Muscarinic drugs regulate the PKG-II-dependent phosphorylation of M3 muscarinic acetylcholine receptors at plasma membranes from airway smooth muscle

Muscarinic agonists induce the activation of the airway smooth muscle (ASM) leading to smooth muscle contraction, important in asthma. This activation is mediated through M-2/M-3 muscarinic acetylcholine receptors (mAChRs). Muscarinic receptor activity, expressed as [H-3]QNB binding at plasma membranes from bovine tracheal smooth muscle (BTSM), increased with cGMP and was augmented significantly cGMP plus ATP but diminished with the PKG-II inhibitor, Sp-8-pCPT-cGMPS. The [H-3]-QNB binding was accelerated by okadaic acid, (OKA), a protein phosphatase (PPase) inhibitor. These two results indicated the involvement of a membrane-bound PPase. Moreover, a cGMP-dependent-[P-32]ATP phosphorylation of plasma membranes from BTSM was stimulated at low concentrations of muscarinic agonist carbamylcholine (CC). However, higher amounts of CC produced a significant decrement of [P-32]-labeling. A selective M(3)mAChR antagonist, 4-DAMP produced a dramatic inhibition of the basal and CC-dependent [P-32]-labeling. The [P-32] labeled membrane sediments were detergent solubilized and immunoprecipitated with specific M(2/)M(3)mAChR antibodies. The M(3)mAChR immuno-precipitates exhibited the highest cGMP-dependent [P-32]-labeling, indicating it is a PKG-II substrate. Experiments using synthetic peptides from the C-terminal of the third intracellular loop (i(3)) of both M(2)mAChR (356-369) and M(3)mAChR (480-493) as external PKG-II substrates resulted in the i(3)M(3)-peptide being heavily phosphorylated. These results indicated that PKG-II phosphorylated the M(3)mAChR at the i(3)M(3) domain ((MSLIKEKK485)-M-480), suggesting that Ser(481) may be the target. Finally, this phosphorylation site seems to be regulated by a membrane-bound PPase linked to muscarinic receptor. These findings are important to understand the role of M(3)mAChR in the patho-physiology of ASM involved in asthma and COPD.