Activation of the Mitogen-activated Protein Kinase Pathway by a Gq/11-coupled Muscarinic Receptor Is Independent of Receptor Internalization

A number of recent studies have demonstrated an essential role for receptor endocytosis in the activation of the mitogen-activated protein (MAP) kinases, Erk-1 and Erk-2 (extracellular activated protein kinases 1 and 2), by growth factor receptors and the G-protein coupled β2-adrenergic receptor. Because ligand-mediated receptor endocytosis and activation of the MAP kinase pathway are common phenomena among G-protein coupled receptors, it has been suggested that the essential role of endocytosis in MAP kinase activation identified for the β2-adrenergic receptor may be universal for all G-protein coupled receptors (Daaka,Y., Luttrell, L. M., Ahn, S., Della Rocca, G. J., Ferguson, S. S. G., Caron, M. G., and Lefkowitz, R. J. (1998) J. Biol. Chem. 273, 685–688). We tested this hypothesis using the Gq/11-coupled m3-muscarinic receptor expressed in Chinese hamster ovary cells and an m3-muscarinic receptor mutant that does not undergo endocytosis. We demonstrate that inhibition of endocytosis by concanavalin A and cytochalasin D does not affect the ability of the wild type m3-muscarinic receptor to activate Erk-1/2. Furthermore, the mutant m3-muscarinic receptor that is unable to undergo endocytosis, activates the MAP kinase pathway in an identical manner to the wild type receptor. We conclude that receptor endocytosis is not universally essential for MAP kinase activation by G-protein coupled receptors. We discuss the possibility that the differential roles played by endocytosis in MAP kinase activation between various receptor subtypes may be linked to the mechanism of upstream activation of Raf-1. A number of recent studies have demonstrated an essential role for receptor endocytosis in the activation of the mitogen-activated protein (MAP) kinases, Erk-1 and Erk-2 (extracellular activated protein kinases 1 and 2), by growth factor receptors and the G-protein coupled β2-adrenergic receptor. Because ligand-mediated receptor endocytosis and activation of the MAP kinase pathway are common phenomena among G-protein coupled receptors, it has been suggested that the essential role of endocytosis in MAP kinase activation identified for the β2-adrenergic receptor may be universal for all G-protein coupled receptors (Daaka,Y., Luttrell, L. M., Ahn, S., Della Rocca, G. J., Ferguson, S. S. G., Caron, M. G., and Lefkowitz, R. J. (1998) J. Biol. Chem. 273, 685–688). We tested this hypothesis using the Gq/11-coupled m3-muscarinic receptor expressed in Chinese hamster ovary cells and an m3-muscarinic receptor mutant that does not undergo endocytosis. We demonstrate that inhibition of endocytosis by concanavalin A and cytochalasin D does not affect the ability of the wild type m3-muscarinic receptor to activate Erk-1/2. Furthermore, the mutant m3-muscarinic receptor that is unable to undergo endocytosis, activates the MAP kinase pathway in an identical manner to the wild type receptor. We conclude that receptor endocytosis is not universally essential for MAP kinase activation by G-protein coupled receptors. We discuss the possibility that the differential roles played by endocytosis in MAP kinase activation between various receptor subtypes may be linked to the mechanism of upstream activation of Raf-1. The mitogen-activated protein (MAP) 1The abbreviations used are: MAP kinase, mitogen-activated protein kinase; CHO, Chinese hamster ovary; Erk, extracellular activated protein kinase; GPCR, G-protein coupled receptor; Ins(1, 4,5)P3, inositol (1,4,5)-trisphosphate; MEK, MAP kinase kinase; PKC, protein kinase C. PLC, phospholipase C; [3H]NMS, [3H]N-methylscopolaminekinases, Erk-1 and Erk-2, are phosphorylated and activated by a protein kinase cascade that can be initiated by both tyrosine kinase growth factor receptors and G-protein coupled receptors (GPCRs) (1Malarkey J. Belham C.M. Paul A. Graham A. Scott P.H. Plevin R. Biochem. J. 1995; 309: 361-375Crossref PubMed Scopus (266) Google Scholar, 2Gutkind S.J. J. Biol. Chem. 1998; 273: 1839-1842Abstract Full Text Full Text PDF PubMed Scopus (692) Google Scholar). Many of the same signal transduction proteins are used by both growth factor receptors and GPCRs in the activation of the MAP kinase pathway. The commonality between these two receptor types has recently been extended to include a role for ligand-mediated receptor endocytosis in the mechanism of MAP kinase activation (3Daaka Y. Luttrell L.M. Ahn S. Della Rocca G.J. Ferguson S.S.G. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1998; 273: 685-688Abstract Full Text Full Text PDF PubMed Scopus (465) Google Scholar, 4Lefkowitz R.J. J. Biol. Chem. 1998; 273: 18677-18680Abstract Full Text Full Text PDF PubMed Scopus (908) Google Scholar). Endocytosis of growth factor receptors, via clathrin-coated pits, is known to play an essential role in Erk-1/2 activation (5Vieira A.V. Lamaze C. Schmid S.L. Science. 1996; 274: 2086-2089Crossref PubMed Scopus (834) Google Scholar, 6Chow J.C. Condorelli G. Smith R.J. J. Biol. Chem. 1998; 273: 4672-4680Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). Recent studies on the β2-adrenergic receptor have also demonstrated that clathrin/dynamin-mediated receptor endocytosis may also be essential in the activation of the MAP kinase pathway by GPCRs (3Daaka Y. Luttrell L.M. Ahn S. Della Rocca G.J. Ferguson S.S.G. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1998; 273: 685-688Abstract Full Text Full Text PDF PubMed Scopus (465) Google Scholar, 4Lefkowitz R.J. J. Biol. Chem. 1998; 273: 18677-18680Abstract Full Text Full Text PDF PubMed Scopus (908) Google Scholar). These studies indicated that ligand-induced β2-adrenergic receptor endocytosis provided a link between the activated Raf-1 complex and MEK, the downstream kinase in the phosphorylation cascade (3Daaka Y. Luttrell L.M. Ahn S. Della Rocca G.J. Ferguson S.S.G. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1998; 273: 685-688Abstract Full Text Full Text PDF PubMed Scopus (465) Google Scholar, 4Lefkowitz R.J. J. Biol. Chem. 1998; 273: 18677-18680Abstract Full Text Full Text PDF PubMed Scopus (908) Google Scholar). Inhibition of endocytosis was sufficient to prevent Raf-1 activation of MEK and thereby block Erk-1/2 phosphorylation mediated by the β2-adrenergic receptor (3Daaka Y. Luttrell L.M. Ahn S. Della Rocca G.J. Ferguson S.S.G. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1998; 273: 685-688Abstract Full Text Full Text PDF PubMed Scopus (465) Google Scholar). Because many GPCRs undergo ligand-induced endocytosis via a clathrin/dynamin-mediated process (7Koening J.A. Edwardson M.J. Trends Pharmacol. Sci. 1997; 18: 276-287Abstract Full Text Full Text PDF PubMed Scopus (333) Google Scholar) and that GPCRs activate the MAP kinase pathway via Raf-1 (2Gutkind S.J. J. Biol. Chem. 1998; 273: 1839-1842Abstract Full Text Full Text PDF PubMed Scopus (692) Google Scholar), it has been suggested that GPCRs generally employ receptor-mediated endocytosis in the mechanism of activation of the MAP kinase pathway (4Lefkowitz R.J. J. Biol. Chem. 1998; 273: 18677-18680Abstract Full Text Full Text PDF PubMed Scopus (908) Google Scholar). This hypothesis, however, has not been extensively tested for receptor subtypes other than the β2-adrenergic receptor. Importantly, different receptor subtypes activate the MAP kinase pathway via distinct mechanisms. These mechanisms depend on the G-protein subtype to which the GPCR is coupled (8Crespo P. Xu N. Simmonds W.F. Gutkind J.S. Nature. 1994; 369: 418-420Crossref PubMed Scopus (766) Google Scholar, 9Koch W.J. Hawes B.E. Allen L.F. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12706-12710Crossref PubMed Scopus (409) Google Scholar, 10Luttrell L.M. Hawes B.E. van Biesen T. Luttrell D.K. Lansing T.J. Lefkowitz R.J. J. Biol. Chem. 1996; 271: 19443-19450Abstract Full Text Full Text PDF PubMed Scopus (494) Google Scholar, 11van Biesen T. Hawes B.E. Raymond J.R. Luttrell L.M. Koch W.J. Lefkowitz R.J. J. Biol. Chem. 1996; 271: 1266-1269Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar, 12Offermanns S. Bombien E. Schultz G. Biochem. J. 1993; 294: 545-550Crossref PubMed Scopus (44) Google Scholar, 13Hawes B.E. van Biesen T. Koch W.J. Luttrell L.M. Lefkowitz R.J. J. Biol. Chem. 1995; 270: 17148-17153Abstract Full Text Full Text PDF PubMed Scopus (413) Google Scholar). Whether the differential mechanisms by which GPCRs activate the MAP kinase pathway contribute to the role played by receptor endocytosis has not yet been examined. To address the question of the possibility that receptor endocytosis is universally essential in the activation of the MAP-kinase pathway by GPCRs, we investigate here the Gq/11-coupled m3-muscarinic receptor expressed in CHO cells. This receptor type shares a number of properties with the β2-adrenergic receptor in that the receptor is rapidly phosphorylated following agonist stimulation (14Tobin A.B. Nahorski S.R. J. Biol. Chem. 1993; 268: 9817-9823Abstract Full Text PDF PubMed Google Scholar) and that it undergoes endocytosis in a clathrin/dynamin-dependent process (15Lee K.B. Pals-Rylaarsdam R. Benovic J.L. Hosey M.M. J. Biol. Chem. 1998; 273: 12967-12972Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 16Vogler O. Bogatkewitsch G.S. Wriske C. Krummenerl P. Jakobs K.H. van Koppen C.J. J. Biol. Chem. 1998; 273: 12155-12160Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Using inhibitors of endocytosis and a mutant receptor deficient in the ability to undergo ligand-mediated endocytosis, we investigate the role receptor endocytosis plays in the activation of Erk-1/2 by the m3-muscarinic receptor. Further, we discuss the possibility that the upstream mechanism of Raf-1 activation may play a role in determining the involvement of receptor endocytosis in GPCR-mediated Erk-1/2 activation. Chinese hamster ovary cells (CHO) were maintained in α-minimal essential media (MEM) supplemented with fetal calf serum (10% v/v), fungizone (2.5 μg/ml), penicillin (100 IU/ml), streptomycin (100 μg/ml) at 37 °C in a humidified incubator containing 5% CO2. CHO-m3 cells expressing recombinant human m3-muscarinic receptors (1.55 ± 0.04 pmol/mg of protein), and CHO cells expressing the m3-muscarinic receptor mutant m3(A349AAA352) (4.72 ± 0.08 pmol/mg protein) were kind gifts from Dr. Wolfgang Sadee and Dr. Jelveh Lameh (School of Pharmacy, University of California, San Francisco, CA). The generation of the m3(A349AAA352) has been previously described (17Moro O. Lameh J. Sadee W. J. Biol. Chem. 1993; 268: 6862-6865Abstract Full Text PDF PubMed Google Scholar). In experiments where endogenous PKC was down-regulated by phorbol 12,13-dibutyrate, cells were exposed to 1 μm phorbol ester overnight prior to experimentation. Furthermore, in experiments where pertussis toxin was used, cells were incubated with 100 ng/ml pertussis toxin overnight before experimentation. Cells grown to confluence in 24-well plates were washed with 250 μl of Krebs-Henseleit buffer (KHB, 10 mmHEPES, pH 7.4, 118 mm NaCl, 4.3 mm KCl, 1.17 mm MgSO4·7H2O, 1.3 mmCaCl2·2H2O, 25 mmNaHCO3, 11.7 mm glucose). Cells were stimulated with 1 mm carbachol for the indicated times. Reactions were terminated by the addition of an equal volume of 1 mtrichloroacetic acid, and Ins(1,4,5)P3 was determined by a radioreceptor assay previously described (18Challiss R.A.J. Batty I.H. Nahorski S.R. Biochem. Biophys. Res. Commun. 1988; 157: 684-691Crossref PubMed Scopus (162) Google Scholar). Cells grown to confluence in 6-well plates were labeled with [32P]orthophosphate (50 μCi/ml) for 1 h. Cells were stimulated with 1 mmcarbachol for the times indicated, and the reactions were terminated by rapid aspiration of media and the addition of 1 ml of ice-cold RIPA buffer (10 mm Tris, 500 mm NaCl, 10 mm EDTA, 1% v/v Nonidet P-40, 0.1% v/v SDS, 0.5% w/v Na-deoxycholate). The mutant m3(A349AAA352) receptor expression levels were ∼3-fold greater than wild type receptors. Therefore, to ensure that equivalent numbers of receptors were immunoprecipitated, the proteins were adjusted so that ∼1 pmol of solubilized receptors were used for each immunoprecipitation. The receptors were immunoprecipitated from precleared lysates using 1 μg of anti-muscarinic m3 receptor antibody (14Tobin A.B. Nahorski S.R. J. Biol. Chem. 1993; 268: 9817-9823Abstract Full Text PDF PubMed Google Scholar). Immunocomplexes were collected on protein A-Sepharose beads, and the beads were washed three times with ice-cold TE buffer (10 mm Tris, pH 7.4, 2.5 mm EDTA). Immunocomplexes were resuspended in 2× SDS-polyacrylamide gel electrophoresis sample buffer and placed in a boiling water bath for 2 min. Proteins were resolved on an 8% SDS-polyacrylamide electrophoresis gel. The gels were stained with 0.2% Coomassie Blue to ensure that there was equal immunoprecipitation. The gels were then dried, and phosphorylated bands were visualized by autoradiography. Confluent CHO cells grown in 24-well plates were serum-starved for 1 h in KHB with or without endocytotic inhibitors and then stimulated with 1 mmcarbachol for the appropriate times. Reactions were terminated by aspiration, and the cells were washed three times with ice-cold KHB. Cells were incubated with ∼0.14 μCi of [3H]N-methylscopolamine (NMS) overnight at 4 °C. Cells were washed two times in ice-cold KHB and solubilized by the addition of 1 ml of ice-cold RIPA buffer, and receptor number was determined by liquid scintillation counting. Nonspecific binding was determined by the inclusion of 10 μm atropine. CHO cells grown to confluence in 6-well plates were serum-starved for 1 h in KHB and then stimulated with the appropriate agents. Stimulations were terminated by aspiration, and cells were incubated for 10 min in lysis buffer (20 mmTris, pH 7.6, 0.5% Nonidet P-40, 250 mm NaCl, 3 mm EDTA, 3 mm EGTA, 1 mmphenylmethylsulfonyl fluoride, 1 mmNa3VO4, 1 mm dithiothreitol, 5 μg/ml benzamidine) at 4 °C. Solubilized CHO cell lysates were pre-cleared by centrifuging at 20,000×g for 5 min. Endogenous MAP kinase was immunoprecipitated using 0.2 μg of anti-Erk-1/2 antiserum (Santa Cruz Biotechnology). Protein MAP kinase was washed two times in lysis buffer and two times in assay buffer (20 mm pH mm pH 10 1 mm dithiothreitol, were resuspended in assay buffer containing 2 μCi μm μm of and reactions were to for min at 37 °C. Reactions were terminated by the addition of and were washed times with and with and with the was determined by liquid scintillation counting. Confluent CHO cells in 6-well plates were serum-starved for 1 h in KHB and stimulated with 1 mm carbachol for the appropriate times. Reactions were terminated by rapidly the and the cells were in lysis buffer mm Tris, pH mm NaCl, 5 mm EDTA, 1 mm EGTA, 1% 1 mm fluoride, μg/ml 1 mm 10 Raf-1 was immunoprecipitated with μg of antiserum from Dr. C. of University of Immunocomplexes were washed two times with ice-cold and two times with ice-cold Raf-1 assay buffer mm pH 10 1 mm dithiothreitol, 1 were resuspended in Raf-1 assay buffer containing μm 2 μCi μg of Raf-1 were for the or of was where of CHO-m3 cells expressing the recombinant human m3-muscarinic receptor with the agonist carbachol 1 in a of cell receptors determined using the binding of the [3H]N-methylscopolamine receptor number determined using binding to a cell from stimulated or cells this not that the of receptor binding at the cell was of receptor endocytosis than receptor receptor endocytosis be following with concanavalin A and by by cytochalasin D of m3-muscarinic receptors in an in Erk-1/2 5 which to min Inhibition of m3-muscarinic receptor endocytosis using concanavalin A and cytochalasin D on m3-muscarinic receptor activation of Erk-1/2 studies have demonstrated that of a in the of the m3-muscarinic receptor in a receptor that was unable to undergo receptor endocytosis (17Moro O. Lameh J. Sadee W. J. Biol. Chem. 1993; 268: 6862-6865Abstract Full Text PDF PubMed Google Scholar). This was in the where expression of the m3(A349AAA352) mutant in CHO cells in a receptor that endocytosis following min of agonist 3 In wild type receptors were by ± following stimulation with carbachol 3 The m3(A349AAA352) receptor however, to the phospholipase pathway by a and of Ins(1,4,5)P3 of the a not in Ins(1,4,5)P3 for the m3(A349AAA352) receptor with wild type receptor Furthermore, the m3(A349AAA352) receptor also rapid phosphorylation 3 which was and was maintained for at min not that phosphorylation of the m3(A349AAA352) receptor was ∼3-fold greater than the wild type receptor. that in the in 3 the receptor number was at the of so that an equal number of wild type and m3(A349AAA352) mutant receptors were immunoprecipitated and the that the m3(A349AAA352) receptor not undergo endocytosis, the m3(A349AAA352) receptor stimulated Erk-1/2 activation with a that was to the wild type receptor The m3(A349AAA352) receptor however, a at 1 min and a at min with the wild type receptor. The between the wild type and m3(A349AAA352) receptor was not different of 4 The wild type and m3(A349AAA352) receptor activation of Erk-1/2 be by the not Furthermore, both the wild type and m3(A349AAA352) receptor Erk-1/2 were at in by of PKC using phorbol ester the wild type and m3(A349AAA352) receptor by and In PKC the Erk-1/2 to phorbol Inhibition of PKC using the wild type and m3(A349AAA352) receptor by and in the Erk-1/2 to phorbol not studies have demonstrated that Gq/11-coupled receptors expressed in CHO cells activated Erk-1/2 via a pertussis G-protein Biesen T. Hawes B.E. Raymond J.R. Luttrell L.M. Koch W.J. Lefkowitz R.J. J. Biol. Chem. 1996; 271: 1266-1269Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar, 13Hawes B.E. van Biesen T. Koch W.J. Luttrell L.M. Lefkowitz R.J. J. Biol. Chem. 1995; 270: 17148-17153Abstract Full Text Full Text PDF PubMed Scopus (413) Google Scholar). This was in the where both m3(A349AAA352) and wild type Erk-1/2 was by pertussis toxin The ability of the receptor to activate the upstream kinase in the MAP kinase was also receptor activation stimulated an in Raf-1 kinase of that at min by a at 10 min and for at min. The that the Gq/11-coupled m3-muscarinic receptor expressed in CHO cells activates the MAP-kinase pathway via a mechanism that is of receptor endocytosis. These with recent on studies on the β2-adrenergic that have suggested that receptor endocytosis is essential for GPCR activation of the MAP-kinase pathway (3Daaka Y. Luttrell L.M. Ahn S. Della Rocca G.J. Ferguson S.S.G. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1998; 273: 685-688Abstract Full Text Full Text PDF PubMed Scopus (465) Google Scholar, 4Lefkowitz R.J. J. Biol. Chem. 1998; 273: 18677-18680Abstract Full Text Full Text PDF PubMed Scopus (908) Google Scholar). is by the that inhibition of m3-muscarinic receptor endocytosis by cytochalasin D and concanavalin A not affect the ability of the receptor to activate Erk-1/2. Furthermore, a receptor mutant that not undergo endocytosis in to agonist was to Erk-1/2 Importantly, with the of receptor endocytosis, the m3(A349AAA352) mutant with many of the with the wild type m3-muscarinic receptor in that it was to to the and undergo The for β2-adrenergic receptor-mediated activation of the MAP-kinase pathway recently by Lefkowitz and is to a for GPCR stimulation of Erk-1/2 (3Daaka Y. Luttrell L.M. Ahn S. Della Rocca G.J. Ferguson S.S.G. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1998; 273: 685-688Abstract Full Text Full Text PDF PubMed Scopus (465) Google Scholar, 4Lefkowitz R.J. J. Biol. Chem. 1998; 273: 18677-18680Abstract Full Text Full Text PDF PubMed Scopus (908) Google Scholar). receptor stimulation in receptor phosphorylation by kinases and protein kinase A Caron M.G. Lefkowitz R.J. PubMed Scopus Google Scholar). previously linked with receptor β2-adrenergic receptor phosphorylation has recently been to play a role in endocytosis and activation of the MAP-kinase pathway. at for the of which an protein the receptor to and the pathway Benovic J.L. Nature. 1996; PubMed Scopus Google Scholar, S.S.G. L. Caron M.G. Science. 1996; 271: PubMed Scopus Google Scholar). Protein kinase A phosphorylation a for the β2-adrenergic receptor to via to the MAP-kinase pathway Y. Luttrell L.M. Lefkowitz R.J. Nature. 1997; PubMed Scopus Google Scholar) in a manner to receptors of and inhibitors of endocytosis, both β2-adrenergic receptor endocytosis and Erk-1/2 activation (3Daaka Y. Luttrell L.M. Ahn S. Della Rocca G.J. Ferguson S.S.G. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1998; 273: 685-688Abstract Full Text Full Text PDF PubMed Scopus (465) Google Scholar). The at which receptor endocytosis with the MAP-kinase pathway is at the activation of by Raf-1 (3Daaka Y. Luttrell L.M. Ahn S. Della Rocca G.J. Ferguson S.S.G. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1998; 273: 685-688Abstract Full Text Full Text PDF PubMed Scopus (465) Google Scholar). has been suggested that the activated Raf-1 complex with the to phosphorylation of (3Daaka Y. Luttrell L.M. Ahn S. Della Rocca G.J. Ferguson S.S.G. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1998; 273: 685-688Abstract Full Text Full Text PDF PubMed Scopus (465) Google Scholar, 4Lefkowitz R.J. J. Biol. Chem. 1998; 273: 18677-18680Abstract Full Text Full Text PDF PubMed Scopus (908) Google Scholar). Because receptor endocytosis is a common among GPCRs (7Koening J.A. Edwardson M.J. Trends Pharmacol. Sci. 1997; 18: 276-287Abstract Full Text Full Text PDF PubMed Scopus (333) Google Scholar) and that GPCR activation of the MAP-kinase pathway Raf-1 (2Gutkind S.J. J. Biol. Chem. 1998; 273: 1839-1842Abstract Full Text Full Text PDF PubMed Scopus (692) Google Scholar), it has been suggested that the role for receptor endocytosis in Erk-1/2 activation described for the β2-adrenergic receptor may generally be to all GPCRs (4Lefkowitz R.J. J. Biol. Chem. 1998; 273: 18677-18680Abstract Full Text Full Text PDF PubMed Scopus (908) Google Scholar). however, the here demonstrate that activation of the MAP-kinase pathway by m3-muscarinic receptors does not receptor endocytosis. receptors coupled via to the pathway have previously been to undergo endocytosis via a pathway (15Lee K.B. Pals-Rylaarsdam R. Benovic J.L. Hosey M.M. J. Biol. Chem. 1998; 273: 12967-12972Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 16Vogler O. Bogatkewitsch G.S. Wriske C. Krummenerl P. Jakobs K.H. van Koppen C.J. J. Biol. Chem. 1998; 273: 12155-12160Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, L.M. Lameh J. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). with these are here that demonstrate the ability of concanavalin A and cytochalasin known to endocytosis S. S. M.J. Pharmacol. 1995; Google Scholar), to m3-muscarinic receptor Furthermore, we have previously that the m3-muscarinic receptor is rapidly phosphorylated following agonist stimulation (14Tobin A.B. Nahorski S.R. J. Biol. Chem. 1993; 268: 9817-9823Abstract Full Text PDF PubMed Google Scholar), by A.B. Nahorski S.R. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). The role phosphorylation plays in m3-muscarinic receptor endocytosis is is to however, that in the we that the process of receptor phosphorylation is not sufficient to ensure endocytosis the m3(A349AAA352) receptor mutant this receptor is unable to In the m3(A349AAA352) receptor was phosphorylated to a ∼3-fold greater than the wild type receptor. The of the m3(A349AAA352) receptor may be by from studies that a role for receptor endocytosis in of GPCRs J. Caron M.G. Ferguson S.S.G. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). endocytosis is for receptor then a mutant receptor unable to undergo endocytosis may in the phosphorylated Whether this is the for the of the m3(A349AAA352) receptor is The m3-muscarinic receptor expressed in CHO-m3 shares in common with the β2-adrenergic receptor that is known to be in β2-adrenergic receptor-mediated MAP-kinase activation. the receptor undergoes rapid phosphorylation following agonist stimulation and is via a clathrin/dynamin-dependent pathway. these the receptors not a common role for receptor endocytosis in MAP-kinase activation. The m3-muscarinic and β2-adrenergic receptors mechanisms of activation of the MAP-kinase pathway at the of the upstream activation of Raf-1. In cell Gq/11-coupled receptors receptors, have been to activate Erk-1/2 in a pertussis via PKC, in a process that is of on Raf-1 Biesen T. Hawes B.E. Raymond J.R. Luttrell L.M. Koch W.J. Lefkowitz R.J. J. Biol. Chem. 1996; 271: 1266-1269Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar, 12Offermanns S. Bombien E. Schultz G. Biochem. J. 1993; 294: 545-550Crossref PubMed Scopus (44) Google Scholar, 13Hawes B.E. van Biesen T. Koch W.J. Luttrell L.M. Lefkowitz R.J. J. Biol. Chem. 1995; 270: 17148-17153Abstract Full Text Full Text PDF PubMed Scopus (413) Google Scholar). The here that the m3-muscarinic Erk-1/2 is by PKC inhibition and is by pertussis that a mechanism is in for the m3-muscarinic receptor expressed in CHO-m3 cells. Furthermore, the ability of the m3-muscarinic receptors to activate Raf-1 in this that the process of Erk-1/2 activation by m3-muscarinic receptors in CHO-m3 cells is mediated via Raf-1 activation of This is by the that the MEK m3-muscarinic receptor-mediated Erk-1/2 activation. In with the Gq/11-coupled receptor the mechanism of Raf-1 activation by β2-adrenergic receptors from pertussis tyrosine kinases that are to proteins in the of and the activation of S.S.G. L. Caron M.G. Science. 1996; 271: PubMed Scopus Google Scholar). The common in the activation of Erk-1/2 by GPCR and growth factor receptors, is that all the activation of Raf-1 (2Gutkind S.J. J. Biol. Chem. 1998; 273: 1839-1842Abstract Full Text Full Text PDF PubMed Scopus (692) Google Scholar). The in the mechanisms in the upstream activation of Raf-1. The tested in this is receptor endocytosis is essential for Raf-1 activation of MEK, which to be the for growth factor receptors (5Vieira A.V. Lamaze C. Schmid S.L. Science. 1996; 274: 2086-2089Crossref PubMed Scopus (834) Google Scholar, 6Chow J.C. Condorelli G. Smith R.J. J. Biol. Chem. 1998; 273: 4672-4680Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar) and the β2-adrenergic receptor (3Daaka Y. Luttrell L.M. Ahn S. Della Rocca G.J. Ferguson S.S.G. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1998; 273: 685-688Abstract Full Text Full Text PDF PubMed Scopus (465) Google Scholar), then it be that this be for GPCRs generally undergo endocytosis and all Erk-1/2 via Raf-1. however, not this hypothesis and demonstrate that receptor endocytosis is not universally essential in the mechanism of GPCR activation of the MAP kinase pathway. The for the in the role played by endocytosis between the various GPCR subtypes may at the differential upstream mechanisms in the activation of via PKC or We Dr. and Paul for and with the MAP kinase