Phosphorylation of the Gq/11-coupled M3-Muscarinic Receptor Is Involved in Receptor Activation of the ERK-1/2 Mitogen-activated Protein Kinase Pathway*

David C. Budd, Gary B. Willars, John E. McDonald, and Andrew B. TobinDagger

From the Department of Cell Physiology and Pharmacology, University of Leicester, P. O. Box 138, Medical Sciences Building, University Road, Leicester, LE1 9HN, United Kingdom

Received for publication, September 27, 2000, and in revised form, November 9, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We investigated the role played by agonist-mediated phosphorylation of the Gq/11-coupled M3-muscarinic receptor in the mechanism of activation of the mitogen-activated protein kinase pathway, ERK-1/2, in transfected Chinese hamster ovary cells. A mutant of the M3-muscarinic receptor, where residues Lys370-Ser425 of the third intracellular loop had been deleted, showed a reduced ability to activate the ERK-1/2 pathway. This reduction was evident despite the fact that the receptor was able to couple efficiently to the phospholipase C second messenger pathway. Importantly, the ERK-1/2 responses to both the wild-type M3-muscarinic receptor and Delta Lys370-Ser425 receptor mutant were dependent on the activity of protein kinase C. Our results, therefore, indicate the existence of two mechanistic components to the ERK-1/2 response, which appear to act in concert. First, the activation of protein kinase C through the diacylglycerol arm of the phospholipase C signaling pathway and a second component, absent in the Delta Lys370-Ser425 receptor mutant, that is independent of the phospholipase C signaling pathway. The reduced ability of the Delta Lys370-Ser425 receptor mutant to activate the ERK-1/2 pathway correlated with an ~80% decrease in the ability of the receptor to undergo agonist-mediated phosphorylation. Furthermore, we have previously shown that M3-muscarinic receptor phosphorylation can be inhibited by a dominant negative mutant of casein kinase 1alpha and by expression of a peptide corresponding to the third intracellular loop of the M3-muscarinic receptor. Expression of these inhibitors of receptor phosphorylation reduced the wild-type M3-muscarinic receptor ERK-1/2 response. We conclude that phosphorylation of the M3-muscarinic receptor on sites in the third intracellular loop by casein kinase 1alpha contributes to the mechanism of receptor activation of ERK-1/2 by working in concert with the diacylglycerol/PKC arm of the phospholipase C signaling pathway.



    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

It is now clear that mitogenic signals mediated by the mitogen-activated protein (MAP)1 kinases, ERK-1 and ERK-2, can be initiated by both receptor-tyrosine kinases (RTKs) and by the heptahelical G-protein-coupled receptors (GPCRs). The activation of the ERK-1/2 pathway by GPCRs is mediated by any one of a number of mechanisms (1) probably reflecting the diversity of receptors within this large gene family. These mechanisms appear quite distinct; for example, ERK-1/2 activation has been shown to proceed via a tyrosine kinase-dependent mechanism for some receptors and a tyrosine kinase-independent manner for others (2, 3). Despite this diversity, common features do exist, the most prominent of which is that GPCRs activate ERK-1/2 by acting initially through "classical" heterotrimeric G-protein signaling pathways (4). For example, stimulation of ERK-1/2 by Gi-coupled receptors, such as M2-muscarinic, and alpha 2A-adrenergic receptors, is pertussis toxin-sensitive indicating a role of Gi-proteins (5-7). It is proposed that liberation of beta gamma -subunits from Gi-proteins is responsible for the initiation of tyrosine phosphorylation (3, 8), possibly by the activation of Src or Src-like tyrosine kinases (9, 10, 11), that ultimately results in Ras-dependent ERK-1/2 activation (3, 6, 7, 12).

Similarly, Gq/11-coupled receptors that stimulate phospholipase C and the subsequent hydrolysis of phosphatidylinositol 4,5-bisphosphate to produce the second messengers inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) and diacylglycerol, activate the ERK-1/2 pathway via Gq/11-heterotrimeric G-proteins. In this case there is evidence for the involvement of both beta gamma -subunits (6, 13, 14) and Galpha q/11-subunits (3, 10, 12, 14, 15). Furthermore, the activation of ERK-1/2 by these receptors appears to be dependent on PKC because inhibition of PKC either abolishes (3, 15-17) or significantly diminishes (18-20) the ERK-1/2 response to Gq/11-coupled receptors. This is particularly apparent for the M3-muscarinic receptor where the ERK-1/2 response is blocked by >85% by either PKC inhibition or PKC down-regulation (21-24).

Studies have also indicated that the Ca2+ mobilization arm of the phospholipase C signaling pathway is important in the activation of ERK-1/2 by Gq/11-coupled receptors. Bradykinin, LPA (25), and alpha 1B-adrenergic (10) receptor-stimulated ERK-1/2 responses were shown to be dependent on changes in intracellular Ca2+. Receptor-mediated Ca2+ mobilization is proposed to activate the Ca2+/PKC-sensitive tyrosine protein kinase, Pyk2 (26), which is thought to act upstream of Ras in the Erk-1/2 pathway (10, 25). In the case of receptors such as the angiotensin AT1 (27), bradykinin (28), CCKA (18), chemokine CXCR-1/2 (19), and purinergic P2Y2 receptors (20, 29), the activation of ERK-1/2 is proposed to be via transactivation of RTKs, a process that is dependent on Ca2+ mobilization and subsequent activation of Pyk2 or related kinases.

These studies indicate that the mechanism for Gq/11-coupled receptor-mediated ERK-1/2 activation is dependent on the coupling of the receptor to Gq/11-heterotrimeric G-proteins and subsequent phospholipase C signaling through Ca2+ mobilization and PKC activation. A further component in the activation of the ERK-1/2 pathway by GPCRs has recently been suggested from studies on the beta 2-adrenergic receptor where receptor phosphorylation has been shown to play a central role. The beta 2-adrenergic receptor is phosphorylated by both PKA and the G-protein coupled receptor kinases (GRKs) (30). PKA phosphorylation of the receptor on sites on the third intracellular loop has been proposed to act as a "molecular switch" coupling the receptor to Gi-proteins and subsequently the activation of the ERK-1/2 pathway via the generation of beta gamma -subunits (31). The beta 2-adrenergic receptor can also be phosphorylated in an agonist-dependent manner by the GRKs, particularly GRK-2. This has classically been considered to result in the recruitment of beta -arrestin and receptor desensitization (30). However, recent studies have shown that beta -arrestin can act as an adaptor protein recruiting activated c-Src to the plasma membrane in a process that is essential in the activation of the ERK-1/2 pathway by the beta 2-adrenergic receptor (32).

In the present paper, we investigate the role played by receptor phosphorylation in the activation of the ERK-1/2 pathway by the Gq/11-coupled M3-muscarinic receptor. This receptor is rapidly phosphorylated on serine following agonist occupation (33). However, in contrast to the beta 2-adrenergic receptor, which is phosphorylated by the GRKs, M3-muscarinic receptors are phosphorylated in an agonist-dependent manner on sites in the third intracellular loop by casein kinase 1alpha (CK1alpha ) (34, 35). Deletion of a region of the third intracellular loop of the human M3-muscarinic receptor (Lys370-Ser425) reduced receptor phosphorylation by ~80% (35). Furthermore, expression of a dominant negative mutant of CK1alpha or a peptide corresponding to the third intracellular loop of the receptor, reduced receptor phosphorylation (35). Using these reagents in the present study, we investigate the role played by agonist-mediated receptor phosphorylation in the activation of the ERK-1/2 pathway.


    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cell Culture-- CHO cell lines were grown in medium consisting of alpha MEM supplemented with 10% fetal calf serum, 100 IU/ml penicillin, 100 µg/ml streptomycin, and 2.5 µg/ml fungizone. Cells were grown in a 5% CO2, 95% air, humidified incubator at 37 °C. The Delta Lys370-Ser425 receptor mutant clone 2 was maintained in blasticidine (5 µg/ml).

Generation of the Dominant Negative Mutant of CK1alpha (F-CK1alpha K46R)-- The dominant negative mutant of CK1alpha (F-CK1alpha K46R) was generated by point mutagenesis of the lysine residue at position 46, which represents the invariant lysine at the ATP binding site of CK1alpha . The lysine residue was mutated to an arginine as described previously (35).

Generation of the Third Intracellular Loop Peptide (3i-Loop Peptide)-- The sequence encoding amino acids Ser345-Leu463 from the third intracellular loop of the M3-muscarinic receptor was cloned into BamHI and EcoRI sites in pcDNA-3 (Invitrogen) as described previously (35).

Generation of the M3-Muscarinic Receptor Deletion Mutant Delta Lys370-- Ser425---Two stably transfected CHO cell lines expressing the M3-muscarinic receptor deletion mutant Delta Lys370-Ser425 were used in the present study. Clone 1 was generated by digestion of the M3-muscarinic receptor coding sequence contained in pcDNA-3 (Invitrogen) with HindIII and then religating the plasmid. This removed the coding sequence for amino acids Lys370-Ser425 inclusive, but maintained the reading frame of the remaining cDNA. This construct was transfected into CHO cells, and clones were selected using medium supplemented with G-418 (200 µg/ml). The second clone used (clone 2) originated from another transfection where the cDNA encoding the Delta Lys370-Ser425 receptor mutant was subcloned into pcDNA-6 (Invitrogen). Clones from this transfection were selected using medium supplemented with blasticidine (5 µg/ml).

Transient Transfections of CHO Cells-- Cells were plated onto 6-well dishes 24 h before transfection. Cells (15-20% confluent) were transfected with either 3 µg of F-CK1alpha K46R or 3i-loop peptide per well using 8 µl of Fugene 6 transfection reagent (Roche Molecular Biochemicals). Cells were used 48 h after transfection. Using a green fluorescent protein construct, we estimated that the transfection efficiency was ~70%.

Quantification of M3-Muscarinic Receptor Expression-- M3-Muscarinic receptor expression on intact plated-down cells was determined using a saturating concentration of the hydrophilic muscarinic antagonist [3H]N-methyl scopolamine ([3H]NMS, ~0.5 nM) as described previously (35). Nonspecific binding was determined in the presence of 20 µM atropine and was < 3% of the total binding.

Mass Ins(1,4,5)P3 Determination-- Cells grown in 24-well dishes were washed with Krebs/HEPES buffer (HEPES (10 mM), NaCl (118 mM), KHPO3 (1.17 mM), KCl (4.3 mM), MgSO4·7 (1.17 mM), CaCl2 (1.3 mM), NaHCO3 (25.0 mM), glucose (11.7 mM), pH 7.4) and challenged with agonist for the appropriate times. Incubations were terminated by rapid aspiration, addition of ice-cold 0.5 M trichloroacetic acid, and transfer to an ice-bath. After 15 min, the supernatant was removed and neutralized by addition of EDTA and freon/tri-n-octylamine as described previously (36). Extracts were brought to pH 7 by addition of NaHCO3 and stored at 4 °C until analysis. Ins(1,4,5)P3 mass measurements were performed using a radio-receptor assay described previously (37).

Erk-1/2 Assay-- CHO cells grown to confluence in 6-well plates were serum-starved for 1 h in Krebs/HEPES buffer and then stimulated with the appropriate agents. Stimulation was terminated by aspiration, and cells were incubated for 10 min in lysis buffer (Tris (20 mM), Nonidet P-40 (0.5%), NaCl (250 mM), EDTA (3 mM), EGTA (3 mM), phenylmethylsulfonyl fluoride (1 mM), Na3VO4 (1 mM), dithiothreitol (1 mM), benzamidine (5 µg/ml), pH 7.6) at 4 °C. Solubilized CHO cell lysates were pre-cleared by centrifuging at 14,000 rpm for 5 min. Endogenous MAP kinase was immunoprecipitated using 0.2 µg of anti-Erk-1/2 antiserum (Santa Cruz). Protein A-Sepharose immobilized MAP kinase was washed twice in lysis buffer and twice in assay buffer (HEPES (20 mM), beta -glycerophosphate (20 mM), MgCl2 (10 mM), dithiothreitol (1 mM), Na3VO4 (50 µM), pH 7.2). Washed pellets were resuspended in assay buffer containing 2 µCi of [32P]ATP, 20 µM ATP, 200 µM EGFr (peptide encompassing region 661-681 of the EGF receptor), and reactions were left to proceed for 20 min at 37 °C. Reactions were terminated by the addition of 25% trichloroacetic acid and spotted onto P81 phosphocellulose paper squares (Whatman). Squares were washed four times with 0.05% orthophosphoric acid and once with acetone, and radioactivity associated with the EGFr was determined by liquid scintillation counting.

Determination of Intracellular Ca2+ Concentrations ([Ca2+]i)-- Confluent monolayers of cells in 175 cm2 flasks were harvested and resuspended in 2.5 ml of Krebs/HEPES buffer. A 0.5-ml aliquot of this was removed for determination of cellular autofluorescence. Fura-2-acetoxymethyl ester (Fura-2-AM, 5 µM) was added to the remaining 2 ml, which was then left for ~40 min at room temperature with gentle mixing. Supernatant containing extracellular Fura-2-AM was removed following gentle centrifugation of the 0.5-ml aliquots. Cells were resuspended in a cuvette containing 3 ml of Krebs/HEPES buffer at 37 °C. Using a Perkin-Elmer LS-5B spectrofluorimeter with a cuvette water jacket to maintain the temperature at 37 °C, emission at 509 nm was recorded following excitation at both 340 and 380 nm. The excitation ratio was recorded every 1 s and converted to [Ca2+]i as previously reported (38) using 0.1% Triton X-100 in the presence of a saturating [Ca2+] to determine Rmax and the addition of EGTA to determine Rmin. Cells were challenged with 10-50 µl of agonist. Initial experiments were conducted in the presence of 1.3 mM extracellular [Ca2+] (as represented in Fig. 4B). In experiments to determine the potency of intracellular Ca2+ mobilization by the full agonist methacholine (represented in Fig. 4C), the experiments were conducted in Ca2+-free medium where the Krebs/HEPES buffer had been supplemented with EGTA to reduce extracellular [Ca2+] to ~100 nM (determined using Fura-2). This was to ensure that the ability of the agonist to mobilize intracellular Ca2+ stores was being measured, because any changes in intracellular Ca2+ concentrations under these conditions would have been the result of release of Ca2+ from intracellular stores with no contribution being made form an influx of extracellular Ca2+.


    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

ERK-1/2 Activation by a Phosphorylation-deficient Mutant of the M3-Muscarinic Receptor-- Previous studies from our laboratory and others (21-24) have shown that M3-muscarinic receptors activate the ERK-1/2 pathway in a PTX-insensitive, PKC-dependent manner. The time course for ERK-1/2 activation peaks at 5 min then falls to a plateau, which is maintained for at least 20 min (21). To test whether receptor phosphorylation plays a role in the regulation of the ERK-1/2 pathway, a mutant M3-muscarinic receptor was used where residues Lys370-Ser425 of the third intracellular loop of the human M3-muscarinic receptor had been deleted. This mutant receptor, termed Delta Lys370-Ser425, had previously been demonstrated to show an ~80% decrease in its ability to undergo agonist-mediated phosphorylation (35). Two stably transfected CHO cell lines were prepared expressing the Delta Lys370-Ser425 receptor at levels comparable with the wild-type controls (Bmax values in fmols of receptor/mg protein: wild type = 908 ± 124, Delta Lys370-Ser425 mutant clone 1 = 782 ± 67, mutant clone 2 = 1209 ± 10).

Concentration-response analysis of CHO cells expressing the wild-type M3-muscarinic receptor (CHO-m3 cells) showed a receptor-mediated ERK-1/2 activation with a half-maximal response (EC50) to the agonist carbachol of 45 ± 1.3 nM (n = 3, ± S.E., Fig. 1.) This is very similar to the EC50 value that we obtained previously using another distinct M3-muscarinic receptor-transfected CHO cell line (21). In contrast to the wild-type receptor, the mutant receptor showed a rightward shift in the ERK-1/2 concentration-response curve to carbachol (Fig. 1). The EC50 values for the two clonal cell lines expressing the Delta Lys370-Ser425 mutant were 660 ± 100 nM and 300 ± 100 nM (n = 3, ± S.E.) for clones 1 and 2, respectively. These EC50 values were significantly different from the wild-type receptor values (p < 0.05, Student's t test). In addition to a reduction in the potency of carbachol, there was also a reduction in the maximal ERK-1/2 response with clone 1 showing a 23 ± 7% reduction and clone 2 a 61 ± 5% reduction (n = 3, ± S.E.) in the maximal carbachol response compared with wild-type receptor controls (Fig. 1). The time course for activation of ERK-1/2 was not, however, significantly different between the control and mutant receptors (data not shown).



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Fig. 1.   Activation of ERK-1/2 by wild-type M3-muscarinic receptors and the deletion mutant Delta Lys370-Ser425. Stably transfected CHO cells expressing either the wild-type human M3-muscarinic receptor or the deletion mutant Delta Lys370-Ser425 were stimulated for 5 min in the presence of varying concentrations of carbachol (CCH). The reaction was terminated by addition of lysis buffer, and ERK-1/2 activity was determined. Shown are the concentration-response curves for wild-type receptor and two separate clones: clone 1 (A), clone 2 (B), expressing the Delta Lys370-Ser425 receptor mutant. The data presented represent the mean ± S.E. for three experiments.

To test for the possibility of clonal variation between the wild-type CHO-m3 cells and mutant receptor cell lines, concentration-response curves for serum-induced ERK-1/2 activation were carried out. The concentration-response curves for serum-activated ERK-1/2 in the mutant receptor CHO cell lines were not significantly different from that of the CHO-m3 cells (Fig. 2). This indicated that there was no clonal difference in the ERK-1/2 pathway stimulated by serum.



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Fig. 2.   Serum-mediated ERK-1/2 responses. Stably transfected CHO cells expressing either the wild-type human M3-muscarinic receptor or the deletion mutant Delta Lys370-Ser425 were stimulated for 20 min in the presence of varying concentrations of fetal calf serum. The reaction was terminated by addition of lysis buffer, and ERK-1/2 activity was determined. Shown are the concentration-response curves for wild-type receptor and two separate clones: clone 1 (A), clone 2 (B), expressing the Delta Lys370-Ser425 receptor mutant. The data represent the mean ± S.E. of at least three experiments.

PKC Dependence of Muscarinic ERK-1/2 Responses-- We have previously shown that the wild-type M3-muscarinic receptor-mediated ERK-1/2 response is dependent on PKC because inhibition of PKC using Ro-318220 or down-regulation of PKC reduced the muscarinic-ERK-1/2 response by >90% (21). The Delta Lys370-Ser425 receptor mutant response also appeared to be sensitive to PKC inhibition in a manner similar to the wild-type receptor. The phorbol 12,13-dibutyrate ERK-1/2 responses in the CHO-m3 cells and cells expressing Delta Lys370-Ser425 receptor mutant (clone 2) were completely inhibited by the PKC inhibitor Ro-318220 (Fig. 3). The ERK-1/2 responses to carbachol in the CHO-m3 cells and the cells expressing the Delta Lys370-Ser425 receptor mutant were inhibited (~90%) by Ro-318220 (Fig. 3).



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Fig. 3.   PKC-dependence of ERK-1/2 responses. Cells were pretreated with either vehicle or the PKC-inhibitor Ro-318220 (10 µM, Ro) for 10 min prior to stimulation with 1 mM carbachol (CCH) or 1 µM phorbol 12,13-dibutyrate (PDBu) or nonstimulated (Control). Stimulation was for 5 min after which reactions were terminated by addition of lysis buffer, and ERK-1/2 activity was determined. The data represent the mean ± S.E. of three experiments.

Coupling of the Delta Lys370-Ser425 Receptor Mutant to the Phospholipase C Signaling Pathway-- We have previously reported that the Delta Lys370-Ser425 receptor mutant showed agonist and antagonist binding characteristics that were not significantly different from the wild-type receptors (35). We have also reported that the Delta Lys370-Ser425 receptor mutant is coupled to the phospholipase C pathway in a manner analogous to the wild-type receptor. For example, the time course of Ins(1,4,5)P3 generation of both the wild-type M3-muscarinic receptor and the Delta Lys370-Ser425 receptor mutant peaks within 5-10 s of agonist stimulation and reaches a plateau phase after 60 s, which is maintained for at least 5 min (35). Significantly, we have shown previously that the Delta Lys370-Ser425 receptor mutant appears to give a more robust Ins(1,4,5)P3 response than the wild-type receptor, suggesting that the receptor may be more efficiently coupled to phospholipase C (35). In the present study, this characteristic is evident by an ~3.0-fold greater production of Ins(1,4,5)P3 at maximal agonist concentration (Fig. 4A).



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Fig. 4.   Coupling of the wild-type M3-muscarinic receptor and Delta Lys370-Ser425 receptor mutant to the phospholipase C pathway. A, Ins(1,4,5)P3 generation was determined in cells that had been stimulated with carbachol (1 mM) for varying times. The data represent the mean ± S.E. of three experiments carried out in duplicate. B, time-course of the changes in free intracellular Ca2+ concentrations ([Ca2+]i) determined in cell suspensions loaded with the Ca2+ indicator Fura2-AM and stimulated with the full agonist methacholine (1 mM). C, concentration-response curve of the peak Ca2+ response following stimulation with methacholine. The data represent the mean ± S.E. of at least three experiments.

We have reported previously that despite the fact that the Delta Lys370-Ser425 receptor mutant was able to drive a larger Ins(1,4,5)P3 response, the potency of the full agonist carbachol to mediate an Ins(1,4,5)P3 response was not significantly different between the mutant and wild-type receptors, which had EC50 values of 9.71 ± 1.9 µM and 7.14 ± 3.2 µM (n = 3, ± S.E.), respectively (35).

The ability of the Delta Lys370-Ser425 receptor mutant (clone 1) to mobilize intracellular Ca2+ was also tested. The time course for receptor-mediated increases in intracellular Ca2+ for both mutant and wild-type receptors were similar (Fig. 4B). Interestingly, in contrast to the Ins(1,4,5)P3 response, there was no significant difference in the magnitude of the Ca2+ mobilization response between the wild-type and Delta Lys370-Ser425 receptor mutant. Similarly, the concentration-response curves for peak Ca2+ mobilization were not significantly different with EC50 values of 166 ± 70 nM and 258 ± 50 nM (n = 3, ± S.E.) for the wild-type M3-muscarinic receptor and Delta Lys370-Ser425 receptor mutant, respectively (Fig. 4C). (Note; in these Ca2+ mobilization experiments the full agonist methacholine was used. Both methacholine and carbachol are full agonists at the M3-muscarinic receptor and produce almost identical responses.)

Effect of the CK1alpha Dominant Negative Mutant (F-CK1alpha -K46R) and the 3i-Loop Peptide on M3-Muscarinic Receptor-mediated ERK-1/2 Activation-- Our previous studies had shown that CK1alpha was able to phosphorylate the M3-muscarinic receptor in an agonist-dependent manner (34). Furthermore, we demonstrated that transient expression of a dominant negative mutant of CK1alpha (F-CK1alpha -K46R) was able to reduce receptor phosphorylation by ~40% (35). In these earlier studies, we also showed that expression of a peptide corresponding to the third intracellular loop of the M3-muscarinic receptor (Ser345-Leu463), named the 3i-loop peptide, resulted in inhibition of receptor phosphorylation by >70% (35). To test the role that receptor phosphorylation might play in ERK-1/2 activation we transiently transfected F-CK1alpha -K46R and the 3i-loop peptide into CHO-m3 cells stably expressing the M3-muscarinic receptor. Expression of F-CK1alpha -K46R and the 3i-loop peptide resulted in the reduction of the carbachol-mediated ERK-1/2 response by 53.9 ± 7.7% and 49.4 ± 3.2%, respectively (Fig. 5A).



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Fig. 5.   Effect of the CK1alpha dominant negative mutant (F-CK1alpha K46R) and the 3i-loop peptide on the M3-muscarinic ERK-1/2 response. CHO-m3 cells stably expressing recombinant M3-muscarinic receptors (A) or native CHO-K1 cells (B), were transiently transfected with the CK1alpha dominant negative mutant, F-CK1alpha K46R (K46R) or the 3i-loop peptide (3i-loop) corresponding to Ser345-Leu463 of the third intracellular loop of the M3-muscarinic receptor or were sham transfected (Control). 48 h after transfection, cells were stimulated with 1 mM carbachol (CCH, A) or 1 µM phorbol 12,13-dibutyrate (PDBu, B), for 5 min. Reactions were terminated using lysis buffer, and ERK-1/2 activity was determined. The data represent the mean ± S.E. of three experiments.

Control experiments were designed to test the ability of F-CK1alpha -K46R or the 3i-loop peptide to inhibit nonreceptor-mediated ERK-1/2 activation. Hence, the effect of transient transfection of F-CK1alpha -K46R or the 3i-loop peptide on the phorbol 12,13-dibutyrate ERK-1/2 response in native CHO-K1 cells was tested. It was found that neither F-CK1alpha -K46R or the 3i-loop peptide had any significant effect on the phorbol ester-mediated ERK-1/2 response in these cells (Fig. 5B).

It is interesting to note that in experiments where phorbol esters were used to stimulate ERK-1/2 activity in CHO-m3 cells the F-CK1alpha -K46R construct inhibited the phorbol ester response by 31% (data not shown). The fact that the F-CK1alpha -K46R construct had very little effect on the phorbol ester response in CHO-K1 cells, but a significant effect in CHO-m3 cells would suggest that in CHO-m3 cells the M3-muscarinic receptor itself might contribute to the phorbol ester ERK-1/2 response. This may be because of the fact that phorbol esters are able to mediate phosphorylation of the agonist-unoccupied M3-muscarinic receptor (33). This and other possibilities are presently under investigation.

Analysis of the ERK-1/2 concentration-response curves to carbachol demonstrated that in addition to reducing the maximal response the 3i-loop peptide and F-CK1alpha -K46R significantly (p < 0.05, Student's t test) reduced the potency of carbachol by 15.7-fold and 1.8-fold, respectively (Fig. 6).



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Fig. 6.   ERK-1/2 concentration-response curves in CHO-m3 cells transiently transfected with the CK1alpha dominant negative mutant (F-CK1alpha K46R) and the 3i-loop peptide. CHO-m3 cells stably expressing recombinant M3-muscarinic receptors were transiently transfected with the CK1alpha dominant negative mutant, F-CK1alpha K46R (K46R) (A), or the 3i-loop peptide (3i-loop peptide, B) corresponding to Ser345-Leu463 of the third intracellular loop of the M3-muscarinic receptor. 48 h after transfection cells were stimulated with varying concentrations of carbachol (CCH) for 5 min. Reactions were terminated using lysis buffer, and ERK-1/2 activity was determined. The data represent the mean ± S.E. of three experiments.



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Despite intensive research, the mechanisms employed by GPCRs in the activation of the ERK-1/2 pathway are generally poorly understood. One reason for this is that GPCRs are able to employ a number of diverse mechanisms in the activation of ERK-1/2 depending on the receptor type and the cellular environment (4). For example, M1-muscarinic receptor ERK-1/2 responses have been shown to operate in both a Ras-dependent (6) and Ras-independent (3) fashion using a mechanism, which in some cell types, employs tyrosine phosphorylation (2) and in others acts in a tyrosine kinase-independent manner (3). To add a further level of complexity, it has now become clear that a number of Gq/11-coupled receptors can simultaneously employ at least two independent mechanisms to activate the ERK-1/2 pathway (20, 24, 39). Despite this diversity there is one overriding common feature in the mechanisms employed by Gq/11-coupled receptors, namely, the involvement of the Gq/11-heterotrimeric G-proteins and the subsequent activation of the phospholipase C signaling pathway. Both the Ins(1,4,5)P3/Ca2+ mobilization and diacylglycerol/PKC arms of the phospholipase C signaling pathway have been implicated to play a role and in many instances appear to provide the primary signal that links receptor activation to the initiation of the ERK-1/2 pathway.

We have shown previously that Gq/11-coupled M3-muscarinic receptors expressed in CHO cells stimulate the ERK-1/2 pathway in a PKC-dependent manner (21). This was confirmed in the present study and is consistent with previous reports from other laboratories (22-24) and would suggest that activation of PKC by the M3-muscarinic receptor is sufficient to stimulate ERK-1/2. This conclusion could be applied to a large number of Gq/11-coupled receptors that show PKC-dependent activation of ERK-1/2, such as prostaglandin F2alpha (15), P2Y2-purinergic (20, 29), CCK (18), M1-muscarinic, alpha 1-adrenergic (3), and bradykinin (17) receptors. Furthermore, the ability of phorbol esters to increase ERK-1/2 activity (40) provides evidence that simply stimulating PKC is sufficient to drive the activation of ERK-1/2.

Thus, one model for ERK-1/2 activation by Gq/11-coupled receptors, including the M3-muscarinic receptor, would be that receptor-mediated PKC activation is sufficient to provide the signal that elicits the ERK-1/2 response.

Our data, however, using the Delta Lys370-Ser425 M3-muscarinic receptor mutant would suggest that this simple model is not correct. Deletion of Lys370-Ser425 in the third intracellular loop of the human M3-muscarinic receptor resulted in a reduction in the ability of the receptor to stimulate ERK-1/2 activity. This reduction was evident despite the fact that the receptor was efficiently coupled to the phospholipase C signaling pathway. In fact this study, consistent with our previous report (35), demonstrates that the Delta Lys370-Ser425 receptor mutant is more efficiently coupled to the phospholipase C pathway than the wild-type receptor. This suggests that simply activating the diacylglycerol/PKC arm of the phospholipase C signaling pathway was not in itself sufficient to drive a full Gq/11-coupled receptor ERK-1/2 response. It is interesting to note that the ERK-1/2 response mediated by the Delta Lys370-Ser425 receptor mutant was still sensitive to PKC inhibition. Thus, the Delta Lys370-Ser425 receptor mutant ERK-1/2 response still has an absolute requirement for the activation of PKC but appears to be unable to employ an additional mechanism that is independent of Gq/11-activated phospholipase C signaling. This additional mechanism (Fig. 7, Mechanism 2) appears to act in concert with PKC to elicit a full ERK-1/2 response.



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Fig. 7.   Scheme of the mechanisms involved in the activation of the ERK-1/2 pathway by M3-muscarinic receptors. Our data have identified two mechanisms involved in the activation of the ERK-1/2 pathway by M3-muscarinic receptors expressed in CHO cells. Mechanism 1 is PKC-dependent and is essential in the activation of ERK-1/2. Inhibition of Mechanism 1 (e.g. inhibition of PKC with Ro-318220) prevents activation of ERK-1/2 despite the fact that Mechanism 2 is still intact. Mechanism 2, therefore, will not elicit an ERK-1/2 response alone. However, Mechanism 2 does operate in concert with Mechanism 1 to give a full ERK-1/2 response. Hence a receptor that is only able to activate Mechanism 1 (i.e. the Delta Lys370-Ser425 receptor mutant or the wild-type M3-muscarinic receptor expressed together with the 3i-loop peptide or F-CK1alpha K46R) will give a less than maximal ERK-1/2 response. CK1alpha , casein kinase 1alpha ; DAG, diacylglycerol; InsP3, inositol 1,4,5-trisphosphate; PIP2, phosphatidylinositol 4,5-bisphosphate; PLC, phospholipase C.

These data, therefore, support a model that identifies two mechanisms in the activation of ERK-1/2 (Fig. 7). Mechanism 1 is PKC-dependent and is absolutely required for ERK-1/2 activation but when operating alone is only able to mediate a partial ERK-1/2 response. Mechanism 2 is PKC-independent and although is unable to elicit an ERK-1/2 response when operating alone, it is able to act in concert with Mechanism 1 to give a full ERK-1/2 response.

The most prominent PKC-independent mechanism assigned to Gq/11-coupled receptor activation of ERK-1/2 is via the activity of the Ca2+-sensitive tyrosine kinase Pyk2 or related kinases (25). Ins(1,4,5)P3-dependent increases in intracellular Ca2+ has been demonstrated to stimulate Pyk2 activity resulting in "transactivation" of RTKs and subsequent activation of the ERK-1/2 pathway (18-20, 27-29). We can, however, eliminate the involvement of this process in the explanation of the results obtained with the Delta Lys370-Ser425 receptor mutant for two reasons. First, the muscarinic receptor ERK-1/2 response in CHO cells is independent of changes in intracellular Ca2+ (22) suggesting that Pyk2 is not involved in the M3-muscarinic receptor response in these cells. Second, GPCR transactivation of RTKs via Pyk2 is a process that involves Ins(1,4,5)P3-mediated increases in intracellular Ca2+ (26). Because the Delta Lys370-Ser425 receptor couples efficiently to the phospholipase C pathway, stimulating Ca2+ mobilization in an identical manner to the wild-type receptor, the involvement of a Ca2+-sensitive mechanism would not explain the lack of responsiveness of this receptor mutant.

Hence, the data presented here identifies a novel component of the M3-muscarinic receptor ERK-1/2 response that is independent of activation of the Gq/11/phospholipase C pathway and dispels the notion that Gq/11-coupled receptors mediate ERK-1/2 activation by solely stimulating PKC or activating tyrosine phosphorylation via Ins(1,4,5)P3-dependent increases in intracellular Ca2+.

We next tested the possibility that the novel component of the M3-muscarinic receptor ERK-1/2 response involved agonist-mediated phosphorylation of the receptor. Our earlier studies had shown that the M3-muscarinic receptor is rapidly phosphorylated on serine in an agonist-dependent manner (33). Extensive studies by our group have identified CK1alpha as a cellular kinase able to phosphorylate the M3-muscarinic receptor (also the M1-muscarinic receptor and rhodopsin) in an agonist-dependent manner (34, 35, 41, 42). These studies established for the first time a mechanism for agonist-dependent phosphorylation of GPCRs that was distinct from that of the GRKs. During these studies we suggested that sites within the third intracellular loop of the M3-muscarinic receptor were important for the phosphorylation of the receptor. To test this we generated the Delta Lys370-Ser425 receptor mutant, which lacked eight potential serine phospho-acceptor sites and the putative CK1alpha binding site (His374-Val391) (35). Consistent with our hypothesis, the Delta Lys370-Ser425 receptor mutant was reduced in its ability to undergo agonist-dependent phosphorylation by ~80% (35).

The reduced ability of the Delta Lys370-Ser425 receptor mutant to undergo agonist-mediated phosphorylation correlates with the reduction in the receptor ERK-1/2 response and suggests that there is a link between receptor phosphorylation and activation of the ERK-1/2 pathway. It is of course possible that deletion of residues Lys370-Ser425 removes a domain involved in the ERK-1/2 response but which is not connected with receptor phosphorylation. This, in itself is an intriguing possibility and one that is being actively tested in our laboratory at the moment. However, our data to date is consistent with the hypothesis that phosphorylation of the M3-muscarinic receptor is involved in the PKC-dependent activation of the ERK-1/2 pathway.

We further investigated the role of receptor phosphorylation in the M3-muscarinic receptor-mediated ERK-1/2 response by inhibiting phosphorylation of the wild-type receptor. We have previously demonstrated that inhibition of CK1alpha -mediated M3-muscarinic receptor phosphorylation could be achieved using either a dominant negative mutant of CK1alpha , F-CK1alpha -K46R, or expression of a region of the third intracellular loop of the M3-muscarinic receptor (3i-loop peptide) that acted as a pseudo-substrate for CK1alpha (35). In the present study, expression of these constructs resulted in rightward shift in the concentration-response curve for carbachol-mediated ERK-1/2 activation and a reduction in the maximal ERK-1/2 response. The effect of these inhibitors of receptor phosphorylation appeared to be specific for the M3-muscarinic-mediated ERK-1/2 response because expression of these constructs in CHO-K1 cells did not greatly affect the phorbol ester-mediated ERK-1/2 response. Furthermore, previously we have shown that F-CK1alpha -K46R did not prevent the receptor from coupling to the phospholipase C pathway but in fact increased the ability of the receptor to activate phospholipase C (35). Thus, the depressed ERK-1/2 response observed in the presence of inhibitors of receptor phosphorylation is receptor specific and produces a response in the wild-type receptor that is very similar to that observed for the phosphorylation-deficient Delta Lys370-Ser425 receptor mutant. These data suggest, therefore, that agonist-mediated phosphorylation of the M3-muscarinic receptor contributes to the mechanism of ERK-1/2 activation.

This conclusion is supported by recent reports linking phosphorylation of the beta 2-adrenergic receptor to the regulation of ERK-1/2 activity. PKA-mediated phosphorylation of the beta 2-adrenergic receptor has been demonstrated to act as a "molecular switch" resulting in the coupling of the receptor to the ERK-1/2 pathway via Gi-protein beta gamma -subunits (31). Furthermore, agonist-mediated GRK-2 phosphorylation has been shown to recruit a beta -arrestin·c-Src complex to the beta 2-adrenergic receptor (32). Preventing the ability of beta -arrestin to interact with c-Src inhibits beta 2-adrenergic receptor-mediated ERK-1/2 activation, suggesting that recruitment of c-Src to the phosphorylated beta 2-adrenergic receptor via beta -arrestin is essential in the mechanism of activation of ERK-1/2 (32). Hence, the data we present here indicates that the M3-muscarinic receptor, in common with the beta 2-adrenergic receptor, employs agonist-mediated receptor phosphorylation in the mechanism of activation of the ERK-1/2 pathway.

In conclusion, we propose that agonist-mediated receptor phosphorylation via CK1alpha initiates a process that acts in concert with PKC to mediate a full M3-muscarinic receptor ERK-1/2 response (Fig. 7). The exact nature of the mechanism initiated by receptor phosphorylation is presently unclear but appears not to involve Gq/11 heterotrimeric G-proteins nor the activation of the phospholipase C second messenger signaling cascade. We are presently pursuing the possibility that phosphorylation of sites in the third intracellular loop of the M3-muscarinic receptor recruits an adaptor protein that is important in the activation of the ERK-1/2 pathway in a manner analogous to beta -arrestin·c-Src and the beta 2-adrenergic receptor.


    ACKNOWLEDGEMENTS

We thank Prof. Nahorski whom, together with Drs. A. B. Tobin and G. B. Willars, initiated the work on the Delta Lys370-Ser425 receptor mutant.


    FOOTNOTES

* This work was supported by Wellcome Trust Grant No. 047600/Z/96.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed. Tel.: 0116-2522935; Fax: 0116-2523996; E-mail: TBA@le.ac.uk.

Published, JBC Papers in Press, November 16, 2000, DOI 10.1074/jbc.M008827200


    ABBREVIATIONS

The abbreviations used are: MAP, mitogen-activated protein; [Ca2+]i, intracellular calcium concentration; CK1alpha , casein kinase 1alpha ; ERK, extracellular-regulated protein kinases; GPCR, G-protein-coupled receptor; GRK, G-protein-coupled receptor kinase; Ins(1, 4,5)P3, inositol (1,4,5)-trisphosphate; PKC, protein kinase C; PKA, cAMP-dependent protein kinase; RTK, receptor-tyrosine kinases; CHO, Chinese hamster ovary cells.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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