ylation from agonist occupation. Such an apparent 'receptor reserve' f or m(3)-muscarinic receptor phosphorylation may be indicative of a mec hanism that is dependent on a small amplification of the receptor sign al, though probably dissociated from the calcium signal.