Receptor Subtype-specific Regulation of Muscarinic Acetylcholine Receptor Sequestration by Dynamin

Sustained stimulation of muscarinic acetylcholine receptors (mAChRs) and other G protein-coupled receptors usually leads to a loss of receptor binding sites from the plasma membrane, referred to as receptor sequestration. Receptor sequestration can occur via endocytosis of clathrin-coated vesicles that bud from the plasma membrane into the cell but may also be accomplished by other, as yet ill-defined, mechanisms. Previous work has indicated that the monomeric GTPase dynamin controls the endocytosis of plasma membrane receptors via clathrin-coated vesicles. To investigate whether mAChRs sequester in a receptor subtype-specific manner via dynamin-dependent clathrin-coated vesicles, we tested the effect of overexpressing the dominant-negative dynamin mutant K44A on m1, m2, m3, and m4 mAChR sequestration in HEK-293 cells. The m1, m2, m3, and m4 mAChRs sequestered rapidly in HEK-293 cells following agonist exposure but displayed dissimilar sequestration pathways. Overexpression of dynamin K44A mutant fully blocked m1 and m3 mAChR sequestration, whereas m2 mAChR sequestration was not affected. Also, m4 mAChRs, which like m2 mAChRs preferentially couple to pertussis toxin-sensitive G proteins, sequestered in a completely dynamin-dependent manner. Following agonist removal, sequestered m1 mAChRs fully reappeared on the cell surface, whereas sequestered m2 mAChRs did not. The distinct sequestration of m2 mAChRs was also apparent in COS-7 and Chinese hamster ovary cells. We conclude that the m2 mAChR displays unique subtype-specific sequestration that distinguishes this receptor from the m1, m3, and m4 subtypes. These results are the first to demonstrate that receptor sequestration represents a new type of receptor subtype-specific regulation within the family of mAChRs. Sustained stimulation of muscarinic acetylcholine receptors (mAChRs) and other G protein-coupled receptors usually leads to a loss of receptor binding sites from the plasma membrane, referred to as receptor sequestration. Receptor sequestration can occur via endocytosis of clathrin-coated vesicles that bud from the plasma membrane into the cell but may also be accomplished by other, as yet ill-defined, mechanisms. Previous work has indicated that the monomeric GTPase dynamin controls the endocytosis of plasma membrane receptors via clathrin-coated vesicles. To investigate whether mAChRs sequester in a receptor subtype-specific manner via dynamin-dependent clathrin-coated vesicles, we tested the effect of overexpressing the dominant-negative dynamin mutant K44A on m1, m2, m3, and m4 mAChR sequestration in HEK-293 cells. The m1, m2, m3, and m4 mAChRs sequestered rapidly in HEK-293 cells following agonist exposure but displayed dissimilar sequestration pathways. Overexpression of dynamin K44A mutant fully blocked m1 and m3 mAChR sequestration, whereas m2 mAChR sequestration was not affected. Also, m4 mAChRs, which like m2 mAChRs preferentially couple to pertussis toxin-sensitive G proteins, sequestered in a completely dynamin-dependent manner. Following agonist removal, sequestered m1 mAChRs fully reappeared on the cell surface, whereas sequestered m2 mAChRs did not. The distinct sequestration of m2 mAChRs was also apparent in COS-7 and Chinese hamster ovary cells. We conclude that the m2 mAChR displays unique subtype-specific sequestration that distinguishes this receptor from the m1, m3, and m4 subtypes. These results are the first to demonstrate that receptor sequestration represents a new type of receptor subtype-specific regulation within the family of mAChRs. Muscarinic acetylcholine receptors (mAChRs) 1The abbreviations used are: mAChR, muscarinic acetylcholine receptor; G protein, guanine nucleotide-binding protein; GRK, G protein-coupled receptor kinase; NMS,N-methylscopolamine; QNB, quinuclidinylbenzilate; CHO, Chinese hamster ovary. belong to the superfamily of plasma membrane receptors that regulate a large number of signal transduction pathways via activation of heterotrimeric GTP-binding proteins (G proteins). The mAChR family consists of five subtypes of cloned mAChRs and can be subdivided into two functional groups: the m1, m3, and m5 subtypes, which preferentially couple to the Gq family of G proteins, and the m2 and m4 subtypes, which effectively activate the Gi family of G proteins. The m1, m2, m3, and m4 receptors are widely expressed in the central nervous system and peripheral tissues, whereas m5 receptors are present in only minute amounts in hippocampus and other areas of the brain (1Hulme E.C. Birdsall N.J.M. Buckley N.J. Annu. Rev. Pharmacol. Toxicol. 1990; 30: 633-673Crossref PubMed Scopus (1096) Google Scholar). Prolonged exposure of mAChRs and other G protein-coupled receptors to agonists usually results in the attenuation of the cellular response. One molecular mechanism of attenuation involves the phosphorylation of the receptors by G protein-coupled receptor kinases (GRKs) and increased binding of the inhibitory protein β-arrestin to the phosphorylated receptors, thereby inhibiting the coupling with G proteins (2Premont R.T. Inglese J. Lefkowitz R.J. FASEB J. 1995; 9: 175-182Crossref PubMed Scopus (472) Google Scholar). Another regulatory mechanism is the sequestration of receptors by which G protein-coupled receptors become inaccessible for hydrophilic membrane-impermeable ligands, including agonists. The sequestration of receptors may represent endocytosis of receptors via clathrin-coated vesicles (3Von Zastrow M. Link R. Daunt D. Barsh G. Kobilka B.K. J. Biol. Chem. 1993; 268: 763-766Abstract Full Text PDF PubMed Google Scholar, 4Tolbert L.M. Lameh J. J. Biol. Chem. 1996; 271: 17335-17342Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar), caveolae (5Dupree P. Parton R.G. Raposo G. Kurzchalia T.V. Simons K. EMBO J. 1993; 12: 1597-1605Crossref PubMed Scopus (403) Google Scholar, 6Roettger B.F. Rentsch R.U. Pinon D. Holicky E. Hadac E. Larkin J.M. Miller L.J. J. Cell Biol. 1995; 128: 1029-1041Crossref PubMed Scopus (204) Google Scholar), or noncoated vesicles (7Raposo G. Dunia I. Delavier-Klutchko C. Kaveri S. Strosberg A.D. Benedetti E.L. Eur. J. Cell Biol. 1989; 50: 340-352PubMed Google Scholar), or may be associated with conformational changes of the receptor in the plasma membrane that make the receptor inaccessible to agonists (8Roettger B.F. Rentsch R.U. Hadac E.M. Hellen E.H. Burghardt T.P. Miller L.J. J. Cell Biol. 1995; 130: 579-590Crossref PubMed Scopus (54) Google Scholar). For some G protein-coupled receptors like the β2-adrenergic receptors, sequestration is required to allow resensitization of desensitized receptors in a presumably subcellular compartment from which they can recycle back to the plasma membrane (9Yu S.S. Lefkowitz R.J. Hausdorff W.P. J. Biol. Chem. 1993; 268: 337-341Abstract Full Text PDF PubMed Google Scholar, 10Pippig S. Andexinger S. Lohse M.J. Mol. Pharmacol. 1995; 47: 666-676PubMed Google Scholar). For other G protein-coupled receptors, including mAChRs and secretin receptors, receptor sequestration is a desensitization mechanism (11Yang J. Williams J.A. Yule D.I. Logsdon C.D. Mol. Pharmacol. 1995; 48: 477-485PubMed Google Scholar, 12Bogatkewitsch G.S. Lenz W. Jakobs K.H. van Koppen C.J. Mol. Pharmacol. 1996; 50: 424-429PubMed Google Scholar, 13Holtmann M.H. Roettger B.F. Pinon D.I. Miller L.J. J. Biol. Chem. 1996; 271: 23566-23571Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). For example, sequestration of m4 mAChRs in CHO cells delays receptor resensitization by more than 2 h, whereas nonsequestered receptors acquire full responsiveness within 10 min after agonist removal (12Bogatkewitsch G.S. Lenz W. Jakobs K.H. van Koppen C.J. Mol. Pharmacol. 1996; 50: 424-429PubMed Google Scholar). A number of monomeric GTPases has been implicated as key regulators of various stages of endocytosis, including the 100-kDa GTPase dynamin. Dynamin is an essential, early acting component of the endocytic pathway that operates via clathrin-coated vesicles. This GTPase controls the formation of clathrin-coated vesicles by regulating the constriction and budding of clathrin-coated pits from the plasma membrane (14Van der Bliek A.M. Redelmeier T.E. Damke H. Tisdale E.J. Meyerowitz E.M. Schmid S.L. J. Cell Biol. 1993; 122: 553-563Crossref PubMed Scopus (589) Google Scholar, 15Takei K. McPherson P.S. Schmid S.L. de Camilli P. Nature. 1995; 374: 186-190Crossref PubMed Scopus (656) Google Scholar). Overexpression of a dominant-negative dynamin mutant (dynamin K44A), which displays reduced GTP binding affinity and hydrolysis, potently inhibits the sequestration of several plasma membrane receptors, including receptors for transferrin and epidermal growth factor. Recently, Zhang et al. (16Zhang J. Ferguson S.S.G. Barak L.S. Ménard L. Caron M.G. J. Biol. Chem. 1996; 271: 18302-18305Abstract Full Text Full Text PDF PubMed Scopus (398) Google Scholar) showed that functional dynamin is required for the internalization of β2-adrenergic receptors in HEK-293 cells via clathrin-coated vesicles. In contrast, angiotensin II AT type 1A receptors, which are structurally and functionally distinct from β2-adrenergic receptors, sequester in a dynamin-independent manner (16Zhang J. Ferguson S.S.G. Barak L.S. Ménard L. Caron M.G. J. Biol. Chem. 1996; 271: 18302-18305Abstract Full Text Full Text PDF PubMed Scopus (398) Google Scholar). Thus, G protein-coupled receptors are able to sequester via dynamin-dependent and dynamin-independent sequestration pathways. Previous radioligand binding studies have suggested that mAChRs may sequester in a receptor subtype-specific manner. For example, whereas both m1 and m2 mAChRs rapidly sequester in Y1 adrenal cells following agonist exposure, only m1 mAChRs sequester upon addition of phorbol ester (17Scherer N.M. Nathanson N.M. Biochemistry. 1990; 29: 8475-8483Crossref PubMed Scopus (26) Google Scholar). Likewise, in JEG-3 cells, agonist-treatment induces m2 but not m1 mAChR sequestration (18Goldman P.S. Schlador M.L. Shapiro R.A. Nathanson N.M. J. Biol. Chem. 1996; 271: 4215-4222Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). Similarly, immunocytochemical studies on the localization of α2-adrenergic receptor subtypes in HEK-293 cells have shown that following agonist binding, α2A-adrenergic receptors do not sequester, whereas α2B-adrenergic receptors are able to undergo agonist-induced sequestration (19Daunt D.A. Hurt C. Hein L. Kallio J. Feng F. Kobilka B.K. Mol. Pharmacol. 1997; 51: 711-720Crossref PubMed Scopus (175) Google Scholar). Thus, within a subfamily of structurally related G protein-coupled receptors, receptors can also differ in their capacity to sequester. The goal of the present work was to analyze the functional role of the GTPase dynamin in mAChR sequestration, and to investigate whether sequestration of the m1, m2, m3, and m4 mAChR subtypes is differentially regulated by dynamin. Here we demonstrate that whereas m1, m3, and m4 mAChR sequestration is regulated by dynamin, m2 mAChRs sequester in a dynamin-independent manner. These results provide strong evidence that receptor sequestration represents a novel type of receptor subtype-specific regulation of mAChRs. [3H]Quinuclidinyl benzilate ([3H]QNB, specific activity 43 Ci/mmol) andN-[3H]methylscopolamine ([3H]NMS, specific activity 84 Ci/mmol) were purchased from New England Nuclear. Mouse anti-dynamin monoclonal antibody and peroxidase-conjugated goat anti-mouse antibody were obtained from Transduction Laboratories and Dianova, respectively. All recombinant DNA procedures were carried out following standard protocols. DNA encoding mouse m1 mAChR (20Shapiro R.A. Scherer N.M. Habecker B.A. Subers E.M. Nathanson N.M. J. Biol. Chem. 1988; 263: 18397-18403Abstract Full Text PDF PubMed Google Scholar), porcine m2 mAChR (21Peralta E.G. Winslow J.G. Peterson G.L. Smith D.H. Ashkenazi A. Ramachandran J. Schimerlik M.I. Capon D.J. Science. 1987; 236: 600-605Crossref PubMed Scopus (334) Google Scholar), human m3 mAChR (22Bonner T.I. Buckley N.J. Young A.C. Brann M.R. Science. 1987; 237: 527-532Crossref PubMed Scopus (1220) Google Scholar), and mouse m4 mAChR (23Van Koppen C.J. Lenz W. Nathanson N.M. Biochim. Biophys. Acta. 1993; 1173: 342-344Crossref PubMed Scopus (14) Google Scholar) were subcloned into pCD-PS expression vector. The cDNAs encoding hemagglutinin-tagged dynamin wild-type and K44A (14Van der Bliek A.M. Redelmeier T.E. Damke H. Tisdale E.J. Meyerowitz E.M. Schmid S.L. J. Cell Biol. 1993; 122: 553-563Crossref PubMed Scopus (589) Google Scholar) were subcloned into pRK5 expression vector. HEK-293 tsA201 cells stably expressing simian virus 40 large T antigen (24Margolskee R.F. McHendry-Rinde B. Horn R. Biotechniques. 1993; 15: 906-911PubMed Google Scholar) and COS-7 cells (American Type Culture Collection) were grown in Dulbecco's modified Eagle's/F-12 medium supplemented with 10% fetal calf serum, penicillin G (100 units/ml), and streptomycin (100 μg/ml) in an atmosphere of 5% CO2. CHO cells (American Type Culture Collection) were grown in α medium containing 10% fetal calf serum, penicillin G (100 units/ml), and streptomycin (100 μg/ml). Cell media were from Life Technologies, Inc. Cells on 150-mm plates were transfected with either 12.5 or 25 μg pCD-PS containing m1 or m2 mAChR DNA, respectively, together with either (unless indicated otherwise) 50 μg of pRK5 dynamin wild-type, pRK5 dynamin K44A, or empty pRK5, using the calcium phosphate method (25Van Koppen C.J. Sell A. Lenz W. Jakobs K.H. Eur. J. Biochem. 1994; 222: 525-531Crossref PubMed Scopus (36) Google Scholar). Transfection efficiency of HEK-293 tsA201 cells was determined by in situstaining for β-galactosidase activity of the cells cotransfected with the constitutively active pSVβ-gal (Promega). Cells on 150-mm plates were washed twice with phosphate-buffered saline (150 mm NaCl, 2.7 mm KCl, 1.5 mmKH2PO4, 6.5 mmNa2HPO4, pH 7.4) and lysed by the addition of 1.0 ml of lysis buffer (1% SDS, 10 mm Tris-HCl, pH 7.4). Lysate was transferred to a and for a were for min to and with lysis buffer to an of protein as by the method of buffer mm Tris-HCl, pH SDS, 10% were to of the and for on protein was was blocked with 10 mm Tris-HCl, pH mm NaCl, and 5% for min in 10 mm Tris-HCl, pH mm NaCl, the was with mouse anti-dynamin monoclonal antibody μg/ml) in buffer for Following for the was with peroxidase-conjugated goat anti-mouse antibody μg/ml) h, the was washed and was by of the was using a after cells from 150-mm plates were on plates and allow to and for The cells were with or for min in 25 mm Dulbecco's modified For of cells were with phosphate-buffered were with 2 in of phosphate-buffered and 2 also of to binding to 10% of of cells were washed with phosphate-buffered in and transferred into which ml of to is expressed as of cell receptors of and receptor number was determined in cell by binding of the muscarinic radioligand as (25Van Koppen C.J. Sell A. Lenz W. Jakobs K.H. Eur. J. Biochem. 1994; 222: 525-531Crossref PubMed Scopus (36) Google Scholar). HEK-293 and CHO cells do not of mAChR as determined by binding to cell To whether mAChR sequestration is regulated in a receptor we first the effect of overexpressing dynamin wild-type and K44A mutant on m1 and m2 mAChR sequestration in HEK-293 tsA201 cells. Transfection of HEK-293 tsA201 cells with 50 μg of DNA encoding the dynamin wild-type and K44A mutant in of the protein the dynamin expression as by and for an efficiency of of m1 HEK-293 tsA201 cells with a of mm for a of min in a 5% loss in cell receptor as with the membrane-impermeable muscarinic was in mAChR number as by binding of the muscarinic to from transfected HEK-293 cells not Overexpression of dynamin K44A m1 mAChR sequestration in a manner. Transfection of cells with μg of pRK5 dynamin K44A 150-mm m1 mAChR sequestration by whereas sequestration was blocked by and following with 50 and μg of pRK5 dynamin K44A DNA, In cells were transfected with 50 μg of pRK5 dynamin wild-type or K44A mutant 150-mm The of receptor sequestration was to a in the than the of mAChR sequestration in both cells and cells overexpressing dynamin K44A was within min of exposure to mm of m1 mAChR sequestration was apparent of as 2 with reduced cell number on cells by receptor sequestration in dynamin cells was only In to dynamin K44A, of dynamin wild-type did not the of m1 mAChR sequestration or the of to receptor sequestration, that expression of dynamin are not A provide strong evidence that m1 mAChR sequestration in HEK-293 tsA201 cells is regulated by and of m1 mAChR sequestration in HEK-293 tsA201 cells. The effect of overexpressing dynamin wild-type and dynamin K44A is HEK-293 tsA201 cells transfected with m1 mAChR in pCD-PS together with pRK5 dynamin wild-type K44A or empty pRK5 were in the and of mm for or min or with or for min was by binding to cells are the of of binding to cells transfected with pRK5, pRK5 dynamin wild-type, and pRK5 dynamin K44A was and protein, We determined whether m2 mAChR sequestration in HEK-293 cells is regulated by dynamin as shown in m2 mAChRs rapidly and cell receptor number was reduced by within min of with mm The loss of cell receptor number a in receptor number as determined by binding to cell not Overexpression of dynamin wild-type or dynamin K44A did not m2 mAChR sequestration in HEK-293 tsA201 cells. In cell m2 mAChRs sequestered with a and the of in receptor sequestration was These results that in HEK-293 tsA201 cells m2 mAChRs sequester by a dynamin-independent The dissimilar sequestration of m1 and m2 mAChRs in HEK-293 tsA201 cells to investigate whether m1 and m2 mAChRs also following agonist-induced receptor sequestration. For m1 and m2 cells were with mm for min and washed to shown in cell mAChR were reduced by 5% and 5% in m1 and m2 cells. h, sequestered binding sites on m1 HEK-293 cells In contrast, of sequestered binding sites on m2 cells was only after removal, only of binding and in the h, was These are with the that m1 and m2 mAChRs distinct pathways of sequestration in HEK-293 cells. To whether the sequestration pathway of m2 mAChRs in HEK-293 cells is a unique to m2 mAChRs, we whether other of the mAChR family this To do HEK-293 tsA201 cells were cotransfected with either m3 or m4 mAChR DNA with dynamin K44A shown in of dynamin K44A completely blocked the sequestration of m3 mAChRs in HEK-293 tsA201 cells, whereas sequestration of m4 mAChRs was by This that the dynamin-independent sequestration of m2 mAChRs is not with a coupling to pertussis toxin-sensitive G proteins. This was by sequestration studies of m2 and m4 mAChRs in COS-7 and CHO cells. In COS-7 cells, of dynamin K44A m2 mAChR sequestration by whereas m4 mAChR sequestration was completely Similarly, sequestration of m4 mAChRs in CHO cells was blocked by by of dynamin K44A, whereas m2 mAChR sequestration was not of mAChR subtypes in HEK-293 and CHO cells and the effect of dynamin from cells transfected with pRK5, from cells transfected with pRK5, from cells transfected with pRK5, from cells transfected with pRK5, 43 from cells transfected with pRK5, from cells transfected with pRK5, from cells transfected with pRK5, in a new In of the role of dynamin in regulating clathrin-coated endocytosis, we whether dynamin is in mAChR sequestration, and in whether the sequestration of the mAChR subtypes m1, m2, m3, and m4 is regulated differentially by this is the first of the regulation of receptor sequestration by dynamin within a family of G protein-coupled mAChR subtypes in HEK-293 cells sequestered rapidly and to a large m2 mAChR sequestration was distinct from that of the other mAChR subtypes. Overexpression of the dominant-negative dynamin K44A mutant fully blocked m1, m3, and m4 mAChR sequestration, whereas m2 mAChR sequestration was not affected. The distinct sequestration of m2 mAChRs in HEK-293 cells was by the that m2 mAChR sequestration was following agonist removal, whereas sequestered m1 mAChRs reappeared completely within The in sequestration m1 and m2 mAChRs was not related to their coupling Also, the m4 mAChR which like the m2 mAChR preferentially to pertussis toxin-sensitive G proteins, sequestered in a dynamin-dependent manner in HEK-293 cells. The distinct sequestration of m2 mAChR was not to HEK-293 cells but also apparent in transfected COS-7 and CHO cells. Overexpression of dynamin K44A in COS-7 cells blocked m4 mAChR sequestration whereas m2 mAChR sequestration was by Similarly, sequestration of m4 mAChRs in CHO cells be fully blocked by overexpressing dynamin K44A, m2 mAChR sequestration was completely the of this and R. J.A. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar) also showed that m2 mAChRs sequester in a dynamin-independent manner in HEK-293 cells. did not investigate the whether the other mAChR subtypes also the dynamin-independent sequestration was the that sequestration of m2 mAChRs in HEK-293 cells was fully m2 mAChR sequestration in COS-7 cells was accomplished via both dynamin-dependent and dynamin-independent pathways. These may a molecular for two studies on the regulation of m2 mAChR sequestration by in COS-7 and HEK-293 cells H. K. H. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar, R. P. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). In COS-7 cells, of a dominant-negative mutant of was to phosphorylation and sequestration by whereas in HEK-293 cells, m2 mAChR phosphorylation but not mAChR sequestration. the of is that are related to the dissimilar sequestration pathways of m2 mAChRs in HEK-293 and COS-7 cells. and J.G. F. Nature. 1996; PubMed Scopus Google Scholar) and Caron and (16Zhang J. Ferguson S.S.G. Barak L.S. Ménard L. Caron M.G. J. Biol. Chem. 1996; 271: 18302-18305Abstract Full Text Full Text PDF PubMed Scopus (398) Google Scholar) have the following early in the sequestration of β2-adrenergic receptors, which like m1 mAChRs sequester via dynamin-dependent clathrin-coated vesicles. Following agonist receptors are phosphorylated by in an manner and the binding of β-arrestin to the phosphorylated receptor the receptor from G In β-arrestin may as a affinity and on a β-arrestin with affinity to the in receptor this is to m2 mAChRs in HEK-293 cells do not sequester via dynamin-dependent clathrin-coated vesicles, yet are phosphorylated by in HEK-293 cells R. P. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar) and are able to β-arrestin J. Biol. Chem. 1993; 268: Full Text PDF PubMed Google Scholar). These studies to conclude that phosphorylation of m2 mAChRs not whether mAChRs sequester via either dynamin-dependent or dynamin-independent pathways. the of the m2 mAChR sequestration pathway is A m2 mAChR sequestration pathway may be the endocytosis via are noncoated plasma membrane which to have the to either into the cell or to in the of the plasma membrane T.V. Parton R.G. 1996; PubMed Scopus Google Scholar). Previous have suggested that receptors in CHO cells as as β2-adrenergic receptors in cells may sequester via caveolae (5Dupree P. Parton R.G. Raposo G. Kurzchalia T.V. Simons K. EMBO J. 1993; 12: 1597-1605Crossref PubMed Scopus (403) Google Scholar, 6Roettger B.F. Rentsch R.U. Pinon D. Holicky E. Hadac E. Larkin J.M. Miller L.J. J. Cell Biol. 1995; 128: 1029-1041Crossref PubMed Scopus (204) Google Scholar). al. Smith R.A. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar) that m2 mAChRs in are to caveolae following agonist This the to that this receptor is required to m2 mAChRs within the caveolae to specific including activation of the of that in the caveolae of m2 mAChRs also sequester and their to hydrophilic studies be to whether m2 mAChRs sequester into caveolae in HEK-293 cells and is the first of a receptor subtype-specific sequestration pathway within the family of mAChRs. In this is to receptor sequestration and regulate receptor (9Yu S.S. Lefkowitz R.J. Hausdorff W.P. J. Biol. Chem. 1993; 268: 337-341Abstract Full Text PDF PubMed Google Scholar, 10Pippig S. Andexinger S. Lohse M.J. Mol. Pharmacol. 1995; 47: 666-676PubMed Google Scholar, J. Williams J.A. Yule D.I. Logsdon C.D. Mol. Pharmacol. 1995; 48: 477-485PubMed Google Scholar, 12Bogatkewitsch G.S. Lenz W. Jakobs K.H. van Koppen C.J. Mol. Pharmacol. 1996; 50: 424-429PubMed Google Scholar, 13Holtmann M.H. Roettger B.F. Pinon D.I. Miller L.J. J. Biol. Chem. 1996; 271: 23566-23571Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar), the m2 subtype-specific sequestration pathway may a regulatory mechanism of receptor distinct from that of the m1, m3, and m4 subtypes. is whether m2 mAChRs are from the dynamin-dependent clathrin-coated sequestration or are by a of as yet regulatory which the m2 mAChRs into the dynamin-independent sequestration The that the m4 which has a in and functional with the m2 (1Hulme E.C. Birdsall N.J.M. Buckley N.J. Annu. Rev. Pharmacol. Toxicol. 1990; 30: 633-673Crossref PubMed Scopus (1096) Google Scholar), as as the m1 and m3 subtypes, sequester via the dynamin-dependent to the The of the molecular that the distinct dynamin-independent m2 mAChR sequestration pathway in HEK-293 and other cells provide new into the regulation of G protein-coupled We and for We are to S. Schmid for the of the human dynamin wild-type and dynamin K44A A. for pRK5, for pCD-PS and the human m3 mAChR M. Nathanson for the mouse m1 mAChR DNA, D. J. Capon for the porcine m2 mAChR and M. M. for HEK-293 tsA201 cells.