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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
    Affiliations
    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
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  • Gary B. Willars
    Affiliations
    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
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  • John E. McDonald
    Affiliations
    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
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  • Andrew B. Tobin
    Correspondence
    To whom correspondence should be addressed.
    Affiliations
    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
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  • Author 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.
Open AccessPublished:February 16, 2001DOI:https://doi.org/10.1074/jbc.M008827200
      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 ΔLys370–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 ΔLys370–Ser425 receptor mutant, that is independent of the phospholipase C signaling pathway. The reduced ability of the ΔLys370–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 1α 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 1α 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.
      MAP
      mitogen-activated protein
      [Ca2+]i
      intracellular calcium concentration
      CK1α
      casein kinase 1α
      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
      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 (
      • Malarkey J.
      • Belham C.M.
      • Paul A.
      • Graham A.
      • Scott P.H.
      • Plevin R.
      ) 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 (
      • Wan Y.
      • Kurosaki T.
      • Huang X-Y.
      ,
      • Hawes B.E.
      • van Biesen T.
      • Koch W.J.
      • Luttrell L.M.
      • Lefkowitz R.J.
      ). 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 (
      • Gutkind J.S.
      ). For example, stimulation of ERK-1/2 by Gi-coupled receptors, such as M2-muscarinic, and α2A-adrenergic receptors, is pertussis toxin-sensitive indicating a role of Gi-proteins (
      • Winitz S.
      • Russell M.
      • Qian N-X.
      • Gardner A.
      • Dwyers L.
      • Johnson G.L.
      ,
      • Crespo P.
      • Xu N.
      • Simmonds W.F.
      • Gutkind J.S.
      ,
      • Koch W.J.
      • Hawes B.E.
      • Allen L.F.
      • Lefkowitz R.J.
      ). It is proposed that liberation of βγ-subunits from Gi-proteins is responsible for the initiation of tyrosine phosphorylation (
      • Hawes B.E.
      • van Biesen T.
      • Koch W.J.
      • Luttrell L.M.
      • Lefkowitz R.J.
      ,
      • Lopez-Ilasaca M.
      • Crespo P.
      • Pellici P.G.
      • Gutkind J.S.
      • Wetzker R.
      ), possibly by the activation of Src or Src-like tyrosine kinases (
      • Luttrell L.M.
      • Hawes B.E.
      • van Biesen T.
      • Luttrell D.K.
      • Lansing T.J.
      • Lefkowitz R.J.
      ,
      • Della Rocca G.J.
      • van Biesen T.
      • Daaka Y.
      • Luttrell D.K.
      • Luttrell L.M.
      • Lefkowitz R.J.
      ,
      • Della Rocca G.J.
      • Maudsley S.
      • Daaka Y.
      • Lefkowitz R.J.
      • Luttrell L.M.
      ), that ultimately results in Ras-dependent ERK-1/2 activation (
      • Hawes B.E.
      • van Biesen T.
      • Koch W.J.
      • Luttrell L.M.
      • Lefkowitz R.J.
      ,
      • Crespo P.
      • Xu N.
      • Simmonds W.F.
      • Gutkind J.S.
      ,
      • Koch W.J.
      • Hawes B.E.
      • Allen L.F.
      • Lefkowitz R.J.
      ,
      • Faure M.
      • Voyno-Yasenetskaya T.A.
      • Bourne H.R.
      ).
      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 βγ-subunits (
      • Crespo P.
      • Xu N.
      • Simmonds W.F.
      • Gutkind J.S.
      ,
      • Palomero T.
      • Barros F.
      • Del Camino D.
      • Viloria C.G.
      • De La Pena P.
      ,
      • Launay J-M.
      • Birraux G.
      • Bondoux D.
      • Callebert J.
      • Choi D-S.
      • Loric S.
      • Maroteaux L.
      ) and Gαq/11-subunits (
      • Hawes B.E.
      • van Biesen T.
      • Koch W.J.
      • Luttrell L.M.
      • Lefkowitz R.J.
      ,
      • Della Rocca G.J.
      • van Biesen T.
      • Daaka Y.
      • Luttrell D.K.
      • Luttrell L.M.
      • Lefkowitz R.J.
      ,
      • Faure M.
      • Voyno-Yasenetskaya T.A.
      • Bourne H.R.
      ,
      • Launay J-M.
      • Birraux G.
      • Bondoux D.
      • Callebert J.
      • Choi D-S.
      • Loric S.
      • Maroteaux L.
      ,
      • Watanabe T.
      • Waga I.
      • Honda Z-i.
      • Kurokawa K.
      • Shimizu T.
      ). Furthermore, the activation of ERK-1/2 by these receptors appears to be dependent on PKC because inhibition of PKC either abolishes (
      • Hawes B.E.
      • van Biesen T.
      • Koch W.J.
      • Luttrell L.M.
      • Lefkowitz R.J.
      ,
      • Watanabe T.
      • Waga I.
      • Honda Z-i.
      • Kurokawa K.
      • Shimizu T.
      ,
      • Zou Y.
      • Komuro I.
      • Aikawa R.
      • Kudoh S.
      • Shiojima I.
      • Hiroi Y.
      • Mizuno T.
      • Yazaki Y.
      ,
      • Velarde V.
      • Ullian M.E.
      • Morinelli T.A.
      • Mayfield R.K.
      • Jaffa A.A.
      ) or significantly diminishes (
      • Tapia J.A.
      • Ferris H.A.
      • Jensen R.T.
      • Garcia L.J.
      ,
      • Venkatakrishnam G.
      • Salgia R.
      • Groopman J.E.
      ,
      • Soltoff S.P.
      • Avraham H.
      • Avraham S.
      • Cantley L.C.
      ) 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 (
      • Budd D.C.
      • Rae A.
      • Tobin A.B.
      ,
      • Wylie P.G.
      • Challiss R.A.J.
      • Blank J.L.
      ,
      • Kim J-Y.
      • Yang M-S.
      • Oh C-D.
      • Kim K-T.
      • Kang S-S.
      • Chun J-S.
      ,
      • Slack B.E.
      ).
      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 (
      • Dikic I.
      • Tokiwa G.
      • Lev S.
      • Courtneidge S.A.
      • Schlessinger J.
      ), and α1B-adrenergic (
      • Della Rocca G.J.
      • van Biesen T.
      • Daaka Y.
      • Luttrell D.K.
      • Luttrell L.M.
      • Lefkowitz R.J.
      ) 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 (
      • Lev S.
      • Moreno H.
      • Martinez R.
      • Canoll P.
      • Peles E.
      • Musacchio J.M.
      • Plowman G.D.
      • Rudy B.
      • Schlessinger J.
      ), which is thought to act upstream of Ras in the Erk-1/2 pathway (
      • Della Rocca G.J.
      • van Biesen T.
      • Daaka Y.
      • Luttrell D.K.
      • Luttrell L.M.
      • Lefkowitz R.J.
      ,
      • Dikic I.
      • Tokiwa G.
      • Lev S.
      • Courtneidge S.A.
      • Schlessinger J.
      ). In the case of receptors such as the angiotensin AT1 (
      • Eguchi S.
      • Numaguchi K.
      • Iwasaki H.
      • Matsumoto T.
      • Yamakawa T.
      • Utsunomiya H.
      • Motley E.D.
      • Kawakatsu H.
      • Owada K.M.
      • Hirata Y.
      • Marumo F.
      • Inagami T.
      ), bradykinin (
      • Zwick E.
      • Daub H.
      • Aoki N.
      • Yamaguchi-Aoki Y.
      • Tinhofer I.
      • Maly K.
      • Ullrich A.
      ), CCKA(
      • Tapia J.A.
      • Ferris H.A.
      • Jensen R.T.
      • Garcia L.J.
      ), chemokine CXCR-1/2 (
      • Venkatakrishnam G.
      • Salgia R.
      • Groopman J.E.
      ), and purinergic P2Y2receptors (
      • Soltoff S.P.
      • Avraham H.
      • Avraham S.
      • Cantley L.C.
      ,
      • Soltoff S.P.
      ), 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 β2-adrenergic receptor where receptor phosphorylation has been shown to play a central role. The β2-adrenergic receptor is phosphorylated by both PKA and the G-protein coupled receptor kinases (GRKs) (
      • Pitcher J.A.
      • Freedman N.J.
      • Lefkowitz R.J.
      ). 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 βγ-subunits (
      • Daaka Y.
      • Luttrell L.M.
      • Lefkowitz R.J.
      ). The β2-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 β-arrestin and receptor desensitization (
      • Pitcher J.A.
      • Freedman N.J.
      • Lefkowitz R.J.
      ). However, recent studies have shown that β-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 β2-adrenergic receptor (
      • Luttrell L.M.
      • Ferguson S.S.G.
      • Daaka Y.
      • Miller W.E.
      • Maudsley S.
      • Della Rocca G.J.
      • Lin F-T
      • Kawakatsu H.
      • Owada K.
      • Luttrell D.K.
      • Caron M.G.
      • Lefkowitz R.J.
      ).
      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 (
      • Tobin A.B.
      • Nahorski S.R.
      ). However, in contrast to the β2-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 1α (CK1α) (
      • Tobin A.B.
      • Totty N.F.
      • Sterlin A.E.
      • Nahorski S.R.
      ,
      • Budd D.C.
      • McDonald J.E.
      • Tobin A.B.
      ). Deletion of a region of the third intracellular loop of the human M3-muscarinic receptor (Lys370–Ser425) reduced receptor phosphorylation by ∼80% (
      • Budd D.C.
      • McDonald J.E.
      • Tobin A.B.
      ). Furthermore, expression of a dominant negative mutant of CK1α or a peptide corresponding to the third intracellular loop of the receptor, reduced receptor phosphorylation (
      • Budd D.C.
      • McDonald J.E.
      • Tobin A.B.
      ). 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.

      DISCUSSION

      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 (
      • Gutkind J.S.
      ). For example, M1-muscarinic receptor ERK-1/2 responses have been shown to operate in both a Ras-dependent (
      • Crespo P.
      • Xu N.
      • Simmonds W.F.
      • Gutkind J.S.
      ) and Ras-independent (
      • Hawes B.E.
      • van Biesen T.
      • Koch W.J.
      • Luttrell L.M.
      • Lefkowitz R.J.
      ) fashion using a mechanism, which in some cell types, employs tyrosine phosphorylation (
      • Wan Y.
      • Kurosaki T.
      • Huang X-Y.
      ) and in others acts in a tyrosine kinase-independent manner (
      • Hawes B.E.
      • van Biesen T.
      • Koch W.J.
      • Luttrell L.M.
      • Lefkowitz R.J.
      ). 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 (
      • Soltoff S.P.
      • Avraham H.
      • Avraham S.
      • Cantley L.C.
      ,
      • Slack B.E.
      ,
      • Xiong L.
      • Lee J.W.
      • Graves L.M.
      • Earp H.S.
      ). 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 (
      • Budd D.C.
      • Rae A.
      • Tobin A.B.
      ). This was confirmed in the present study and is consistent with previous reports from other laboratories (
      • Wylie P.G.
      • Challiss R.A.J.
      • Blank J.L.
      ,
      • Kim J-Y.
      • Yang M-S.
      • Oh C-D.
      • Kim K-T.
      • Kang S-S.
      • Chun J-S.
      ,
      • Slack B.E.
      ) 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 F (
      • Watanabe T.
      • Waga I.
      • Honda Z-i.
      • Kurokawa K.
      • Shimizu T.
      ), P2Y2-purinergic (
      • Soltoff S.P.
      • Avraham H.
      • Avraham S.
      • Cantley L.C.
      ,
      • Soltoff S.P.
      ), CCK (
      • Tapia J.A.
      • Ferris H.A.
      • Jensen R.T.
      • Garcia L.J.
      ), M1-muscarinic, α1-adrenergic (
      • Hawes B.E.
      • van Biesen T.
      • Koch W.J.
      • Luttrell L.M.
      • Lefkowitz R.J.
      ), and bradykinin (
      • Velarde V.
      • Ullian M.E.
      • Morinelli T.A.
      • Mayfield R.K.
      • Jaffa A.A.
      ) receptors. Furthermore, the ability of phorbol esters to increase ERK-1/2 activity (
      • Cobb M.H.
      • Goldsmith E.J.
      ) 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 ΔLys370–Ser425M3-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 (
      • Budd D.C.
      • McDonald J.E.
      • Tobin A.B.
      ), demonstrates that the ΔLys370–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 ΔLys370–Ser425 receptor mutant was still sensitive to PKC inhibition. Thus, the ΔLys370–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.
      Figure thumbnail gr7
      Figure 7Scheme 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 ΔLys370–Ser425 receptor mutant or the wild-type M3-muscarinic receptor expressed together with the 3i-loop peptide or F-CK1αK46R) will give a less than maximal ERK-1/2 response. CK1α, casein kinase 1α;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 (
      • Dikic I.
      • Tokiwa G.
      • Lev S.
      • Courtneidge S.A.
      • Schlessinger J.
      ). 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 (
      • Tapia J.A.
      • Ferris H.A.
      • Jensen R.T.
      • Garcia L.J.
      ,
      • Venkatakrishnam G.
      • Salgia R.
      • Groopman J.E.
      ,
      • Soltoff S.P.
      • Avraham H.
      • Avraham S.
      • Cantley L.C.
      ,
      • Eguchi S.
      • Numaguchi K.
      • Iwasaki H.
      • Matsumoto T.
      • Yamakawa T.
      • Utsunomiya H.
      • Motley E.D.
      • Kawakatsu H.
      • Owada K.M.
      • Hirata Y.
      • Marumo F.
      • Inagami T.
      ,
      • Zwick E.
      • Daub H.
      • Aoki N.
      • Yamaguchi-Aoki Y.
      • Tinhofer I.
      • Maly K.
      • Ullrich A.
      ,
      • Soltoff S.P.
      ). We can, however, eliminate the involvement of this process in the explanation of the results obtained with the ΔLys370–Ser425 receptor mutant for two reasons. First, the muscarinic receptor ERK-1/2 response in CHO cells is independent of changes in intracellular Ca2+ (
      • Wylie P.G.
      • Challiss R.A.J.
      • Blank J.L.
      ) 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+ (
      • Lev S.
      • Moreno H.
      • Martinez R.
      • Canoll P.
      • Peles E.
      • Musacchio J.M.
      • Plowman G.D.
      • Rudy B.
      • Schlessinger J.
      ). Because the ΔLys370–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 (
      • Tobin A.B.
      • Nahorski S.R.
      ). Extensive studies by our group have identified CK1α as a cellular kinase able to phosphorylate the M3-muscarinic receptor (also the M1-muscarinic receptor and rhodopsin) in an agonist-dependent manner (
      • Tobin A.B.
      • Totty N.F.
      • Sterlin A.E.
      • Nahorski S.R.
      ,
      • Budd D.C.
      • McDonald J.E.
      • Tobin A.B.
      ,
      • Tobin A.B.
      • Keys B.
      • Nahorski S.R.
      ,
      • Waugh M.G.
      • Challiss R.A.J.
      • Berstein G.
      • Nahorski S.R.
      • Tobin A.B.
      ). 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 ΔLys370–Ser425 receptor mutant, which lacked eight potential serine phospho-acceptor sites and the putative CK1α binding site (His374–Val391) (
      • Budd D.C.
      • McDonald J.E.
      • Tobin A.B.
      ). Consistent with our hypothesis, the ΔLys370–Ser425receptor mutant was reduced in its ability to undergo agonist-dependent phosphorylation by ∼80% (
      • Budd D.C.
      • McDonald J.E.
      • Tobin A.B.
      ).
      The reduced ability of the ΔLys370–Ser425receptor 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 CK1α-mediated M3-muscarinic receptor phosphorylation could be achieved using either a dominant negative mutant of CK1α, F-CK1α-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 CK1α (
      • Budd D.C.
      • McDonald J.E.
      • Tobin A.B.
      ). 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-CK1α-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 (
      • Budd D.C.
      • McDonald J.E.
      • Tobin A.B.
      ). 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 ΔLys370–Ser425receptor 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 β2-adrenergic receptor to the regulation of ERK-1/2 activity. PKA-mediated phosphorylation of the β2-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 βγ-subunits (
      • Daaka Y.
      • Luttrell L.M.
      • Lefkowitz R.J.
      ). Furthermore, agonist-mediated GRK-2 phosphorylation has been shown to recruit a β-arrestin·c-Src complex to the β2-adrenergic receptor (
      • Luttrell L.M.
      • Ferguson S.S.G.
      • Daaka Y.
      • Miller W.E.
      • Maudsley S.
      • Della Rocca G.J.
      • Lin F-T
      • Kawakatsu H.
      • Owada K.
      • Luttrell D.K.
      • Caron M.G.
      • Lefkowitz R.J.
      ). Preventing the ability of β-arrestin to interact with c-Src inhibits β2-adrenergic receptor-mediated ERK-1/2 activation, suggesting that recruitment of c-Src to the phosphorylated β2-adrenergic receptor via β-arrestin is essential in the mechanism of activation of ERK-1/2 (
      • Luttrell L.M.
      • Ferguson S.S.G.
      • Daaka Y.
      • Miller W.E.
      • Maudsley S.
      • Della Rocca G.J.
      • Lin F-T
      • Kawakatsu H.
      • Owada K.
      • Luttrell D.K.
      • Caron M.G.
      • Lefkowitz R.J.
      ). Hence, the data we present here indicates that the M3-muscarinic receptor, in common with the β2-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 CK1α 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 β-arrestin·c-Src and the β2-adrenergic receptor.

      Acknowledgments

      We thank Prof. Nahorski whom, together with Drs. A. B. Tobin and G. B. Willars, initiated the work on the ΔLys370–Ser425 receptor mutant.

      REFERENCES

        • Malarkey J.
        • Belham C.M.
        • Paul A.
        • Graham A.
        • Scott P.H.
        • Plevin R.
        Biochem. J. 1995; 309: 361-375
        • Wan Y.
        • Kurosaki T.
        • Huang X-Y.
        Nature. 1996; 380: 541-544
        • Hawes B.E.
        • van Biesen T.
        • Koch W.J.
        • Luttrell L.M.
        • Lefkowitz R.J.
        J. Biol. Chem. 1995; 270: 17148-17153
        • Gutkind J.S.
        J. Biol. Chem. 1998; 273: 1839-1842
        • Winitz S.
        • Russell M.
        • Qian N-X.
        • Gardner A.
        • Dwyers L.
        • Johnson G.L.
        J. Biol. Chem. 1993; 268: 19196-19199
        • Crespo P.
        • Xu N.
        • Simmonds W.F.
        • Gutkind J.S.
        Nature. 1994; 369: 418-420
        • Koch W.J.
        • Hawes B.E.
        • Allen L.F.
        • Lefkowitz R.J.
        Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12706-12710
        • Lopez-Ilasaca M.
        • Crespo P.
        • Pellici P.G.
        • Gutkind J.S.
        • Wetzker R.
        Science. 1997; 275: 394-397
        • Luttrell L.M.
        • Hawes B.E.
        • van Biesen T.
        • Luttrell D.K.
        • Lansing T.J.
        • Lefkowitz R.J.
        J. Biol. Chem. 1996; 271: 19443-19450
        • Della Rocca G.J.
        • van Biesen T.
        • Daaka Y.
        • Luttrell D.K.
        • Luttrell L.M.
        • Lefkowitz R.J.
        J. Biol. Chem. 1997; 272: 19125-19132
        • Della Rocca G.J.
        • Maudsley S.
        • Daaka Y.
        • Lefkowitz R.J.
        • Luttrell L.M.
        J. Biol. Chem. 1999; 274: 13978-13984
        • Faure M.
        • Voyno-Yasenetskaya T.A.
        • Bourne H.R.
        J. Biol. Chem. 1994; 269: 7851-7854
        • Palomero T.
        • Barros F.
        • Del Camino D.
        • Viloria C.G.
        • De La Pena P.
        Mol. Pharmacol. 1998; 53: 613-622
        • Launay J-M.
        • Birraux G.
        • Bondoux D.
        • Callebert J.
        • Choi D-S.
        • Loric S.
        • Maroteaux L.
        J. Biol. Chem. 1996; 271: 3141-3147
        • Watanabe T.
        • Waga I.
        • Honda Z-i.
        • Kurokawa K.
        • Shimizu T.
        J. Biol. Chem. 1995; 270: 8984-8990
        • Zou Y.
        • Komuro I.
        • Aikawa R.
        • Kudoh S.
        • Shiojima I.
        • Hiroi Y.
        • Mizuno T.
        • Yazaki Y.
        J. Biol. Chem. 1996; 271: 33592-33597
        • Velarde V.
        • Ullian M.E.
        • Morinelli T.A.
        • Mayfield R.K.
        • Jaffa A.A.
        Am. J. Physiol. 1999; 277: C253-C261
        • Tapia J.A.
        • Ferris H.A.
        • Jensen R.T.
        • Garcia L.J.
        J. Biol. Chem. 1999; 274: 31261-31271
        • Venkatakrishnam G.
        • Salgia R.
        • Groopman J.E.
        J. Biol. Chem. 2000; 275: 6868-6875
        • Soltoff S.P.
        • Avraham H.
        • Avraham S.
        • Cantley L.C.
        J. Biol. Chem. 1998; 273: 2653-2660
        • Budd D.C.
        • Rae A.
        • Tobin A.B.
        J. Biol. Chem. 1999; 274: 12355-12360
        • Wylie P.G.
        • Challiss R.A.J.
        • Blank J.L.
        Biochem. J. 1999; 338: 619-628
        • Kim J-Y.
        • Yang M-S.
        • Oh C-D.
        • Kim K-T.
        • Kang S-S.
        • Chun J-S.
        Biochem. J. 1999; 337: 275-280
        • Slack B.E.
        Biochem. J. 2000; 348: 381-387
        • Dikic I.
        • Tokiwa G.
        • Lev S.
        • Courtneidge S.A.
        • Schlessinger J.
        Nature. 1996; 383: 547-550
        • Lev S.
        • Moreno H.
        • Martinez R.
        • Canoll P.
        • Peles E.
        • Musacchio J.M.
        • Plowman G.D.
        • Rudy B.
        • Schlessinger J.
        Nature. 1995; 276: 737-745
        • Eguchi S.
        • Numaguchi K.
        • Iwasaki H.
        • Matsumoto T.
        • Yamakawa T.
        • Utsunomiya H.
        • Motley E.D.
        • Kawakatsu H.
        • Owada K.M.
        • Hirata Y.
        • Marumo F.
        • Inagami T.
        J. Biol. Chem. 1998; 273: 8890-8896
        • Zwick E.
        • Daub H.
        • Aoki N.
        • Yamaguchi-Aoki Y.
        • Tinhofer I.
        • Maly K.
        • Ullrich A.
        J. Biol. Chem. 1997; 272: 24767-24770
        • Soltoff S.P.
        J. Biol. Chem. 1998; 273: 23110-23117
        • Pitcher J.A.
        • Freedman N.J.
        • Lefkowitz R.J.
        Annu. Rev. Biochem. 1998; 67: 653-692
        • Daaka Y.
        • Luttrell L.M.
        • Lefkowitz R.J.
        Nature. 1997; 390: 88-91
        • Luttrell L.M.
        • Ferguson S.S.G.
        • Daaka Y.
        • Miller W.E.
        • Maudsley S.
        • Della Rocca G.J.
        • Lin F-T
        • Kawakatsu H.
        • Owada K.
        • Luttrell D.K.
        • Caron M.G.
        • Lefkowitz R.J.
        Science. 1999; 283: 655-660
        • Tobin A.B.
        • Nahorski S.R.
        J. Biol. Chem. 1993; 268: 9817-9823
        • Tobin A.B.
        • Totty N.F.
        • Sterlin A.E.
        • Nahorski S.R.
        J. Biol. Chem. 1997; 272: 20844-20849
        • Budd D.C.
        • McDonald J.E.
        • Tobin A.B.
        J. Biol. Chem. 2000; 275: 19667-19675
        • Tobin A.B.
        • Lambert D.G.
        • Nahorski S.R.
        Mol. Pharmacol. 1992; 42: 1042-1048
        • Challiss R.A.J.
        • Batty I.H.
        • Nahorski S.R.
        Biochem. Biophys. Res. Commun. 1988; 157: 684-691
        • Grynkiewicz G.
        • Poenie M.
        • Tsien R.Y.
        J. Biol. Chem. 1985; 260: 3440-3450
        • Xiong L.
        • Lee J.W.
        • Graves L.M.
        • Earp H.S.
        EMBO J. 1998; 17: 2574-2583
        • Cobb M.H.
        • Goldsmith E.J.
        J. Biol. Chem. 1995; 270: 14843-14846
        • Tobin A.B.
        • Keys B.
        • Nahorski S.R.
        J. Biol. Chem. 1996; 271: 3907-3916
        • Waugh M.G.
        • Challiss R.A.J.
        • Berstein G.
        • Nahorski S.R.
        • Tobin A.B.
        J. Biochem. 1999; 338: 175-183