Phosphorylation and Regulation of a Gq/11-coupled Receptor by Casein Kinase 1α

Agonist-mediated receptor phosphorylation by one or more of the members of the G-protein receptor kinase (GRK) family is an established model for G-protein-coupled receptor (GPCR) phosphorylation resulting in receptor desensitization. Our recent studies have, however, suggested that an alternative route to GPCR phosphorylation may be an operation involving casein kinase 1α (CK1α). In the current study we investigate the involvement of CK1α in the phosphorylation of the human m3-muscarinic receptor in intact cells. We show that expression of a catalytically inactive mutant of CK1α, designed to act in a dominant negative manner, inhibits agonist-mediated receptor phosphorylation by ∼40% in COS-7 and HEK-293 cells. Furthermore, we present evidence that a peptide corresponding to the third intracellular loop of the m3-muscarinic receptor (Ser345-Leu463) is an inhibitor of CK1α due to its ability to both act as a pseudo-substrate for CK1α and form a high affinity complex with CK1α. Expression of this peptide was able to reduce both basal and agonist-mediated m3-muscarinic receptor phosphorylation in intact cells. These results support the notion that CK1α is able to mediate GPCR phosphorylation in an agonist-dependent manner and that this may provide a novel mechanism for GPCR phosphorylation. The functional role of phosphorylation was investigated using a mutant of the m3-muscarinic receptor that showed an ∼80% reduction in agonist-mediated phosphorylation. Surprisingly, this mutant underwent agonist-mediated desensitization suggesting that, unlike many GPCRs, desensitization of the m3-muscarinic receptor is not mediated by receptor phosphorylation. The inositol (1,4,5)-trisphosphate response did, however, appear to be dramatically potentiated in the phosphorylation-deficient mutant indicating that phosphorylation may instead control the magnitude of the initial inositol phosphate response. Agonist-mediated receptor phosphorylation by one or more of the members of the G-protein receptor kinase (GRK) family is an established model for G-protein-coupled receptor (GPCR) phosphorylation resulting in receptor desensitization. Our recent studies have, however, suggested that an alternative route to GPCR phosphorylation may be an operation involving casein kinase 1α (CK1α). In the current study we investigate the involvement of CK1α in the phosphorylation of the human m3-muscarinic receptor in intact cells. We show that expression of a catalytically inactive mutant of CK1α, designed to act in a dominant negative manner, inhibits agonist-mediated receptor phosphorylation by ∼40% in COS-7 and HEK-293 cells. Furthermore, we present evidence that a peptide corresponding to the third intracellular loop of the m3-muscarinic receptor (Ser345-Leu463) is an inhibitor of CK1α due to its ability to both act as a pseudo-substrate for CK1α and form a high affinity complex with CK1α. Expression of this peptide was able to reduce both basal and agonist-mediated m3-muscarinic receptor phosphorylation in intact cells. These results support the notion that CK1α is able to mediate GPCR phosphorylation in an agonist-dependent manner and that this may provide a novel mechanism for GPCR phosphorylation. The functional role of phosphorylation was investigated using a mutant of the m3-muscarinic receptor that showed an ∼80% reduction in agonist-mediated phosphorylation. Surprisingly, this mutant underwent agonist-mediated desensitization suggesting that, unlike many GPCRs, desensitization of the m3-muscarinic receptor is not mediated by receptor phosphorylation. The inositol (1,4,5)-trisphosphate response did, however, appear to be dramatically potentiated in the phosphorylation-deficient mutant indicating that phosphorylation may instead control the magnitude of the initial inositol phosphate response. G-protein-coupled receptor casein kinase 1α G-protein-coupled receptor kinase 4,5)P3, inositol (1,4,5) trisphosphate Chinese hamster ovary polyacrylamide gel electrophoresis glutathioneS-transferase It is now well established that G-protein-coupled receptor (GPCR)1 phosphorylation is a general phenomenon that controls specific key signaling properties of receptors. Originally associated with receptor desensitization (1.Tobin A.B. Pharmacol. Ther. 1997; 75: 135-151Crossref PubMed Scopus (47) Google Scholar, 2.Pitcher J.A. Freedman N.J. Lefkowitz R.J. Annu. Rev. Biochem. 1998; 67: 653-692Crossref PubMed Scopus (1070) Google Scholar), GPCR phosphorylation has now been implicated in a number of processes including receptor internalization (3.Tsuga H. Kameyama K. Haga T. Kurise H. Nagao T. J. Biol. Chem. 1994; 269: 32522-32527Abstract Full Text PDF PubMed Google Scholar, 4.Ruiz-Gomez A. Mayor Jr., F. J. Biol. Chem. 1997; 272: 9601-9604Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 5.Tsuga H. Okuno E. Kameyama K. Haga T. Mol. Pharmacol. 1998; 284: 1218-1226Google Scholar, 6.Tsuga H. Kameyama K. Haga T. Honma T. Lameh J. Sadee W. J. Biol. Chem. 1998; 273: 5323-5330Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar) and as a molecular switch that determines coupling to specific signaling pathways (7.Daaka Y. Luttrell L.M. Lefkowitz R.J. Nature. 1997; 390: 88-91Crossref PubMed Scopus (1073) Google Scholar, 8.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: 655-660Crossref PubMed Scopus (1264) Google Scholar). The receptor-specific kinases involved are generally considered to belong to the G-protein-coupled receptor kinase (GRK) family which are characterized by their sequence homology to rhodopsin kinase (GRK-1) and that include the extensively studied β-adrenergic receptor kinases 1 and 2 (GRK-2 and -3, respectively) (2.Pitcher J.A. Freedman N.J. Lefkowitz R.J. Annu. Rev. Biochem. 1998; 67: 653-692Crossref PubMed Scopus (1070) Google Scholar, 9.Lefkowitz R.J. Cell. 1993; 74: 409-412Abstract Full Text PDF PubMed Scopus (403) Google Scholar). Reconstitution experiments using purified, or partially purified, receptors have demonstrated that in addition to the β2-adrenergic receptor a number of GPCRs including muscarinic ((10–12), substance P (13.Kwatra M.M. Schwinn D.A. Schreurs J. Blank J.L. Kim C.M. Benovic J.L. Krausse J.E. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1993; 268: 9161-9164Abstract Full Text PDF PubMed Google Scholar), bradykinin B2 (14.Blaukat A. Alla S.A. Lohse M.J. Muller-Esterl W. J. Biol. Chem. 1996; 271: 32366-32374Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar), and adenosine A3receptors (15.Palmer T.M. Benovic J.L. Stiles G.L. J. Biol. Chem. 1995; 270: 29607-29613Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar) can act as GRK substrates. Furthermore, a GRK-2 dominant negative mutant (16.Kong G. Penn R. Benovic J.L. J. Biol. Chem. 1994; 269: 13084-13087Abstract Full Text PDF PubMed Google Scholar) has been widely employed to probe the role of endogenous GRK-2 in the regulation of GPCRs (3.Tsuga H. Kameyama K. Haga T. Kurise H. Nagao T. J. Biol. Chem. 1994; 269: 32522-32527Abstract Full Text PDF PubMed Google Scholar, 17.Diviani D. Lattion A.-L. Kunapuli P. Pronin A. Benovic J.L. Cotecchia S. J. Biol. Chem. 1996; 271: 5049-5058Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar, 18.Oppermann M. Freedman N.J. Alexander W.R. Lefkowitz R.J. J. Biol. Chem. 1996; 271: 13266-13272Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar, 19.Freedman N.J. Ament A.S. Oppermann M. Stoffel R.H. Exum S.T. Lefkowitz R.J. J. Biol. Chem. 1997; 272: 17734-17743Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). These studies, and others, have led to the proposal that the GRKs, and in particular GRK-2, have a broad receptor substrate specificity and are able to phosphorylate and regulate GPCRs coupled to both adenylyl cyclase via Gs/i and those coupled to the phospholipase C pathway via Gq/11. In contrast to this model of GRK-mediated phosphorylation of GPCRs, our studies on the Gq/11-coupled m3-muscarinic receptor have suggested that there may be an alternative mechanism mediating agonist-dependent receptor phosphorylation. This receptor is rapidly phosphorylated on serine following agonist addition (20.Tobin A.B. Nahorski S.R. J. Biol. Chem. 1993; 268: 9817-9823Abstract Full Text PDF PubMed Google Scholar) with a time course that closely correlates with receptor desensitization as measured by diminished inositol (1,4,5)-trisphosphate (Ins(1,4,5)P3) and intracellular calcium responses (21.Tobin A.B. Lambert D.G. Nahorski S.R. Mol. Pharmacol. 1992; 42: Google Scholar). of the kinase involved in this phosphorylation a role for kinase kinase and kinase (20.Tobin A.B. Nahorski S.R. J. Biol. Chem. 1993; 268: 9817-9823Abstract Full Text PDF PubMed Google Scholar). m3-muscarinic receptor to receptor kinase and that this was to by and that to GRK-2 A.B. Nahorski S.R. 1993; PubMed Scopus Google Scholar). These the suggesting that the m3-muscarinic receptor was phosphorylated by a kinase that was using a of the third intracellular loop of the m3-muscarinic receptor as a pseudo-substrate for the muscarinic receptor we able to a kinase that in experiments was able to phosphorylate the m3-muscarinic receptor in an agonist-dependent manner A.B. Nahorski S.R. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). sequence this kinase as casein kinase 1α A.B. Nahorski S.R. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). the ability of CK1α to receptor phosphorylation was not to the m3-muscarinic receptor both rhodopsin and the receptor to be in that phosphorylated in a manner A.B. Nahorski S.R. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar, M.G. G. Nahorski S.R. A.B. J. Biochem. 1999; Google Scholar). These in studies suggested that CK1α may act as a kinase for specific GPCRs, an alternative and route to GPCR phosphorylation that of the In the present study we this by using a catalytically inactive mutant of CK1α and a peptide corresponding to the third intracellular loop of the m3-muscarinic receptor to endogenous CK1α These experiments provide evidence for a role of CK1α in the phosphorylation and regulation of the m3-muscarinic and in of with and in a casein kinase 1α that been the with the and A.B. Nahorski S.R. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar) was as a for the The was This a in the sequence of CK1α. The resulting was in and a on the The was by The sequence the third intracellular loop of the m3-muscarinic receptor was using the and and and in The resulting was in of HEK-293 or COS-7 or to The m3-muscarinic receptor sequence in was with and This the sequence for the of the of the in this study have been A.B. Nahorski S.R. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). and of the m3-muscarinic receptor specific in the third intracellular loop of the m3-muscarinic receptor has been (20.Tobin A.B. Nahorski S.R. J. Biol. Chem. 1993; 268: 9817-9823Abstract Full Text PDF PubMed Google Scholar). The was a peptide corresponding to of the This sequence is in the muscarinic receptor and the resulting be to with of the muscarinic receptor The peptide was to and using of the using showed that the with the and muscarinic receptors. This was in the phosphorylation of the mutant was The was a peptide corresponding to the of CK1α of the using showed that was able to with CK1α and endogenous CK1α present in COS-7 and the of with m3-muscarinic receptor in or with m3-muscarinic receptor or loop peptide The was using a of In experiments to inositol (1,4,5)-trisphosphate HEK-293 and well was with of with the loop peptide using the the peptide in and for expression by using the m3-muscarinic receptor that was this peptide (20.Tobin A.B. Nahorski S.R. J. Biol. Chem. 1993; 268: 9817-9823Abstract Full Text PDF PubMed Google Scholar). The resulting in experiments the m3-muscarinic receptor was in and in with for or the was and for a by of the and of 1 of on for and by was and the on for on and the with In the of receptor the was in 1 of and an of the was corresponding to a of receptors as by This that for the number of receptors was on the on in the of muscarinic receptors or for the loop The and to and the of phosphorylation was with a model receptor expression for was by with of a of muscarinic receptor for with and was by of in was in the of and was of the in with and with agonist for the by addition of and to an the was and by addition of and as (21.Tobin A.B. Lambert D.G. Nahorski S.R. Mol. Pharmacol. 1992; 42: Google Scholar). to by addition of and using a Nahorski S.R. Biochem. PubMed Scopus Google Scholar). The and one was in of by a in a was by for The was and in the receptor with for 1 on and in in and by The of CK1α was by using the casein kinase HEK-293 with or with 1 of 2 1 1 The was for and the the was for 1 by for The on and with and with kinase 1 The complex was in a kinase by the in kinase of The to for and by the addition of on a gel which with to and an was investigate the kinase for m3-muscarinic receptor phosphorylation was CK1α, we the ability of a catalytically inactive form of CK1α to agonist-mediated m3-muscarinic receptor phosphorylation. in CK1α to the the of kinases K. T.M. S. A. 1993; PubMed Scopus (163) Google Scholar). we a to that be to in a catalytically inactive Expression of was in HEK-293 by for the that was the to the the mutant a molecular the endogenous CK1α. by with a CK1α that both endogenous CK1α and mutant we that and endogenous CK1α 1 results for in COS-7 not In to HEK-293 with or both of which the with the In kinases on that the kinase 1 m3-muscarinic receptors HEK-293 or COS-7 phosphorylated in an agonist-dependent manner by endogenous of the m3-muscarinic receptor with in a in agonist-mediated receptor phosphorylation by and in HEK-293 and COS-7 In experiments with a of agonist for that we have results in phosphorylation of the receptor (20.Tobin A.B. Nahorski S.R. J. Biol. Chem. 1993; 268: 9817-9823Abstract Full Text PDF PubMed Google Scholar). In receptor expression was and the of receptor to the gel was that receptor on the of the with the receptor not the of m3-muscarinic receptor we in the of receptor expression control and due to in studies our demonstrated that a a of the third intracellular loop of the m3-muscarinic receptor was able to muscarinic receptor phosphorylation in A.B. Nahorski S.R. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google we the ability of this peptide to m3-muscarinic receptor phosphorylation in intact cells. Expression of the of corresponding to loop of the m3-muscarinic receptor was by using an m3-muscarinic receptor that was this peptide (20.Tobin A.B. Nahorski S.R. J. Biol. Chem. 1993; 268: 9817-9823Abstract Full Text PDF PubMed Google Scholar) of m3-muscarinic receptors with the loop peptide in COS-7 in a in agonist-mediated m3-muscarinic receptor phosphorylation by the basal phosphorylation in COS-7 was by the loop peptide In HEK-293 m3-muscarinic receptor phosphorylation was by expression of the loop in this by of the loop peptide basal and agonist-mediated phosphorylation of the m3-muscarinic the m3-muscarinic receptor or with the loop peptide with and with for m3-muscarinic receptor phosphorylation was by using an m3-muscarinic receptor number of muscarinic receptors was to an and an was a gel an using COS-7 cells. In this m3-muscarinic receptor a gel an using HEK-293 cells. In this m3-muscarinic receptor a gel an or that the loop peptide with the m3-muscarinic In the m3-muscarinic receptor The are of The of molecular are in We a that the loop peptide This was with the m3-muscarinic receptor and receptor phosphorylation with with the In experiments expression of the loop peptide agonist-mediated phosphorylation of the m3-muscarinic receptor by Furthermore, basal phosphorylation was in the of the loop peptide The loop peptide in was this phosphorylation was not by m3-muscarinic receptor The results in COS-7 and HEK-293 not CK1α in a associated with the muscarinic receptor of a that the third intracellular loop sequence This in to a of 2 was not to of CK1α not of to the of CK1α the and of not the ability of the to with CK1α present in or CK1α partially not of the in of CK1α of in a that was to with CK1α the of the in the is to be CK1α its molecular and CK1α that is associated with The in is to be CK1α and an that with the CK1α of the of CK1α and the third intracellular loop of the m3-muscarinic of the muscarinic receptor of the in the of the serine of the in the of CK1α experiments with of The are of The of molecular are in to CK1α, Our studies have demonstrated that phosphorylation of the is that of the A.B. Nahorski S.R. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). It to the in the intact m3-muscarinic receptor with the to a receptor to agonist-mediated phosphorylation. a was a m3-muscarinic receptor a in the third intracellular The was to have a receptor expression as the control the m3-muscarinic receptor mutant expression receptor expression of this not agonist or properties of the for and for the mutant and m3-muscarinic for agonist to in the of and for the mutant and m3-muscarinic the m3-muscarinic receptor-specific this study was the was that the mutant may have that by this this an alternative was a peptide the muscarinic receptor family in the intracellular loop of the This both and m3-muscarinic receptors as by not and was using the in studies, was that the of agonist-mediated phosphorylation of the receptor the was dramatically with receptor that the mutant with the muscarinic receptor that The did, however, with the m3-muscarinic receptor as by and in control The of are not These experiments on the m3-muscarinic receptor or the of the m3-muscarinic receptor response to agonist showed a of by a with (21.Tobin A.B. Lambert D.G. Nahorski S.R. Mol. Pharmacol. 1992; 42: Google Scholar, 1994; PubMed Scopus Google Scholar). with studies was the that the response be by following a with agonist (21.Tobin A.B. Lambert D.G. Nahorski S.R. Mol. Pharmacol. 1992; 42: Google Scholar, 1994; PubMed Scopus Google Scholar). The of the response on agonist of the mutant was to the receptor the that agonist-mediated phosphorylation of the mutant was dramatically this receptor was by following a agonist however, a in the magnitude of the responses the mutant and m3-muscarinic receptor The control receptor response was of with of in the of the This in on of the mutant receptor was to the response the responses for the mutant and receptors the of the mutant in of the magnitude of the there was in the of the the receptor and the mutant for and mutant receptors and The ability of the mutant to show of inositol phosphate was in HEK-293 cells. experiments are the of the In experiments the mutant response was the receptor response not The that the of the mutant was not as as that in the may be due to the and however, the is the the mutant to a response the responses to agonist was in HEK-293 with the m3-muscarinic receptor or with a with agonist and a the response to of the m3-muscarinic receptor was by In with the m3-muscarinic receptor and the catalytically inactive kinase a that agonist-mediated receptor phosphorylation the response was following agonist the response in with the m3-muscarinic receptor and the mutant was the response of with the m3-muscarinic receptor It that of resulting in receptor phosphorylation has on the ability of the inositol phosphate response to be in an the magnitude of the response. This is with the the the which showed receptor an inositol phosphate response with receptors The present study in evidence that CK1α is in for agonist-mediated receptor phosphorylation of the m3-muscarinic receptor in Furthermore, phosphorylation not to mediate receptor desensitization instead may control the magnitude of the response. This a the widely model for GPCR phosphorylation and desensitization mediated by the in studies our have established that CK1α is able to mediate phosphorylation of a number of GPCRs A.B. Nahorski S.R. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar, M.G. G. Nahorski S.R. A.B. J. Biochem. 1999; Google Scholar) suggesting that CK1α may a the GRKs, for receptor phosphorylation in intact cells. In the present study we have of the that, many GPCRs, the m3-muscarinic receptor is phosphorylated in an agonist-dependent manner by endogenous receptor kinases in a number of and HEK-293 investigate CK1α was one of endogenous receptor we the ability of a catalytically inactive mutant of CK1α to m3-muscarinic receptor phosphorylation. a to an in of the of CK1α, to be for K. T.M. S. A. 1993; PubMed Scopus (163) Google Scholar), the of the kinase was in in particular GRK-2, have been to dominant negative (16.Kong G. Penn R. Benovic J.L. J. Biol. Chem. 1994; 269: 13084-13087Abstract Full Text PDF PubMed Google Scholar). Furthermore, of this in has been to in a dominant negative mutant able to endogenous phosphorylation of in Nature. 1999; PubMed Scopus Google Scholar). We that endogenous CK1α was for m3-muscarinic receptor phosphorylation expression of the mutant receptor phosphorylation by in a dominant negative We show that in to the endogenous CK1α in a reduction in the of agonist-mediated m3-muscarinic receptor suggesting that a of the receptor phosphorylation was mediated by endogenous CK1α. We to this by an alternative inhibitor to CK1α. studies have that a peptide corresponding to the third intracellular loop of the m3-muscarinic receptor in a was able to m3-muscarinic receptor phosphorylation in A.B. Nahorski S.R. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). This was to the ability of the to act as a pseudo-substrate for CK1α and with the receptor for endogenous CK1α present in the in the present study we that this peptide a high affinity complex with CK1α, and this may to its In to investigate the involvement of CK1α in the phosphorylation of m3-muscarinic receptors in intact we the ability of the third intracellular loop peptide loop to receptor phosphorylation in Expression of the loop peptide in a reduction in both basal and agonist-mediated m3-muscarinic receptor phosphorylation. The peptide phosphorylated suggesting that, as in the the peptide was as a pseudo-substrate for CK1α. is that the loop peptide may be receptor phosphorylation by with kinases CK1α, the is with the and that m3-muscarinic receptor phosphorylation in intact is in by CK1α. the m3-muscarinic receptor is not the GPCR to be phosphorylated by casein kinase in intact cells. In a recent phosphorylation of the receptor of was to be mediated by casein kinase and this was associated with control of receptor and H. J. Biol. 1998; PubMed Scopus Google Scholar). The that agonist-mediated phosphorylation of the m3-muscarinic receptor in the present study was not by or the loop peptide that the are not in high or that phosphorylation is mediated by kinases in addition to CK1α. In the of the GRK-2 dominant negative in have demonstrated that a of the dominant negative mutant was to β2-adrenergic receptor phosphorylation by the of G-protein (16.Kong G. Penn R. Benovic J.L. J. Biol. Chem. 1994; 269: 13084-13087Abstract Full Text PDF PubMed Google Scholar). In the present study we able to a of expression that was to endogenous CK1α. This of expression may have been to the endogenous kinase and is that CK1α is one of a number of kinases for agonist-mediated phosphorylation. for evidence for the involvement of the in muscarinic receptor phosphorylation. The adenylyl receptor was one of the receptors to be to be an in substrate for the K. Haga T. J. Biol. Chem. 1992; Full Text PDF PubMed Google Scholar). on the third intracellular loop is to mediate receptor internalization H. Kameyama K. Haga T. Honma T. Lameh J. Sadee W. J. Biol. Chem. 1998; 273: 5323-5330Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, H. Kameyama K. Haga K. Haga T. J. Biochem. 1994; PubMed Scopus Google Scholar). m3-muscarinic receptors in have been to be phosphorylated by GRK-2 and not and Kunapuli P. Benovic J.L. M. Mol. Pharmacol. 1994; Scholar). peptide corresponding to the third intracellular loop of the m3-muscarinic receptor has been to be a substrate for GRK-2 in in studies G. Benovic J.L. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). Furthermore, the receptor is phosphorylated in an agonist-dependent manner following with GRK-2 K. Kameyama K. Haga T. K. H. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). These in studies that have the to phosphorylate members of the muscarinic receptor family coupled via both and evidence that this is the in has to be in with studies our the that the m3-muscarinic receptor may be phosphorylated by CK1α and one or more of the studies in our the phosphorylation on the m3-muscarinic receptor may that be to CK1α and the involvement of receptor The functional of receptor phosphorylation investigated in a receptor the in the third intracellular loop been with the receptor this mutant showed an ∼80% in agonist-mediated receptor phosphorylation. The the in this study to in the the reduction in the ability of the mutant to agonist-mediated receptor phosphorylation be by the of the be that this serine that may act as Furthermore, the results are with our studies using we showed that of or a the of the third intracellular loop in a reduction in the of phosphorylation mediated by CK1α A.B. Nahorski S.R. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). of the m3-muscarinic receptor response a response to agonist the response was by a with we have that due to the time course of agonist-mediated m3-muscarinic receptor and the of evidence in the receptor phosphorylation to GPCR that phosphorylation was the mechanism the desensitization of the (20.Tobin A.B. Nahorski S.R. J. Biol. Chem. 1993; 268: 9817-9823Abstract Full Text PDF PubMed Google Scholar, M.G. G. Nahorski S.R. A.B. J. Biochem. 1999; Google Scholar, A.B. Nahorski S.R. Pharmacol. 1993; Full Text PDF PubMed Scopus Google Scholar). our the mutant receptor showed a response with a to the Furthermore, of the mutant in desensitization of the These that agonist-mediated phosphorylation of the m3-muscarinic receptor was not involved in the desensitization of the response. It however, that the of phosphorylation that in the mutant mediated by one or more of the may be to desensitization of the response. studies have a number of GPCRs that desensitization. desensitization of the and the bradykinin B2 receptors are not associated with receptor phosphorylation H. R. J. Biol. Chem. 1993; 268: Full Text PDF PubMed Google Scholar, H. R. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar, Muller-Esterl W. Nahorski S.R. J. 1999; PubMed Google Scholar). of receptor phosphorylation and desensitization has been for the receptor of of the not desensitization of the adenylyl and cyclase responses M.J. J. D. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). The that desensitization of the m3-muscarinic receptor is a of of the receptor is in our In the present study the mutant a more response with the receptor This was not due to in agonist for the receptor in the of the agonist may an coupling of the receptor to Gq/11. in the magnitude of the response was in m3-muscarinic receptor phosphorylation was by with suggesting that receptor phosphorylation mediated by CK1α was able to control the magnitude of the response. inositol phosphate has been for of the T. T. Kameyama K. Haga K. Haga T. K. T. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar) and receptors J. A. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar) been In phospholipase C to be by resulting in It that receptor phosphorylation may a role in the magnitude of the inositol phosphate response mediated by m3-muscarinic receptors and phospholipase In we show that of CK1α agonist-mediated phosphorylation of the m3-muscarinic These support our studies suggesting that CK1α is a receptor kinase involved in GPCR phosphorylation. Furthermore, by using a receptor mutant that agonist-mediated we show that phosphorylation is not associated with desensitization of the response may instead be involved in the magnitude of the inositol phosphate response. We for of the and Nahorski for many and