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

Abstract Muscarinic agonists induce the activation of the airway smooth muscle (ASM) leading to smooth muscle contraction, important in asthma. This activation is mediated through M2/M3 muscarinic acetylcholine receptors (mAChRs). Muscarinic receptor activity, expressed as [3H]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 [3H]-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-[32P]γATP 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 [32P]-labeling. A selective M3mAChR antagonist, 4-DAMP produced a dramatic inhibition of the basal and CC-dependent [32P]-labeling. The [32P] labeled membrane sediments were detergent solubilized and immunoprecipitated with specific M2/M3mAChR antibodies. The M3mAChR immuno-precipitates exhibited the highest cGMP-dependent [32P]-labeling, indicating it is a PKG-II substrate. Experiments using synthetic peptides from the C-terminal of the third intracellular loop (i3) of both M2mAChR (356–369) and M3mAChR (480–493) as external PKG-II substrates resulted in the i3M3-peptide being heavily phosphorylated. These results indicated that PKG-II phosphorylated the M3mAChR at the i3M3 domain (480MSLIKEKK485), suggesting that Ser481 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 M3mAChR in the patho-physiology of ASM involved in asthma and COPD.


Introduction
Muscarinic agonists induce the activation of the airway smooth muscle (ASM) leading to smooth muscle contraction, related to asthma. This activation is mediated through the M 2 and M 3 muscarinic acetylcholine receptors subtypes (mAChRs) (1)(2)(3). Muscarinic agonists increase intracellular cGMP levels and contractility in ASM from guinea pigs (4) and bovine tracheal smooth muscle (BTSM) (5). Likewise, muscarinic agonists acting on both M 2 /M 3 mAChRs in BTSM isolated strips induce the generation of two cGMP signal peaks at 20-s and 60-s (6). The first signal (20-s) produced by the activation of M 2 mAChR coupled to a Gi/o protein induces the transient translocation of NO-sensitive soluble guanylyl cyclase (NO-sGC) from cytoplasm to plasma membranes (7,8). The second signal (60-s), produced by the stimulation of the M 3 mAChR coupled to a Gq 16 protein leads to the activation of a membrane-bound natriuretic peptide receptorguanylyl cyclase B (NPR-GC-B) (9)(10)(11)(12). We have previously shown that [ 3 H]QNB binding studies on a plasma membranes fraction from BTSM demonstrate a high activity of M 2 / M 3 mAChR subtypes (13). Functional studies have shown that the M 2 /M 3 mAChR subtypes are present in these BTSM plasma membranes (7,8,11,12). Recently, we showed that cGMP affects the M 3 mAChR functionality expressed as [ 3 H]QNB binding activity (14).
Using a plasma membranes fractions from BTSM, we demonstrate that cGMP, via PKG-II, phosphorylated the M 3 mAChR affected its functionality, expressed by an increment in the B max for [ 3 H]QNB binding activity and displaying a positive co-operativity. Moreover, okadaic acid (OKA), (a protein phosphatase (PPase inhibitor) induced a faster [ 3 H]QNB binding, suggesting the involvement of a membrane-bound PPase. Furthermore, a cGMP-dependent [ 32 P]-phosphorylation of membrane protein was specific for the M 3 mAChR. This [ 32 P]-membrane labeling was affected by muscarinic agonists such as carbamylcholine (CC) displaying agonist-dependent phospho/dephosphorylation reactions. Conversely, 4-DAMP, a selective M 3 mAChR antagonist, inhibited both the basal and cGMP-dependent membrane protein [ 32 P]-phosphorylations, supporting the involvement of an unknown PPase.
In this work, a putative cGMP regulatory feedback mechanism on the mAChR activity at plasma membranes from ASM was studied.

Methods
The following compounds were purchased from Sigma Chemical Co. Synthetic tetradecapeptides derived from the C-terminal of third intracellular loop (i3) of mAChR subtypes M 2 (amino acid sequence: 356 KQNIVARKIVKMTK 369 ) and M 3 ( 480 MSLIKEKKAAQTLS 493 ) were prepared in the Synthetic Peptide Unit of Tropical Medicine Institute, of Universidad Central de Venezuela. Both peptides were purified by highperformance liquid chromatography (HPLC) and the amino acid sequence was checked by mass spectrometry.

Plasma membrane preparation
The plasma membrane fraction (P 1 ) was prepared from BTSM as previously described (15). Aliquots (2-3 mg membrane protein/ml) were suspended in a Buffer containing 0.3 M sucrose, 0.5 mM DTT, 20 mM Tris-HCl (pH 7.2), frozen in liquid N 2 and stored at À80 C until use.

Measurement of muscarinic acetylcholine receptor activity
The mAChR activity was evaluated using the [ 3 H]QNB binding studies, which were performed as described previously (13). Briefly, PM (P 1 ) fraction was diluted with 80 volumes of hypotonic buffer containing 20 mM Tris-HCl (pH 7.2), 0.5 mM DTT and centrifuged at 150 000 Â g for 30 min, washed and suspended in small volume of incubation buffer (50 mM Tris-HCl, pH 7.8), prior to use. The [ 3 H]QNB binding assay was started by adding membrane protein (2-5 mg) in incubation buffer of 50 mM Tris-HCl (pH 7.8), and L-[ 3 H]QNB (1250 nM) to a final volume of 120 ml. Different compounds to be tested were added to the incubation media. After 1 h at 37 C, the incubation mixture was placed onto a pre-centrifuged Sephadex G-50 column (3 ml) equilibrated with 0.3 M sucrose-50 mM Tris-HCl (pH 7.6) and immediately centrifuged at 700 Â g for 1.5 min to remove free [ 3 H]QNB. The column effluent containing 97-98% of the bound [ 3 H]QNB was transferred to vials containing the liquid scintillation cocktail. Radioactivity was measured in a RackBeta liquid scintillation counter LKB, Wallac 1214/ 1219 and all samples counted with approximately the same efficiency (30%). Specific binding was calculated by subtracting non-specific binding (less than 1% of total binding, measured with 1 mM atropine), from the total binding (16). In all binding experiments, no more than 5% of the fixed radioligand concentration was allowed to bind to the membranes to avoid ligand depletion. Similar amounts of active receptors were employed in these binding assays. The values of B max , K D and Hill coefficient n H were calculated as described (17).

Protein kinase G and [ 32 P]-incorporation into membrane protein and synthetic peptides assays
Protein kinase G (PKG) activity and [ 32 P]-incorporation into membranes proteins were measured in plasma membrane fractions using endogenous substrates or synthetic peptides as described (18) and modified (19). Briefly, the incubation medium (50 ml), contained 5 mM MgCl 2 , 20 mM KPi (pH 7.0), 100 mM of the cocktail of phosphatase inhibitors (OKA, NIPP-1) and 0.1-1 mM [ 32 P] gATP (3 mCi/assay). In the assays using synthetic peptides from i 3 M 2 /M 3 AChRs, which were based on mAChRs M 2 [Bos Taurus] NCBI reference sequence NP_001074202.1 and mAChRs M 3 [Bos Taurus] NCBI reference sequence NP_776695.1. The peptides were synthesized and purified by HPLC and later used as exogenous substrates to 32 P-ATP-dependent membrane phosphorylation. To calculate these values, basal 32 P-endogenous labeling was subtracted.
The phosphotransfer reaction was allowed to proceed for 10-15 min at 37 C, at which time it was terminated by spotting aliquots onto P81 phosphocellulose papers (GE Healthcare Bio-Sciences, Pittsburgh, PA), and immediately dropped into ice-cold 5% TCA (100 ml). Under gentle agitation at 4 C, discs were washed 4 times (100 ml icecold 5% TCA). The paper discs were washed with 100% cold ethanol and some 50 ml of cold ether was used to remove possible [ 32 P]-labeled lipids. Discs were allowed to dry under air stream and 32 P was counted using Cêrenkov radiation (20) in a liquid scintillation counter. dephostatin, OKA and NIPP-1 Bovine Thymus recombinant) and other compounds as indicated. [ 32 P]-labeled plasma membranes were separated from the excess of [g 32 P]ATP by centrifugation at 12 000 Â g for 15 min at 4 C in an Eppendorf Õ (Hamburg, Germany) centrifuge. [ 32 P]-labeled membranes were washed twice with a solution containing, 20 mM EDTA, 1 mM PMSF, 20 mM KPi (pH 7.2) and 100 mM protein phosphatase inhibitor kit (Buffer I). [ 32 P]labeled membranes (5 mg/ml) were solubilized by incubation at 4 C for 15 min in a mixture containing 5 mM MgCl 2 , 1 mM DTT, 1 mM EDTA, 0.1 mM PMSF, 20 mM KPi, pH 7.2 (Buffer II). One ninth volume of a detergent mixture containing 0.1% Digitonin-0.02% sodium cholate was added to selected solubilized mAChRs (21). Detergent-solubilized proteins were recovered after centrifugation at 150 000 Â g for 30 min. The sediment was again re-extracted using the same procedure and both detergent-solubilized supernatants were pooled. The digitonin/cholate-solubilized material was incubated with protein A/G-agarose beads for 1 h at 4 C. The pre-clarified supernatant was incubated overnight with specific anti-M 3 or anti-M 2 mAChR antibodies at 4 C. Immunoprecipitates were collected upon the addition of protein A/G-agarose beads for 6 h at 4 C. The beads were then collected by low speed centrifugation and washed three times using detergent-free buffer II. An aliquot of these [ 32 P]-immunoprecipitates was used to measure [ 32 P] labeling in a RackBeta liquid scintillation counter LKB, Wallac 1214/1219.

Protein measurement
The amount of protein was quantified using bovine serum albumin (BSA) as standard (22).

Data analysis
A computer-assisted non-linear regression program (InPlot, Graph Pad Õ software, La Jolla, CA) was used to analyze binding and competition experiments results (13).

Results
The effect of cGMP on [ 3 H]QNB antagonist binding at plasma membranes from BTSM The effect of increasing concentrations of cGMP on the [ 3 H]QNB binding activity as an expression of mAChRs functionality was evaluated in the presence of 5 mM ATP and 10 mM IBMX, a non-selective phosphodiesterase (PDE) inhibitor ( Figure 1). The binding of [ 3 H]QNB to plasma membranes from BTSM reached a maximum around 50 nM cGMP. It remained constant at higher cyclic nucleotide concentrations, giving an ED 50 for cGMP of 0.5 Â 10 À9 M. IBMX was included in all assays, otherwise bimodal behavior was observed (data not shown) due to the existence of PDE activity in this plasma membrane fraction. To establish the nature of this cGMP-dependent increase in [ 3 H]QNB binding, saturation experiments were performed to estimate the kinetic parameters of this process, such as the maximal binding activity (B max ), the dissociation constant (K D and the Hill coefficient (n H ) associated with the cGMP effect ( Table 1). The K D values for ATP plus cGMP remained unchanged but the B max and n H parameters were markedly affected. Thus, B max values (pmoles [ 3 H]QNB/mg of protein) increased significantly from 1.20 ± 0.12, in the presence of cGMP or, 1.34 ± 0.15 in the presence of ATP to 1.98 ± 0.19 for the condition containing ATP plus cGMP. This increment represents more than 65% in comparison to cGMP alone and higher than 40% to only ATP. In addition, n H values shifted significantly from 1.2 ± 0.1 in the assays with either cGMP or ATP to 1.9 ± 0.3 for the condition having ATP plus cGMP. These kinetic parameters indicated that cGMP in the presence of ATP is affecting the mAChR functionality.

Effects of the inhibitor (Rp-8-pCPT-cGMPS) of PKG-II on the [ 3 H]QNB binding activity
These changes induced by ATP plus cGMP in the [ 3 H]QNB binding kinetic parameters may be through the activation of a PKG-II activity, associated with these plasma membranes. In this sense, the effects of a specific inhibitor (Rp-8-pCPT-cGMPS) of PKG on the [ 3 H]QNB binding activity was evaluated. In these experiments, the [ 3 H]QNB binding, induced by cGMP (50 nM), was completely abolished with a dose-dependent titration of this PKG-II inhibitor ( Figure 2). These results indicated that the cGMP activator effect on the [ 3 H]QNB binding is mediated via a PKG-II isoenzyme anchored to the plasma membrane fraction from BTSM.

Effect of OKA on [ 3 H]QNB binding
If a phosphorylation of these mAChRs is occurring, a dephosphorylation process must also exist. Thus, protein phosphatase inhibitors such as OKA may affect the

Effect of cGMP on endogenous 32 P phosphorylation of plasma membranes proteins
The ATP requirement for this cGMP effect implies that a phospho-transfer reaction is taking place (Figure 4). This was measured as a 32 P incorporation from [g 32 P]ATP into plasma membrane proteins. A cGMP titration on the 32 P-endogenous membrane protein phosphorylation gave maximal 32 P incorporation at approximately 5 Â 10 À7 M of cGMP with a ED 50 of 1 Â 10 À9 M for cGMP. The observations indicated that a PKG-II might be involved. To further examine the character of the PKG-II associated with the BTSM plasma membranes, the protein kinase activity was measured as the [ 32 P]-phosphorylation of membrane protein components. Consequently, the [ 32 P]-labeling was increased significantly from 3.25 ± 0.23 pmoles/10 min for the basal condition to 4.95 ± 0.27 pmoles/ 10 min in the presence of cGMP ( Table 2). The detergentsolubilization procedure extracted more than 66% of the total [ 32 P]-labeled material. The difference was statistically significant between the two conditions, which was almost twice in the detergent-solubilized membrane proteins.
To identify specific [ 32 P]-labeled polypeptides, a more detailed [ 32 P]-labeling study was performed. The [ 32 P]labeling difference between the muscarinic receptors was more significant in the immunoprecipitates using specific anti-M 2 and -anti-M 3 mAChR antibodies. Both receptor subtypes were labeled with [g 32 P]ATP ( Table 2). The M 3 mAChR was preferentially and significantly phosphorylated in the presence of 50 nM cGMP, being more than 80% of the 32 P solubilized material immunoprecipated by the M 3 mAChR antibodies, in comparison only 20% of the phosphorylated M 2 mAChR was pull down by the M 2 mAChRs antibodies. These data indicate    (17). (*)The difference in the B max and n H for the assays with both ATP and cGMP was statistically significant at p50.01.
that cGMP, via a PKG-II, phosphorylates the M 3 mAChR subtype. In these experimental conditions, the [ 32 P]-labeled M 3 mAChR represents more than the 55% of the total [ 32 P]labeled plasma membrane proteins from BTSM. Another biochemical approach was undertaken to identify the M 3 mAChR motifs and the putative amino acids involved in this PKG-II phosphorylation. Thus, an experimental approach using synthetic peptides based on amino acids segments containing the putative consensus phosphorylation sequences for PKG [(R/K)2-3,-X-S*/T*]. These motifs were located close to the C-terminal from the third intracellular loop (i 3 ) of the M 2 and M 3 mAChR subtypes ( Figure 5). These peptides have the following amino acid sequences: M 3 ( 480 MSLIKEKKAAQTLS 493 ) and a close related in the M 2 ( 356 KQNIVARKIVKMTK 369 ). These synthetic peptides were phosphorylated in a concentration-dependent manner, by the PKG-II, in the presence of 50 nM cGMP and1 mM [g 32 P]ATP and 10 mM IBMX.
The M 3 mAChR synthetic peptide was greater phosphorylated than the M 2 mAChR peptide by the membrane-bound protein PKG-II. The V max for the M 3 mAChR synthetic peptide was 0.75 ± 0.03 pmoles P/min/mg protein, which was 4.6 times higher than for the M 2 mAChR synthetic peptide (0.16 ± 0.03 pmoles P/min/mg protein). It is important to emphasize that these kinetic values are relative due to the fact that this 32 P-incorporation is the result of a competition between the endogenous mAChRs and the exogenous synthetic peptides. These findings indicate that the intracellular i 3 -loop region from the M 3 mAChRs is specifically phosphorylated by a PKG-II anchored to the plasma membranes.

Modulation by muscarinic agonists and antagonists on cGMP-dependent 32 P-ATP phosphorylation
It was important to establish, whether or not, the cGMP effect on 32 P phosphorylations was affected by muscarinic   compounds. In the following experiments, the protein phosphatase inhibitor kit (containing cypermethrin, dephostatin, OKA and NIPP-1 Bovine Thymus recombinant) was omitted for these 32 P-phosphorylation assays. A CC titration on 32 P protein membranes labeling is shown in Figure 6. It can be seen that this muscarinic agonist induces a ''two opposite responses'' expressed by a significant rise in 32 P-phosphorylation of membrane proteins to a concentration of 1 Â 10 À8 M ( Figure 6). However, increasing concentrations of CC produced a dramatic decrease in 32 P phosphorylation reaching basal levels at 1 Â 10 À5 M CC. Similarly, in the presence of 50 nM cGMP, the 32 P incorporation into plasma membrane proteins followed a similar ''two opposite responses'' reaching a maximal at a CC concentration of 1 Â 10 À8 M analogous behavior to the basal condition. However, after this maximal 32 P incorporation peak, at higher CC concentrations, muscarinic agonist-dose-dependent dephosphorylations proceed, reaching the same basal 32 P labeling at 1 Â 10 À5 M CC.
To differentiate between the M 2 /M 3 AChR subtypes being phosphorylated/dephosphorylated, which are present in these plasma membranes fractions, a selective (M 1 ,M 3 ,M 5 AChRs) antagonist, such as 4-DAMP was used. A 4-DAMP titration was performed, with both the cGMP-dependent and the basal 32 P-phosphorylating activities (Figure 7). Thus, 4-DAMP, in a dose-dependent fashion induced a dramatic inhibition in the basal activity, which was more pronounced for the cGMP-dependent 32 P-labeling. As a result, in the presence of cGMP, an IC 50 for 4-DAMP of 1.0 ± 0.1 nM was estimated. The last results suggest the existence of a putative muscarinic receptor-linked PPase activity.

Discussion
The muscarinic activation of ASM contraction (4-6) involves both muscarinic receptors, M 2 /M 3 mAChRs, coupled to NPR-GC-B, contributing to the generation of the so-called ''membrane-associated cGMP pool'' (7,8,11,12). This ''membrane-associated cGMP pool'' has a different regulation to the ''soluble cGMP pool'' produced by the NO releasing agents acting on a NO-sGC implicated in the ASM relaxation (23). The role of this ''membrane-associated cGMP pool'', as a regulatory feedback mechanism, on the mAChRs, anchored to the plasma membranes, was evaluated in this work.
Classically, the effect of cGMP has been studied in intact tissue/cells using cell-permeable cGMP analogs, for example, 8-Bromo-cGMP or the participation of PKG activator, Sp-8-pCPT-cGMPS (24) or PKG inhibitor, Rp-8-pCPT-cGMPS (25). Thus, in intact cells using these tools, it is difficult to discriminate between the cyclic GMP protein kinases (cGKs) substrates under the control of either PKG-I isoforms or membrane-bound PKG-II enzymes (26,27). To overcome this complex experimental task, we used a broken cell system such as a plasma membranes fraction from BTSM, exhibiting the following advantages: 1. It contains a high M 3/ M 2 AChR biological activity as described (11-13). 2. It contains an  active PKG-II isoform, previously identified (14). 3. These membranes are depleted of PKG-I isoenzymes and other soluble cGKs substrates and other PKs.
Previously, we have shown that cGMP may regulate mAChR binding via PKG-II activation (14). In the present work, more compelling evidences are given to demonstrate that indeed PKG-II, at plasma membranes from BTSM, phosphorylates the M 3 mAChR, in a G-protein independent manner, and regulates its receptor activity. Moreover, this phosphorylation was affected by OKA. Interestingly, muscarinic agonists and antagonists regulated this cGMP-dependent phosphorylation, suggesting a relevant role on the M 3 mAChR.
Cyclic GMP via PKG-II phosphorylation induced the following biological actions on the mAChR binding activity here described: 1. The increase of B max for [ 3 H]QNB binding activity (460%) indicating that ''new [ 3 H]-QNB binding sites'' are displayed. Nonetheless, this cGMP effect on B max , was obliterated by the 4-DAMP alkylation of M 3 mAChR subtype as previously reported (14). 2. A doubling of the Hill coefficients (n H ), which increased from 1.0 to almost 2.0, suggesting a positive co-operativity or homodimer formation of M 3 mAChRs as previously postulated (14). 3. The PPase inhibitor OKA induced a faster [ 3 H]QNB binding at these M 3 mAChRs indicating a dephosphorylation-linked process is present in these plasma membrane fractions.
These ''new M 3 mAChRs'' that exhibited similar affinity constants (K D ), can be excluded as newly synthesized or exposed ''recycled or hidden'' M 3 mAChRs from endosomes vesicles since our assays were performed with isolated plasma membrane fragments. The appearance of these ''new M 3 mAChR'' may be related to some complex molecular mechanism, possibly via a two-step isomerization, of the mAChRs induced by antagonist binding (28,29). Molecular biology studies using point mutations and irreversible affinity labeling of the M 1 mAChR led to the proposal of a tandem two-site model (30). The possibility that the receptor binds two ligand molecules is relevant to the pharmacology and new therapeutic approaches. It is possible that the PKG-II phosphorylation of M 3 mAChR induced a similar molecular mechanism of homodimer/oligomer formation as previously proposed (14). This rationale is supported by the fact that, the M 3 mAChR displays a greater propensity to form a homodimer/oligomer structure at higher density receptor population (31,32). Recent studies demonstrated the formation of M 3 mAChR dimers in vivo (33,34), which may reflect the situation in these plasma membranes from BTSM, which have higher mAChRs amounts (13,15).
The M 3 mAChRs belong to the class A GPCR regulated by three principal mechanisms: Desensitization, internalization, and down-regulation. Internalization and down-regulation are ruled out in our experiments using isolated plasma membranes fractions. Thus, the receptor desensitization is the unique mechanism to explain these effects induced by cGMP via a PKG-II on mAChR functionality.
In relation to the muscarinic receptor desensitization, it was previously reported that cGMP plus ATP affected the agonist-antagonist muscarinic binding activities (14). Thus, the binding sites for the high affinity-agonist, CC, disappeared as an expression of receptor desensitization.
In comparison, 4-DAMP, an M 3 selective antagonist, displayed high and low affinity-binding sites. In contrast, a nonselective antagonist atropine and the M 2 -selective antagonists such as methoctramine and gallamine, revealed only one low affinity binding site, which was not affected by cGMP plus ATP. Moreover, the 4-DAMP-mustard alkylation of the mAChRs blocked the cGMP effect indicating that the M 3 mAChR is the main receptor target of cGMP (14).
The involvement of a PKG-II on the desensitization of the muscarinic receptors was further established by the ability of cGMP (activator of PKG) in a dose-dependent manner (35) to increase the [ 3 H]QNB binding and 32 P-labeling in the plasma membranes from BTSM. Interestingly, the ED 50 results for cGMP (1 Â 10 À9 M) was similar for both the rise of the [ 3 H]QNB binding and the 32 P-labeling in these plasma membranes. Moreover, the effect of the cGMP analogs such as Rp-8-pCPT-cGMPS, a PKG-specific inhibitor (24) suppressed the increase induced by cGMP on the [ 3 H]-QNB binding activity. Similar behavior towards the inhibitor Rp-8-pCPT-cGMPS has been reported for the native (25) and recombinant PKG-II (26). Furthermore, PKG-II has previously been established as a membrane-bound enzyme in plasma membranes from BTSM (14) as well as in other biological systems (27).
The M 3 mAChR phosphorylation, via PKG-II is a G-protein independent phosphorylation. Other G-protein independent phosphorylations have been involved in the regulation of the muscarinic-antagonist binding to rat cerebral synaptic membranes (36). In the case of the phosphorylation by PKA and PKC of the agonist-unbound M 3 mAChRs, induces receptor uncoupling from G-proteins (37), which has been reported in SHSY5Y cell line (38).
Our previous observations using 32 P-autoradiographs (14) and the specific immunoprecipitation assays support the argument that M 3 mAChR is specifically phosphorylated by PKG-II. Several agonist-dependent phosphorylations of M 3 mAChR have been reported (39). An M 3 mAChR hyperphosphorylation occurs following agonist occupation, linked to desensitization of muscarinic receptors. This usually occurs at serine (Ser) and threonine (Thr) residues contained on the i 3 -loop and C-terminal tail, which has been described for these GPCRs (40)(41)(42). Most of these phosphorylations by the kinases GRK, PKA, PKC, and CK1 occur at phosphorylation consensus sites (40,43) located in the i 3 -loop and C-terminal tail domains of the mAChRs (40,44). The classic consensus phosphorylation sequences for PKG is [(R/K)2-3,-X-S*/T*], which describes 75% of the sites surveyed (45).
Nevertheless, there are only a few well-characterized proteins preferentially phosphorylated by PKG-II. One is the inositol 1,4,5-trisphosphate receptor (IP 3 R), which generates the IP 3 Rtide (GRRESLTSFG) and the cAMP response element binding protein (CREB) which contains a CREBtide (KRREILSRRPSYR) (26). In both specific peptides, Ser (S) residues are phosphorylated and the cluster of two or more positive cluster charges such as Arg (R) or Lys (K) seems to be the consensus sequences for PKG-II. Taking into account, these observations, a possible PKG-II phosphorylation site may be located in the i 3 -loop of the M 3 mAChRs extending from Thr 450 to Q 490 . This contains the peptide M 3 AChR ( 480 MSLIKEKKAAQTLS 493 ), which was heavily phosphorylated by PKG-II located in the sarcolemma from BTSM. The known consensus sequence for phosphorylation of PKG is in the domain of i 3 M 3 mAChR, specifically 480 MSLIKEKK 485 (45). Based on the data presented here it is proposed that the specific amino acid residue, susceptible to phosphorylation by PKG-II is that of Ser 481 .
Whether or not, this PKG-II-dependent M 3 mAChR phosphorylation has a relevant biological function was also explored. We found the effect was mediated mainly by M 3 mAChRs (Table 2).
Consequently, we evaluated, in the absence of PPase inhibitors, the effect of muscarinic drugs, specifically muscarinic agonist, CC, and the M 3 mAChR selective antagonist, 4-DAMP, on these cGMP-dependent 32 P-membrane proteins.
In this sense, the 32 P labeling of plasma membrane proteins showed a ''bell shaped'' as a dose-response curve for muscarinic agonists. Thus, a rise of the 32 P-labeling, induced by CC was maximal at a concentration of 1 Â 10 À8 M. The agonist-dependent membrane protein phosphorylations may be linked to M 3 mAChR activation, especially as a response to agonist occupation by becoming rapidly hyperphosphorylated at intracellular domains as described (40)(41)(42)(43). However, in the plasma membrane fragments, an agonist-dependent dephosphorylation process was observed at higher doses of CC (41 Â 10 À8 M). This ''two opposite responses'' phenomenon on these dephospho/phosphorylation processes can be interpreted using a model based on the interactions of a ligand with two different receptors that mediate opposite effects (one stimulatory and one inhibitory) (46). Similar pharmacological muscarinic agonist ''two opposite responses'' behavior has been previously described to explain the two opposite mAChRs signal transducing mechanisms, regulating a G-protein-coupled NPR-GC-B (11,12).
Surprisingly, 4-DAMP, a selective M 3 mAChR antagonist, increased this dephosphorylation activity, reducing both the basal and cGMP-dependent phosphorylations. This was shown in the presence of cGMP, with an IC 50 of 1.0 ± 0.1 nM for 4-DAMP, which was in the expected nM range for a specific inhibition of M 3 mAChRs (47). This inhibition of 32 P-phosphorylation of membrane proteins was similar to that observed at higher muscarinic agonist doses (41 Â 10 À8 M).
Two mechanisms can be proposed to explain the dramatic inhibition of 4-DAMP on 32 P-labeling. First, 4-DAMP may be acting as an ''inverse-agonist'' on M 3 mAChR, changing the receptor conformation, especially at the i 3 -loop of the M 3 mAChRs, which is less susceptible to phosphorylation by PKG-II. Similar ''inverse-agonist'' behavior, reducing the constitutive phosphorylation of the mutant N514Y M 3 mAChR, has been reported for some muscarinic antagonists such as atropine, 4-DAMP and pirenzepine (48). Second, 4-DAMP may also be acting as an ''inverse agonist'' on the M 2 mAChR. Thus, it is possible that M 2 mAChR activates a putative membrane-bound PPase leading to a profound decrease in 32 P-labeling in plasma membranes. The existence of a membrane-bound PPase activity, involved in these muscarinic actions, is supported by the effect of OKA, a classic inhibitor of PPase activity, which reduced the time required to achieve maximal saturation (B max ) of [ 3 H] QNB binding from 60 min to 10 min.
The PPase family has been described, as four-major serine/ threonine-specific PPases present in animal cells. Moreover, OKA provides important clues to the physiological roles of these PP-1, PP-2A and PP-2B enzymes (49,50). PPases have been suggested to be involved in the modulation of ionic currents by muscarinic agents in several neuronal and nonneuronal cells. In hippocampal pyramidal neurons, a PPase linked to mAChRs is involved in the cholinergic suppression of the Ca 2+ -activated K + current (sIAHP) (51). Another nonneuronal system, under muscarinic action linked to PPase, is the ability of acetylcholine to decrease the cAMP-dependent currents through cardiac L-type Ca 2+ channels at guinea-pig ventricular myocytes (52). These authors neither identify the muscarinic receptor subtype nor firmly establish the biochemical nature of the PPase involved in these muscarinic actions.
It has been claimed that M 3 mAChR dephosphorylation regulates the receptor interactions with G proteins (53). The muscarinic receptor signaling regulator named SET is a PPase 2A inhibitor, which binds to the C-terminal of the i 3 -loop-M 3 mAChR (54,55) decreasing receptor engagement with G proteins.
All of the above evidences suggest that PPase 2A, which is also a membrane-bound enzyme (56) may be the PPase involved in M 3 mAChR dephosphorylation. Further research will establish the validity of this proposal.
A recent study indicates that the binding site of both SET and PP2A on M 3 mAChR occurs at the i 3 -loop ( 474 ITKRKRMSLIKEKKAAQ 490 ). SET specifically binds to the site 476 KRKR 479 in close vicinity to a domain 484 KEKKAAQ 490 involved in G protein coupling and activation (55,57,58). Our results based on the synthetic peptide 480 MSLIKEKKAAQTLS 493 led us to propose that the domain 480 MSLIKEKK 485 is the putative phosphorylation site on PKG-II. This domain contains the S 481 , which is located between these two relevant regulatory binding sites, suggesting an important biological function for the PKG-II response.
The involvement of PKG-II as a cGMP-dependent M 3 mAChR phosphorylation, is a novel mechanism, present in ASM cells to guarantee a feedback control of cGMP on M 3 mAChR activation. This post-translational reversible modification at M 3 mAChRs may act as a feedback mechanism to terminate the cGMP-dependent muscarinic signal transduction cascades at the sarcolemma of BTSM.
Finally, the M 3 mAChR, a prototype of class A GPCR that preferentially couples to the family of G proteins, is involved in numerous important physiological functions in ASM. These are the cholinergic tone that contributes to airflow obstruction and chronic airway inflammation in asthma and COPD, where anti-cholinergics are effective bronchodilators by blocking this muscarinic receptor subtype (59). This work supports the existence of ''cGMP linked muscarinic signal transducing signalosome'' machinery comprised of M 3 mAChRs, NPR-GC-B, PKG-II and cGMP-PDE located in the plasma membrane of ASM. This signalosome involves a ''membrane-associated cGMP pool'' as a product of NPR-GC-B (6-12), as a second messenger which streams down to activate a membrane-bound PKG-II, which then phosphorylates the M 3 mAChR, inducing the desensitization of this M 3 mAChR subtype, in an inhibitory feedback mechanism (14). Furthermore, the presence of a cGMP-PDE in these plasma membranes from BTSM can shut down this ''cGMP linked muscarinic signal transducing signalosome'' (unpublished results).

Conclusions
This work supports the existence of a ''muscarinic signal transducing signalosome'' machinery comprised of M 3 mAChRs, NPR-GC-B, PKG-II and a putative PPase located in the plasma membrane of ASM. This signalosome involves a ''membrane-associated cGMP pool'' (6-12) as second messenger, which streams down to activate a membrane-bound PKG-II, which then phosphorylates the M 3 mAChR at the i 3 -loop extending from Thr 450 -Q 490 , and containing the peptide M 3 mAChR ( 480 MSLIKEKKAAQ TLS 493 ). The latter was heavily phosphorylated by PKG-II, inducing the desensitization of this M 3 mAChR subtype, in a feedback mechanism at plasma membrane level (14). The M 3 mAChR, a prototypic class A GPCR, which preferentially couples to the family of G proteins, is involved in numerous important physiological functions in ASM. Interestingly, M 3 mAChR in ASM, the main subject of this work, is involved in the cholinergic tone, which contributes to airflow obstruction and chronic airway inflammation in asthma and COPD. It is known that anti-cholinergics are effective bronchodilators by blocking this muscarinic receptor subtype (59). Thus, understanding how the M 3 mAChR functions at the molecular level is of considerable relevance for designing novel classes of drugs that can modulate M 3 mAChR function for therapeutic purposes in pathological conditions such as asthma and COPD.