Relationship between agonist binding, phosphorylation and immunoprecipitation of the m3-muscarinic receptor, and second messenger responses.
1. Phosphoinositidase C-linked m3-muscarinic receptors expressed in Chinese hamster ovary cells (CHO-m3 cells) are phosphorylated on serine following agonist stimulation. 2. m3-Muscarinic receptor phosphorylation is concentration-dependent requiring a carbachol concentration of 13.2 microM for half maximal stimulation. 3. The phosphorylation concentration-response curve lies to the left of the curve for carbachol binding to muscarinic receptors (KD = 100 microM) in membranes from CHO-m3 cells. In contrast, receptor phosphorylation closely correlates with receptor-mediated phosphoinositidase C activation (EC50 for inositol 1,4,5 trisphosphate accumulation during the peak and plateau phases were 7.14 microM and 5.92 microM respectively) but not with rapid agonist-mediated calcium elevation (EC50 = 0.32 microM) measured in fura-2-AM loaded cells. 4. These data suggest a dissociation of receptor phosphorylation from agonist occupation. Such an apparent 'receptor reserve' for m3-muscarinic receptor phosphorylation may be indicative of a mechanism that is dependent on a small amplification of the receptor signal, though probably dissociated from the calcium signal.
Leicester University, Dept. Of Cell Physiology and Pharmacology.
A B
AB
Tobin
G B
GB
Willars
N T
NT
Burford
S R
SR
Nahorski
D000818:Animals; D001667:Binding, Competitive; D016466:CHO Cells; D002217:Carbachol / Q000494:pharmacology*; D002478:Cells, Cultured; D006224:Cricetinae; D004305:Dose-Response Relationship, Drug; D010766:Phosphorylation; D011233:Precipitin Tests; D011976:Receptors, Muscarinic / Q000187:drug effects* / Q000502:physiology*; D015290:Second Messenger Systems / Q000502:physiology*; D015398:Signal Transduction / Q000502:physiology*; D013997:Time Factors
D011976:Receptors, Muscarinic; D002217:Carbachol