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Essential Role of Dynamin in Internalization of M2Muscarinic Acetylcholine and Angiotensin AT1AReceptors

2000; Elsevier BV; Volume: 275; Issue: 29 Linguagem: Inglês

10.1074/jbc.m001736200

ISSN

1083-351X

Autores

Yvonne Werbonat, Nina Kleutges, Karl H. Jakobs, Chris J. van Koppen,

Tópico(s)

Neuroscience and Neuropharmacology Research

Resumo

Most G protein-coupled receptors (GPCRs), including the M1 muscarinic acetylcholine receptor (mAChR), internalize in clathrin-coated vesicles, a process that requires dynamin GTPase. The observation that some GPCRs like the M2 mAChR and the angiotensin AT1A receptor (AT1AR) internalize irrespective of expression of dominant-negative K44A dynamin has led to the proposal that internalization of these GPCRs is dynamin-independent. Here, we report that, contrary to what is postulated, internalization of M2mAChR and AT1AR in HEK-293 cells is dynamin-dependent. Expression of N272 dynamin, which lacks the GTP-binding domain, or K535M dynamin, which is not stimulatable by phosphatidylinositol 4,5-bisphosphate, strongly inhibits internalization of M1 and M2 mAChRs and AT1ARs. Expression of kinase-defective K298M c-Src or Y231F,Y597F dynamin (which cannot be phosphorylated by c-Src) reduces M1 mAChR internalization. Similarly, c-Src inhibitor PP1 as well as the generic tyrosine kinase inhibitor genistein strongly inhibit M1 mAChR internalization. In contrast, M2 mAChR internalization is not (or is only slightly) reduced by expression of these constructs or treatment with PP1 or genistein. Thus, dynamin GTPases are not only essential for M1 mAChR but also for M2 mAChR and AT1AR internalization in HEK-293 cells. Our findings also indicate that dynamin GTPases are differentially regulated by c-Src-mediated tyrosine phosphorylation. Most G protein-coupled receptors (GPCRs), including the M1 muscarinic acetylcholine receptor (mAChR), internalize in clathrin-coated vesicles, a process that requires dynamin GTPase. The observation that some GPCRs like the M2 mAChR and the angiotensin AT1A receptor (AT1AR) internalize irrespective of expression of dominant-negative K44A dynamin has led to the proposal that internalization of these GPCRs is dynamin-independent. Here, we report that, contrary to what is postulated, internalization of M2mAChR and AT1AR in HEK-293 cells is dynamin-dependent. Expression of N272 dynamin, which lacks the GTP-binding domain, or K535M dynamin, which is not stimulatable by phosphatidylinositol 4,5-bisphosphate, strongly inhibits internalization of M1 and M2 mAChRs and AT1ARs. Expression of kinase-defective K298M c-Src or Y231F,Y597F dynamin (which cannot be phosphorylated by c-Src) reduces M1 mAChR internalization. Similarly, c-Src inhibitor PP1 as well as the generic tyrosine kinase inhibitor genistein strongly inhibit M1 mAChR internalization. In contrast, M2 mAChR internalization is not (or is only slightly) reduced by expression of these constructs or treatment with PP1 or genistein. Thus, dynamin GTPases are not only essential for M1 mAChR but also for M2 mAChR and AT1AR internalization in HEK-293 cells. Our findings also indicate that dynamin GTPases are differentially regulated by c-Src-mediated tyrosine phosphorylation. G protein-coupled receptor angiotensin AT1A receptor bovine serum albumin muscarinic acetylcholine receptor N-methylscopolamine phosphatidylinositol 4,5-bisphosphate HEPES-buffered saline Dulbecco's modified Eagle's medium For most G protein-coupled receptors (GPCRs),1 receptor internalization is thought to be initiated by phosphorylation of the receptor by G protein-coupled receptor kinases and binding of the cytosolic protein β-arrestin to the phosphorylated receptor (1.Goodman Jr., O.B. Krupnick J.G. Santini F. Gurevich V.V. Penn R.B. Gagnon A.W. Keen J.H. Benovic J.L. Nature. 1996; 383: 447-450Crossref PubMed Scopus (1153) Google Scholar). β-Arrestin then sterically inhibits further interaction of the receptor with heterotrimeric G proteins and binds with high affinity to clathrin heavy chains (1.Goodman Jr., O.B. Krupnick J.G. Santini F. Gurevich V.V. Penn R.B. Gagnon A.W. Keen J.H. Benovic J.L. Nature. 1996; 383: 447-450Crossref PubMed Scopus (1153) Google Scholar). Through this interaction, GPCRs are believed to be targeted to clathrin-coated pits. Following transformation of the clathrin-coated pit into a clathrin-coated vesicle, the clathrin-coated vesicle pinches off from the plasma membrane. This process is catalyzed by the 100-kDa GTPase dynamin, which probably activates (as yet largely unknown) effectors of the fission machinery (2.Sever S. Muhlberg A.B. Schmid S.L. Nature. 1999; 398: 481-486Crossref PubMed Scopus (313) Google Scholar). Three closely related mammalian dynamin isoforms have been identified: neuronal dynamin-1, ubiquitously expressed dynamin-2, and dynamin-3, which is expressed in testes, neurons, and lung (3.Schmid S.L. McNiven M.A. De Camilli P. Curr. Opin. Cell Biol. 1998; 10: 504-512Crossref PubMed Scopus (354) Google Scholar). Comparison of the primary sequence shows that all three dynamin isoforms contain three highly conserved GTP-binding motifs (i.e. elements I, II, and III). A Lys44 → Ala substitution in the first of the three putative GTP-binding motifs yields a dominant-negative dynamin mutant, which displays strongly impaired GTPase activity and is predicted to have a greatly reduced GTP binding affinity (4.Van 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 (586) Google Scholar). The two other GTP-binding motifs in dynamin are likely to be involved in GTP binding as well. Mutation of the third GTP-binding motif (substitution Lys206 → Asp in element III) or removal of all three GTP-binding motifs (amino acids 1–271 in dynamin-1) drastically reduces clathrin-coated vesicle-mediated internalization (4.Van 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 (586) Google Scholar, 5.Herskovits J.S. Burgess C.C. Obar R.A. Vallee R.B. J. Cell Biol. 1993; 122: 565-578Crossref PubMed Scopus (394) Google Scholar, 6.Achiriloaie M. Barylko B. Albanesi J.P. Mol. Cell. Biol. 1999; 19: 1410-1415Crossref PubMed Scopus (143) Google Scholar). A second important regulator of dynamin function is phosphatidylinositol 4,5-bisphosphate (PIP2) (6.Achiriloaie M. Barylko B. Albanesi J.P. Mol. Cell. Biol. 1999; 19: 1410-1415Crossref PubMed Scopus (143) Google Scholar, 7.Lin H.C. Gilman A.G. J. Biol. Chem. 1996; 271: 27979-27982Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 8.Barylko B. Binns D. Lin K.-M. Atkinson M.A.L. Jameson D.M. Yin H.L. Albanesi J.P. J. Biol. Chem. 1998; 273: 3791-3797Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 9.Klein D.E. Lee A. Frank D.W. Marks M.S. Lemmon M.A. J. Biol. Chem. 1998; 273: 27725-27733Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar). All three dynamin isoforms contain a pleckstrin homology domain that is able to bind PIP2. Binding of PIP2 to dynamin not only strongly increases the GTPase activity of dynamin but may also serve to target dynamin to the plasma membrane, allowing subsequent dynamin self-assembly at the neck of the clathrin-coated vesicle (6.Achiriloaie M. Barylko B. Albanesi J.P. Mol. Cell. Biol. 1999; 19: 1410-1415Crossref PubMed Scopus (143) Google Scholar, 7.Lin H.C. Gilman A.G. J. Biol. Chem. 1996; 271: 27979-27982Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 8.Barylko B. Binns D. Lin K.-M. Atkinson M.A.L. Jameson D.M. Yin H.L. Albanesi J.P. J. Biol. Chem. 1998; 273: 3791-3797Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 9.Klein D.E. Lee A. Frank D.W. Marks M.S. Lemmon M.A. J. Biol. Chem. 1998; 273: 27725-27733Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar). Expression of the dynamin mutant K535M, which is not stimulatable by PIP2, effectively blocks transferrin receptor internalization in clathrin-coated vesicles (6.Achiriloaie M. Barylko B. Albanesi J.P. Mol. Cell. Biol. 1999; 19: 1410-1415Crossref PubMed Scopus (143) Google Scholar).A large number of recent studies indicate that most GPCRs, including M1, M3, and M4 muscarinic acetylcholine receptors (mAChRs) in HEK-293 cells, internalize in clathrin-coated vesicles in a dynamin-dependent manner. This evidence is primarily based on the inhibitory effect of the dominant-negative inhibitor of dynamin-mediated internalization, K44A dynamin (10.Vögler 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 (78) Google Scholar, 11.Vögler O. Nolte B. Voss M. Schmidt M. Jakobs K.H. Van Koppen C.J. J. Biol. Chem. 1999; 274: 12333-12338Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 12.Pals-Rylaarsdam R. Gurevich V.V. Lee K.B. Ptasiensky J.A. Benovic J.L. Hosey M.M. J. Biol. Chem. 1997; 272: 23682-23689Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 13.Zhang 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). In contrast, M2 mAChRs internalize in a clathrin-independent manner and irrespective of expression of K44A dynamin in HEK-293 cells (10.Vögler 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 (78) Google Scholar, 12.Pals-Rylaarsdam R. Gurevich V.V. Lee K.B. Ptasiensky J.A. Benovic J.L. Hosey M.M. J. Biol. Chem. 1997; 272: 23682-23689Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). Likewise, internalization of angiotensin AT1A receptors (AT1ARs) (13.Zhang 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), dopamine D2 receptors (14.Vickery R.G. von Zastrow M. J. Cell Biol. 1999; 144: 31-43Crossref PubMed Scopus (197) Google Scholar), and secretin receptors (15.Walker J.K.L. Premont R.T. Barak L.S. Caron M.G. Shetzline M.A. J. Biol. Chem. 1999; 274: 31515-31523Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar) is also insensitive to expression of K44A dynamin. This has led to the proposal that internalization of these GPCRs is dynamin-independent. However, in light of the notion that the binding of GTP to dynamin probably involves binding to all three GTP- binding motifs in the GTP-binding pocket, we reasoned that a dynamin mutant lacking all three GTP-binding motifs might be a more appropriate dominant negative dynamin mutant to determine whether internalization of a particular GPCR is dynamin-dependent. Indeed, we here demonstrate that internalization of M2 mAChR and AT1AR is strongly inhibited by expression of N272 dynamin, which lacks the complete GTP-binding domain. Also, expression of K535M dynamin, which lacks PIP2-stimulated GTPase activity, significantly blocks internalization of these GPCR species.DISCUSSIONIn the past few years, the question whether dynamin plays an essential role in the internalization of a particular GPCR has been mostly analyzed by using K44A dynamin as dominant-negative mutant. While internalization of most GPCRs is blocked by expression of K44A dynamin, some GPCRs like the M2 mAChRs, D2dopamine receptors, secretin receptors, and AT1ARs internalize irrespective of K44A dynamin expression, suggesting that internalization of these GPCRs is dynamin-independent (10.Vögler 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 (78) Google Scholar, 12.Pals-Rylaarsdam R. Gurevich V.V. Lee K.B. Ptasiensky J.A. Benovic J.L. Hosey M.M. J. Biol. Chem. 1997; 272: 23682-23689Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 13.Zhang 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, 14.Vickery R.G. von Zastrow M. J. Cell Biol. 1999; 144: 31-43Crossref PubMed Scopus (197) Google Scholar, 15.Walker J.K.L. Premont R.T. Barak L.S. Caron M.G. Shetzline M.A. J. Biol. Chem. 1999; 274: 31515-31523Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). We now report that, contrary to what is currently postulated, internalization of M2 mAChR and AT1AR is dynamin-dependent. Coexpression of the dominant-negative dynamin mutants N272 and K535M strongly inhibited M2 mAChR and AT1AR internalization in HEK-293 tsA201 cells. These findings imply that N272 and K535M dynamin are more appropriate dominant-negative dynamin mutants than K44A dynamin. In this context, it will be interesting to determine whether fluid-phase endocytosis (5.Herskovits J.S. Burgess C.C. Obar R.A. Vallee R.B. J. Cell Biol. 1993; 122: 565-578Crossref PubMed Scopus (394) Google Scholar,20.Damke H. Baba T. Warnock D.E. Schmid S.L. J. Cell Biol. 1994; 127: 915-934Crossref PubMed Scopus (1034) Google Scholar) and internalization of ricin (21.Simpson J.C. Smith D.C. Roberts L.M. Lord J.M. Exp. Cell Res. 1998; 239: 293-300Crossref PubMed Scopus (63) Google Scholar) are affected by expression of N272 or K535M dynamin also, because these trafficking processes are not blocked by K44A dynamin and are thus considered to be dynamin-independent. It is intriguing that N272 dynamin, which lacks all three GTP-binding motifs, inhibits internalization of both M1 and M2 mAChR, while K44A dynamin, which lacks only the first GTP-binding motif, blocks only M1mAChR internalization. It is possible that K44A dynamin selectively sequesters away an essential component of the M1 but not of the M2 mAChR internalization pathway. Another potential explanation relates to the fact that K44A dynamin is able to coassemble with wild-type dynamin (22.Warnock D.E. Hinshaw J.E. Schmid S.L. J. Biol. Chem. 1996; 271: 22310-22314Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). Since dynamin assembly and interaction of dynamin with other proteins requires the C terminus of the dynamin, which varies among the dynamin isoforms (23.Smirnova E. Shurland D.-L. Newman-Smith E.D. Pishvaee B. Van der Bliek A.M. J. Biol. Chem. 1999; 274: 14942-14947Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar), different internalization pathways may use different dynamin isoforms. As a result, different internalization pathways may display differential sensitivity toward interference of K44A dynamin. Perhaps assembled GTP-bound K44A dynamin is sufficiently active to catalyze the budding of M2 mAChR- and AT1AR-containing vesicles from the plasma membrane but is not able to support internalization of M1 mAChRs in clathrin-coated vesicles.In the present study, we observed that mAChR and AT1AR internalization in HEK-293 cells is strongly inhibited by expression of K535M dynamin, a dynamin mutant, which lacks the putative PIP2 binding site (6.Achiriloaie M. Barylko B. Albanesi J.P. Mol. Cell. Biol. 1999; 19: 1410-1415Crossref PubMed Scopus (143) Google Scholar). At present, it is unknown at which stage of the vesicle budding process PIP2 binding to dynamin is essential. It has been postulated that, after recruitment of dynamin to the clathrin-coated pit, dynamin's interaction with the plasma membrane is strengthened by the binding of dynamin's pleckstrin homology domain with PIP2 in the plasma membrane (8.Barylko B. Binns D. Lin K.-M. Atkinson M.A.L. Jameson D.M. Yin H.L. Albanesi J.P. J. Biol. Chem. 1998; 273: 3791-3797Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). In addition, PIP2 binding might promote self-assembly of the dynamin molecules at the neck of the clathrin-coated pit and stimulate dynamin's GTPase activity (8.Barylko B. Binns D. Lin K.-M. Atkinson M.A.L. Jameson D.M. Yin H.L. Albanesi J.P. J. Biol. Chem. 1998; 273: 3791-3797Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 9.Klein D.E. Lee A. Frank D.W. Marks M.S. Lemmon M.A. J. Biol. Chem. 1998; 273: 27725-27733Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar). An alternative possibility is that lysine 535 in the pleckstrin homology domain of dynamin serves to promote interaction of dynamin with proteins rather than with PIP2 (6.Achiriloaie M. Barylko B. Albanesi J.P. Mol. Cell. Biol. 1999; 19: 1410-1415Crossref PubMed Scopus (143) Google Scholar). Regardless of the mechanism, our study clearly underscores the relevance of dynamin's lysine 535 residue in GPCR internalization. Identification of the binding partners of dynamin's pleckstrin homology domain will be important for understanding the mechanisms of GPCR internalization.In analogy with recent studies on the regulation of internalization of β2-adrenergic receptors in HEK-293 cells (16.Ahn S. Maudsley S. Luttrell L.M. Lefkowitz R.J. Daaka Y. J. Biol. Chem. 1999; 274: 1185-1188Abstract Full Text Full Text PDF PubMed Scopus (243) Google Scholar, 24.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: 655-661Crossref PubMed Scopus (1250) Google Scholar), we show that internalization of M1 mAChRs is strongly reduced by inhibition of c-Src activity and by overexpression of Y231F,Y597F dynamin, which cannot be phosphorylated by c-Src. Since M1 mAChRs internalize in clathrin-coated vesicles in a β-arrestin-dependent manner, we propose that, in analogy to β2-adrenergic receptors, internalization of M1 mAChRs involves β-arrestin-mediated targeting of receptor in the clathrin-coated pit and activation of c-Src by β-arrestin. c-Src then phosphorylates dynamin, a process that is required for M1 mAChR internalization in HEK-293 cells. Whether tyrosine phosphorylation activates dynamin or allows activation of dynamin by other molecules remains to be determined. In contrast, dynamin-mediated internalization of M2 mAChRs was found not to be inhibited by expression of kinase-defective K298M c-Src or treatment of the cells with the specific c-Src inhibitor PP1. Thus, c-Src does not play a role in M2 mAChR internalization. These findings are supported by recent studies showing that M2 mAChR internalization in HEK-293 cells is β-arrestin-independent (11.Vögler O. Nolte B. Voss M. Schmidt M. Jakobs K.H. Van Koppen C.J. J. Biol. Chem. 1999; 274: 12333-12338Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 12.Pals-Rylaarsdam R. Gurevich V.V. Lee K.B. Ptasiensky J.A. Benovic J.L. Hosey M.M. J. Biol. Chem. 1997; 272: 23682-23689Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). However, treatment of the cells with the generic tyrosine kinase, genistein, or coexpression of Y231F,Y597F dynamin did slightly reduce M2 mAChR internalization. This suggests that M2 mAChR internalization is regulated to a very limited extent by phosphorylation of dynamin by tyrosine kinases other than c-Src. On the basis of the present and previous findings (11.Vögler O. Nolte B. Voss M. Schmidt M. Jakobs K.H. Van Koppen C.J. J. Biol. Chem. 1999; 274: 12333-12338Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar), we propose that M2 mAChR internalization in HEK-293 cells is catalyzed by a dynamin isoform that differs from the dynamin isoform involved in clathrin-mediated M1 mAChR internalization. Much remains to be learned about the internalization pathway of M2 mAChR (and AT1AR) in HEK-293 cells. We have observed that pretreatment of HEK-293 cells with 0.45m sucrose fully blocks M2 (and M1) mAChR internalization in HEK-293 cells. 2Y. Werbonat, N. Kleutges, K. H. Jakobs, and C. J. van Koppen, unpublished observations. Yet expression of a dominant-negative clathrin mutant or β-arrestin V53D, which inhibits clathrin-mediated internalization of GPCRs, blocks M1 but not M2 mAChR internalization (11.Vögler O. Nolte B. Voss M. Schmidt M. Jakobs K.H. Van Koppen C.J. J. Biol. Chem. 1999; 274: 12333-12338Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). Similarly, expression of the amphiphysin SH3 domain, which blocks the targeting of dynamin to clathrin-coated pits, inhibits M1 but not M2mAChR internalization in HEK-293 cells.2 On the basis of these findings, we conclude that M2 mAChR internalization in HEK-293 cells is clathrin-independent and that the inhibitory effect of sucrose on receptor internalization is not specific for clathrin-mediated internalization. In this respect, it is important to note that hypertonic sucrose treatment of HEK-293 cells does not only block vesicle formation at the plasma membrane but may also induce other cellular responses including MAP kinase activation (25.Schramm N.L. Limbird L.E. J. Biol. Chem. 1999; 274: 24935-24940Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar), which may inhibit receptor internalization indirectly (26.Pitcher J.A. Tesmer J.J.G. Freeman J.L.R. Capel W.D. Stone W.C. Lefkowitz R.J. J. Biol. Chem. 1999; 274: 34531-34534Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). Interestingly, similar findings have been obtained recently with the secretin receptor. Internalization of secretin receptors in HEK-293 cells is unaffected by expression of dynamin K44A and β-arrestin V53D but is sensitive to sucrose pretreatment (15.Walker J.K.L. Premont R.T. Barak L.S. Caron M.G. Shetzline M.A. J. Biol. Chem. 1999; 274: 31515-31523Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Presently, it is unknown through which vesicles AT1AR internalizes in HEK-293 cells. In adrenal glomerulosa and Chinese hamster ovary cells, AT1AR may internalize through clathrin-coated vesicles, although this has only been inferred from biochemical (and not morphological) experiments using hypertonic sucrose treatment or potassium depletion (27.Hunyady L. J. Am. Soc. Nephrol. 1999; 10: S47-S56PubMed Google Scholar). In vascular smooth muscle cells, however, AT1ARs have been found to internalize through noncoated pits, possibly caveolae, as well as coated pits (27.Hunyady L. J. Am. Soc. Nephrol. 1999; 10: S47-S56PubMed Google Scholar, 28.Ishizaka N. Griendling K.K. Lassègue B. Alexander R.W. Hypertension. 1998; 32: 459-466Crossref PubMed Scopus (167) Google Scholar). Thus, the internalization of AT1AR and other GPCRs (10.Vögler 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 (78) Google Scholar) appears to differ among different cell types. The identification of the budding vesicles through which AT1AR and M2 mAChR internalize in HEK-293 cells and other cell types will provide important information on the molecular mechanisms of GPCR internalization. For most G protein-coupled receptors (GPCRs),1 receptor internalization is thought to be initiated by phosphorylation of the receptor by G protein-coupled receptor kinases and binding of the cytosolic protein β-arrestin to the phosphorylated receptor (1.Goodman Jr., O.B. Krupnick J.G. Santini F. Gurevich V.V. Penn R.B. Gagnon A.W. Keen J.H. Benovic J.L. Nature. 1996; 383: 447-450Crossref PubMed Scopus (1153) Google Scholar). β-Arrestin then sterically inhibits further interaction of the receptor with heterotrimeric G proteins and binds with high affinity to clathrin heavy chains (1.Goodman Jr., O.B. Krupnick J.G. Santini F. Gurevich V.V. Penn R.B. Gagnon A.W. Keen J.H. Benovic J.L. Nature. 1996; 383: 447-450Crossref PubMed Scopus (1153) Google Scholar). Through this interaction, GPCRs are believed to be targeted to clathrin-coated pits. Following transformation of the clathrin-coated pit into a clathrin-coated vesicle, the clathrin-coated vesicle pinches off from the plasma membrane. This process is catalyzed by the 100-kDa GTPase dynamin, which probably activates (as yet largely unknown) effectors of the fission machinery (2.Sever S. Muhlberg A.B. Schmid S.L. Nature. 1999; 398: 481-486Crossref PubMed Scopus (313) Google Scholar). Three closely related mammalian dynamin isoforms have been identified: neuronal dynamin-1, ubiquitously expressed dynamin-2, and dynamin-3, which is expressed in testes, neurons, and lung (3.Schmid S.L. McNiven M.A. De Camilli P. Curr. Opin. Cell Biol. 1998; 10: 504-512Crossref PubMed Scopus (354) Google Scholar). Comparison of the primary sequence shows that all three dynamin isoforms contain three highly conserved GTP-binding motifs (i.e. elements I, II, and III). A Lys44 → Ala substitution in the first of the three putative GTP-binding motifs yields a dominant-negative dynamin mutant, which displays strongly impaired GTPase activity and is predicted to have a greatly reduced GTP binding affinity (4.Van 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 (586) Google Scholar). The two other GTP-binding motifs in dynamin are likely to be involved in GTP binding as well. Mutation of the third GTP-binding motif (substitution Lys206 → Asp in element III) or removal of all three GTP-binding motifs (amino acids 1–271 in dynamin-1) drastically reduces clathrin-coated vesicle-mediated internalization (4.Van 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 (586) Google Scholar, 5.Herskovits J.S. Burgess C.C. Obar R.A. Vallee R.B. J. Cell Biol. 1993; 122: 565-578Crossref PubMed Scopus (394) Google Scholar, 6.Achiriloaie M. Barylko B. Albanesi J.P. Mol. Cell. Biol. 1999; 19: 1410-1415Crossref PubMed Scopus (143) Google Scholar). A second important regulator of dynamin function is phosphatidylinositol 4,5-bisphosphate (PIP2) (6.Achiriloaie M. Barylko B. Albanesi J.P. Mol. Cell. Biol. 1999; 19: 1410-1415Crossref PubMed Scopus (143) Google Scholar, 7.Lin H.C. Gilman A.G. J. Biol. Chem. 1996; 271: 27979-27982Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 8.Barylko B. Binns D. Lin K.-M. Atkinson M.A.L. Jameson D.M. Yin H.L. Albanesi J.P. J. Biol. Chem. 1998; 273: 3791-3797Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 9.Klein D.E. Lee A. Frank D.W. Marks M.S. Lemmon M.A. J. Biol. Chem. 1998; 273: 27725-27733Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar). All three dynamin isoforms contain a pleckstrin homology domain that is able to bind PIP2. Binding of PIP2 to dynamin not only strongly increases the GTPase activity of dynamin but may also serve to target dynamin to the plasma membrane, allowing subsequent dynamin self-assembly at the neck of the clathrin-coated vesicle (6.Achiriloaie M. Barylko B. Albanesi J.P. Mol. Cell. Biol. 1999; 19: 1410-1415Crossref PubMed Scopus (143) Google Scholar, 7.Lin H.C. Gilman A.G. J. Biol. Chem. 1996; 271: 27979-27982Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 8.Barylko B. Binns D. Lin K.-M. Atkinson M.A.L. Jameson D.M. Yin H.L. Albanesi J.P. J. Biol. Chem. 1998; 273: 3791-3797Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 9.Klein D.E. Lee A. Frank D.W. Marks M.S. Lemmon M.A. J. Biol. Chem. 1998; 273: 27725-27733Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar). Expression of the dynamin mutant K535M, which is not stimulatable by PIP2, effectively blocks transferrin receptor internalization in clathrin-coated vesicles (6.Achiriloaie M. Barylko B. Albanesi J.P. Mol. Cell. Biol. 1999; 19: 1410-1415Crossref PubMed Scopus (143) Google Scholar). A large number of recent studies indicate that most GPCRs, including M1, M3, and M4 muscarinic acetylcholine receptors (mAChRs) in HEK-293 cells, internalize in clathrin-coated vesicles in a dynamin-dependent manner. This evidence is primarily based on the inhibitory effect of the dominant-negative inhibitor of dynamin-mediated internalization, K44A dynamin (10.Vögler 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 (78) Google Scholar, 11.Vögler O. Nolte B. Voss M. Schmidt M. Jakobs K.H. Van Koppen C.J. J. Biol. Chem. 1999; 274: 12333-12338Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 12.Pals-Rylaarsdam R. Gurevich V.V. Lee K.B. Ptasiensky J.A. Benovic J.L. Hosey M.M. J. Biol. Chem. 1997; 272: 23682-23689Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 13.Zhang 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). In contrast, M2 mAChRs internalize in a clathrin-independent manner and irrespective of expression of K44A dynamin in HEK-293 cells (10.Vögler 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 (78) Google Scholar, 12.Pals-Rylaarsdam R. Gurevich V.V. Lee K.B. Ptasiensky J.A. Benovic J.L. Hosey M.M. J. Biol. Chem. 1997; 272: 23682-23689Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). Likewise, internalization of angiotensin AT1A receptors (AT1ARs) (13.Zhang 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), dopamine D2 receptors (14.Vickery R.G. von Zastrow M. J. Cell Biol. 1999; 144: 31-43Crossref PubMed Scopus (197) Google Scholar), and secretin receptors (15.Walker J.K.L. Premont R.T. Barak L.S. Caron M.G. Shetzline M.A. J. Biol. Chem. 1999; 274: 31515-31523Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar) is also insensitive to expression of K44A dynamin. This has led to the proposal that internalization of these GPCRs is dynamin-independent. However, in light of the notion that the binding of GTP to dynamin probably involves binding to all three GTP- binding motifs in the GTP-binding pocket, we reasoned that a dynamin mutant lacking all three GTP-binding motifs might be a more appropriate dominant negative dynamin mutant to determine whether internalization of a particular GPCR is dynamin-dependent. Indeed, we here demonstrate that internalization of M2 mAChR and AT1AR is strongly inhibited by expression of N272 dynamin, which lacks the complete GTP-binding domain. Also, expression of K535M dynamin, which lacks PIP2-stimulated GTPase activity, significantly blocks internalization of these GPCR species. DISCUSSIONIn the past few years, the question whether dynamin plays an essential role in the internalization of a particular GPCR has been mostly analyzed by using K44A dynamin as dominant-negative mutant. While internalization of most GPCRs is blocked by expression of K44A dynamin, some GPCRs like the M2 mAChRs, D2dopamine receptors, secretin receptors, and AT1ARs internalize irrespective of K44A dynamin expression, suggesting that internalization of these GPCRs is dynamin-independent (10.Vögler 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 (78) Google Scholar, 12.Pals-Rylaarsdam R. Gurevich V.V. Lee K.B. Ptasiensky J.A. Benovic J.L. Hosey M.M. J. Biol. Chem. 1997; 272: 23682-23689Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 13.Zhang 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, 14.Vickery R.G. von Zastrow M. J. Cell Biol. 1999; 144: 31-43Crossref PubMed Scopus (197) Google Scholar, 15.Walker J.K.L. Premont R.T. Barak L.S. Caron M.G. Shetzline M.A. J. Biol. Chem. 1999; 274: 31515-31523Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). We now report that, contrary to what is currently postulated, internalization of M2 mAChR and AT1AR is dynamin-dependent. Coexpression of the dominant-negative dynamin mutants N272 and K535M strongly inhibited M2 mAChR and AT1AR internalization in HEK-293 tsA201 cells. These findings imply that N272 and K535M dynamin are more appropriate dominant-negative dynamin mutants than K44A dynamin. In this context, it will be interesting to determine whether fluid-phase endocytosis (5.Herskovits J.S. Burgess C.C. Obar R.A. Vallee R.B. J. Cell Biol. 1993; 122: 565-578Crossref PubMed Scopus (394) Google Scholar,20.Damke H. Baba T. Warnock D.E. Schmid S.L. J. Cell Biol. 1994; 127: 915-934Crossref PubMed Scopus (1034) Google Scholar) and internalization of ricin (21.Simpson J.C. Smith D.C. Roberts L.M. Lord J.M. Exp. Cell Res. 1998; 239: 293-300Crossref PubMed Scopus (63) Google Scholar) are affected by expression of N272 or K535M dynamin also, because these trafficking processes are not blocked by K44A dynamin and are thus considered to be dynamin-independent. It is intriguing that N272 dynamin, which lacks all three GTP-binding motifs, inhibits internalization of both M1 and M2 mAChR, while K44A dynamin, which lacks only the first GTP-binding motif, blocks only M1mAChR internalization. It is possible that K44A dynamin selectively sequesters away an essential component of the M1 but not of the M2 mAChR internalization pathway. Another potential explanation relates to the fact that K44A dynamin is able to coassemble with wild-type dynamin (22.Warnock D.E. Hinshaw J.E. Schmid S.L. J. Biol. Chem. 1996; 271: 22310-22314Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). Since dynamin assembly and interaction of dynamin with other proteins requires the C terminus of the dynamin, which varies among the dynamin isoforms (23.Smirnova E. Shurland D.-L. Newman-Smith E.D. Pishvaee B. Van der Bliek A.M. J. Biol. Chem. 1999; 274: 14942-14947Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar), different internalization pathways may use different dynamin isoforms. As a result, different internalization pathways may display differential sensitivity toward interference of K44A dynamin. Perhaps assembled GTP-bound K44A dynamin is sufficiently active to catalyze the budding of M2 mAChR- and AT1AR-containing vesicles from the plasma membrane but is not able to support internalization of M1 mAChRs in clathrin-coated vesicles.In the present study, we observed that mAChR and AT1AR internalization in HEK-293 cells is strongly inhibited by expression of K535M dynamin, a dynamin mutant, which lacks the putative PIP2 binding site (6.Achiriloaie M. Barylko B. Albanesi J.P. Mol. Cell. Biol. 1999; 19: 1410-1415Crossref PubMed Scopus (143) Google Scholar). At present, it is unknown at which stage of the vesicle budding process PIP2 binding to dynamin is essential. It has been postulated that, after recruitment of dynamin to the clathrin-coated pit, dynamin's interaction with the plasma membrane is strengthened by the binding of dynamin's pleckstrin homology domain with PIP2 in the plasma membrane (8.Barylko B. Binns D. Lin K.-M. Atkinson M.A.L. Jameson D.M. Yin H.L. Albanesi J.P. J. Biol. Chem. 1998; 273: 3791-3797Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). In addition, PIP2 binding might promote self-assembly of the dynamin molecules at the neck of the clathrin-coated pit and stimulate dynamin's GTPase activity (8.Barylko B. Binns D. Lin K.-M. Atkinson M.A.L. Jameson D.M. Yin H.L. Albanesi J.P. J. Biol. Chem. 1998; 273: 3791-3797Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 9.Klein D.E. Lee A. Frank D.W. Marks M.S. Lemmon M.A. J. Biol. Chem. 1998; 273: 27725-27733Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar). An alternative possibility is that lysine 535 in the pleckstrin homology domain of dynamin serves to promote interaction of dynamin with proteins rather than with PIP2 (6.Achiriloaie M. Barylko B. Albanesi J.P. Mol. Cell. Biol. 1999; 19: 1410-1415Crossref PubMed Scopus (143) Google Scholar). Regardless of the mechanism, our study clearly underscores the relevance of dynamin's lysine 535 residue in GPCR internalization. Identification of the binding partners of dynamin's pleckstrin homology domain will be important for understanding the mechanisms of GPCR internalization.In analogy with recent studies on the regulation of internalization of β2-adrenergic receptors in HEK-293 cells (16.Ahn S. Maudsley S. Luttrell L.M. Lefkowitz R.J. Daaka Y. J. Biol. Chem. 1999; 274: 1185-1188Abstract Full Text Full Text PDF PubMed Scopus (243) Google Scholar, 24.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: 655-661Crossref PubMed Scopus (1250) Google Scholar), we show that internalization of M1 mAChRs is strongly reduced by inhibition of c-Src activity and by overexpression of Y231F,Y597F dynamin, which cannot be phosphorylated by c-Src. Since M1 mAChRs internalize in clathrin-coated vesicles in a β-arrestin-dependent manner, we propose that, in analogy to β2-adrenergic receptors, internalization of M1 mAChRs involves β-arrestin-mediated targeting of receptor in the clathrin-coated pit and activation of c-Src by β-arrestin. c-Src then phosphorylates dynamin, a process that is required for M1 mAChR internalization in HEK-293 cells. Whether tyrosine phosphorylation activates dynamin or allows activation of dynamin by other molecules remains to be determined. In contrast, dynamin-mediated internalization of M2 mAChRs was found not to be inhibited by expression of kinase-defective K298M c-Src or treatment of the cells with the specific c-Src inhibitor PP1. Thus, c-Src does not play a role in M2 mAChR internalization. These findings are supported by recent studies showing that M2 mAChR internalization in HEK-293 cells is β-arrestin-independent (11.Vögler O. Nolte B. Voss M. Schmidt M. Jakobs K.H. Van Koppen C.J. J. Biol. Chem. 1999; 274: 12333-12338Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 12.Pals-Rylaarsdam R. Gurevich V.V. Lee K.B. Ptasiensky J.A. Benovic J.L. Hosey M.M. J. Biol. Chem. 1997; 272: 23682-23689Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). However, treatment of the cells with the generic tyrosine kinase, genistein, or coexpression of Y231F,Y597F dynamin did slightly reduce M2 mAChR internalization. This suggests that M2 mAChR internalization is regulated to a very limited extent by phosphorylation of dynamin by tyrosine kinases other than c-Src. On the basis of the present and previous findings (11.Vögler O. Nolte B. Voss M. Schmidt M. Jakobs K.H. Van Koppen C.J. J. Biol. Chem. 1999; 274: 12333-12338Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar), we propose that M2 mAChR internalization in HEK-293 cells is catalyzed by a dynamin isoform that differs from the dynamin isoform involved in clathrin-mediated M1 mAChR internalization. Much remains to be learned about the internalization pathway of M2 mAChR (and AT1AR) in HEK-293 cells. We have observed that pretreatment of HEK-293 cells with 0.45m sucrose fully blocks M2 (and M1) mAChR internalization in HEK-293 cells. 2Y. Werbonat, N. Kleutges, K. H. Jakobs, and C. J. van Koppen, unpublished observations. Yet expression of a dominant-negative clathrin mutant or β-arrestin V53D, which inhibits clathrin-mediated internalization of GPCRs, blocks M1 but not M2 mAChR internalization (11.Vögler O. Nolte B. Voss M. Schmidt M. Jakobs K.H. Van Koppen C.J. J. Biol. Chem. 1999; 274: 12333-12338Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). Similarly, expression of the amphiphysin SH3 domain, which blocks the targeting of dynamin to clathrin-coated pits, inhibits M1 but not M2mAChR internalization in HEK-293 cells.2 On the basis of these findings, we conclude that M2 mAChR internalization in HEK-293 cells is clathrin-independent and that the inhibitory effect of sucrose on receptor internalization is not specific for clathrin-mediated internalization. In this respect, it is important to note that hypertonic sucrose treatment of HEK-293 cells does not only block vesicle formation at the plasma membrane but may also induce other cellular responses including MAP kinase activation (25.Schramm N.L. Limbird L.E. J. Biol. Chem. 1999; 274: 24935-24940Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar), which may inhibit receptor internalization indirectly (26.Pitcher J.A. Tesmer J.J.G. Freeman J.L.R. Capel W.D. Stone W.C. Lefkowitz R.J. J. Biol. Chem. 1999; 274: 34531-34534Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). Interestingly, similar findings have been obtained recently with the secretin receptor. Internalization of secretin receptors in HEK-293 cells is unaffected by expression of dynamin K44A and β-arrestin V53D but is sensitive to sucrose pretreatment (15.Walker J.K.L. Premont R.T. Barak L.S. Caron M.G. Shetzline M.A. J. Biol. Chem. 1999; 274: 31515-31523Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Presently, it is unknown through which vesicles AT1AR internalizes in HEK-293 cells. In adrenal glomerulosa and Chinese hamster ovary cells, AT1AR may internalize through clathrin-coated vesicles, although this has only been inferred from biochemical (and not morphological) experiments using hypertonic sucrose treatment or potassium depletion (27.Hunyady L. J. Am. Soc. Nephrol. 1999; 10: S47-S56PubMed Google Scholar). In vascular smooth muscle cells, however, AT1ARs have been found to internalize through noncoated pits, possibly caveolae, as well as coated pits (27.Hunyady L. J. Am. Soc. Nephrol. 1999; 10: S47-S56PubMed Google Scholar, 28.Ishizaka N. Griendling K.K. Lassègue B. Alexander R.W. Hypertension. 1998; 32: 459-466Crossref PubMed Scopus (167) Google Scholar). Thus, the internalization of AT1AR and other GPCRs (10.Vögler 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 (78) Google Scholar) appears to differ among different cell types. The identification of the budding vesicles through which AT1AR and M2 mAChR internalize in HEK-293 cells and other cell types will provide important information on the molecular mechanisms of GPCR internalization. In the past few years, the question whether dynamin plays an essential role in the internalization of a particular GPCR has been mostly analyzed by using K44A dynamin as dominant-negative mutant. While internalization of most GPCRs is blocked by expression of K44A dynamin, some GPCRs like the M2 mAChRs, D2dopamine receptors, secretin receptors, and AT1ARs internalize irrespective of K44A dynamin expression, suggesting that internalization of these GPCRs is dynamin-independent (10.Vögler 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 (78) Google Scholar, 12.Pals-Rylaarsdam R. Gurevich V.V. Lee K.B. Ptasiensky J.A. Benovic J.L. Hosey M.M. J. Biol. Chem. 1997; 272: 23682-23689Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 13.Zhang 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, 14.Vickery R.G. von Zastrow M. J. Cell Biol. 1999; 144: 31-43Crossref PubMed Scopus (197) Google Scholar, 15.Walker J.K.L. Premont R.T. Barak L.S. Caron M.G. Shetzline M.A. J. Biol. Chem. 1999; 274: 31515-31523Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). We now report that, contrary to what is currently postulated, internalization of M2 mAChR and AT1AR is dynamin-dependent. Coexpression of the dominant-negative dynamin mutants N272 and K535M strongly inhibited M2 mAChR and AT1AR internalization in HEK-293 tsA201 cells. These findings imply that N272 and K535M dynamin are more appropriate dominant-negative dynamin mutants than K44A dynamin. In this context, it will be interesting to determine whether fluid-phase endocytosis (5.Herskovits J.S. Burgess C.C. Obar R.A. Vallee R.B. J. Cell Biol. 1993; 122: 565-578Crossref PubMed Scopus (394) Google Scholar,20.Damke H. Baba T. Warnock D.E. Schmid S.L. J. Cell Biol. 1994; 127: 915-934Crossref PubMed Scopus (1034) Google Scholar) and internalization of ricin (21.Simpson J.C. Smith D.C. Roberts L.M. Lord J.M. Exp. Cell Res. 1998; 239: 293-300Crossref PubMed Scopus (63) Google Scholar) are affected by expression of N272 or K535M dynamin also, because these trafficking processes are not blocked by K44A dynamin and are thus considered to be dynamin-independent. It is intriguing that N272 dynamin, which lacks all three GTP-binding motifs, inhibits internalization of both M1 and M2 mAChR, while K44A dynamin, which lacks only the first GTP-binding motif, blocks only M1mAChR internalization. It is possible that K44A dynamin selectively sequesters away an essential component of the M1 but not of the M2 mAChR internalization pathway. Another potential explanation relates to the fact that K44A dynamin is able to coassemble with wild-type dynamin (22.Warnock D.E. Hinshaw J.E. Schmid S.L. J. Biol. Chem. 1996; 271: 22310-22314Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). Since dynamin assembly and interaction of dynamin with other proteins requires the C terminus of the dynamin, which varies among the dynamin isoforms (23.Smirnova E. Shurland D.-L. Newman-Smith E.D. Pishvaee B. Van der Bliek A.M. J. Biol. Chem. 1999; 274: 14942-14947Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar), different internalization pathways may use different dynamin isoforms. As a result, different internalization pathways may display differential sensitivity toward interference of K44A dynamin. Perhaps assembled GTP-bound K44A dynamin is sufficiently active to catalyze the budding of M2 mAChR- and AT1AR-containing vesicles from the plasma membrane but is not able to support internalization of M1 mAChRs in clathrin-coated vesicles. In the present study, we observed that mAChR and AT1AR internalization in HEK-293 cells is strongly inhibited by expression of K535M dynamin, a dynamin mutant, which lacks the putative PIP2 binding site (6.Achiriloaie M. Barylko B. Albanesi J.P. Mol. Cell. Biol. 1999; 19: 1410-1415Crossref PubMed Scopus (143) Google Scholar). At present, it is unknown at which stage of the vesicle budding process PIP2 binding to dynamin is essential. It has been postulated that, after recruitment of dynamin to the clathrin-coated pit, dynamin's interaction with the plasma membrane is strengthened by the binding of dynamin's pleckstrin homology domain with PIP2 in the plasma membrane (8.Barylko B. Binns D. Lin K.-M. Atkinson M.A.L. Jameson D.M. Yin H.L. Albanesi J.P. J. Biol. Chem. 1998; 273: 3791-3797Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). In addition, PIP2 binding might promote self-assembly of the dynamin molecules at the neck of the clathrin-coated pit and stimulate dynamin's GTPase activity (8.Barylko B. Binns D. Lin K.-M. Atkinson M.A.L. Jameson D.M. Yin H.L. Albanesi J.P. J. Biol. Chem. 1998; 273: 3791-3797Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 9.Klein D.E. Lee A. Frank D.W. Marks M.S. Lemmon M.A. J. Biol. Chem. 1998; 273: 27725-27733Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar). An alternative possibility is that lysine 535 in the pleckstrin homology domain of dynamin serves to promote interaction of dynamin with proteins rather than with PIP2 (6.Achiriloaie M. Barylko B. Albanesi J.P. Mol. Cell. Biol. 1999; 19: 1410-1415Crossref PubMed Scopus (143) Google Scholar). Regardless of the mechanism, our study clearly underscores the relevance of dynamin's lysine 535 residue in GPCR internalization. Identification of the binding partners of dynamin's pleckstrin homology domain will be important for understanding the mechanisms of GPCR internalization. In analogy with recent studies on the regulation of internalization of β2-adrenergic receptors in HEK-293 cells (16.Ahn S. Maudsley S. Luttrell L.M. Lefkowitz R.J. Daaka Y. J. Biol. Chem. 1999; 274: 1185-1188Abstract Full Text Full Text PDF PubMed Scopus (243) Google Scholar, 24.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: 655-661Crossref PubMed Scopus (1250) Google Scholar), we show that internalization of M1 mAChRs is strongly reduced by inhibition of c-Src activity and by overexpression of Y231F,Y597F dynamin, which cannot be phosphorylated by c-Src. Since M1 mAChRs internalize in clathrin-coated vesicles in a β-arrestin-dependent manner, we propose that, in analogy to β2-adrenergic receptors, internalization of M1 mAChRs involves β-arrestin-mediated targeting of receptor in the clathrin-coated pit and activation of c-Src by β-arrestin. c-Src then phosphorylates dynamin, a process that is required for M1 mAChR internalization in HEK-293 cells. Whether tyrosine phosphorylation activates dynamin or allows activation of dynamin by other molecules remains to be determined. In contrast, dynamin-mediated internalization of M2 mAChRs was found not to be inhibited by expression of kinase-defective K298M c-Src or treatment of the cells with the specific c-Src inhibitor PP1. Thus, c-Src does not play a role in M2 mAChR internalization. These findings are supported by recent studies showing that M2 mAChR internalization in HEK-293 cells is β-arrestin-independent (11.Vögler O. Nolte B. Voss M. Schmidt M. Jakobs K.H. Van Koppen C.J. J. Biol. Chem. 1999; 274: 12333-12338Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 12.Pals-Rylaarsdam R. Gurevich V.V. Lee K.B. Ptasiensky J.A. Benovic J.L. Hosey M.M. J. Biol. Chem. 1997; 272: 23682-23689Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). However, treatment of the cells with the generic tyrosine kinase, genistein, or coexpression of Y231F,Y597F dynamin did slightly reduce M2 mAChR internalization. This suggests that M2 mAChR internalization is regulated to a very limited extent by phosphorylation of dynamin by tyrosine kinases other than c-Src. On the basis of the present and previous findings (11.Vögler O. Nolte B. Voss M. Schmidt M. Jakobs K.H. Van Koppen C.J. J. Biol. Chem. 1999; 274: 12333-12338Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar), we propose that M2 mAChR internalization in HEK-293 cells is catalyzed by a dynamin isoform that differs from the dynamin isoform involved in clathrin-mediated M1 mAChR internalization. Much remains to be learned about the internalization pathway of M2 mAChR (and AT1AR) in HEK-293 cells. We have observed that pretreatment of HEK-293 cells with 0.45m sucrose fully blocks M2 (and M1) mAChR internalization in HEK-293 cells. 2Y. Werbonat, N. Kleutges, K. H. Jakobs, and C. J. van Koppen, unpublished observations. Yet expression of a dominant-negative clathrin mutant or β-arrestin V53D, which inhibits clathrin-mediated internalization of GPCRs, blocks M1 but not M2 mAChR internalization (11.Vögler O. Nolte B. Voss M. Schmidt M. Jakobs K.H. Van Koppen C.J. J. Biol. Chem. 1999; 274: 12333-12338Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). Similarly, expression of the amphiphysin SH3 domain, which blocks the targeting of dynamin to clathrin-coated pits, inhibits M1 but not M2mAChR internalization in HEK-293 cells.2 On the basis of these findings, we conclude that M2 mAChR internalization in HEK-293 cells is clathrin-independent and that the inhibitory effect of sucrose on receptor internalization is not specific for clathrin-mediated internalization. In this respect, it is important to note that hypertonic sucrose treatment of HEK-293 cells does not only block vesicle formation at the plasma membrane but may also induce other cellular responses including MAP kinase activation (25.Schramm N.L. Limbird L.E. J. Biol. Chem. 1999; 274: 24935-24940Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar), which may inhibit receptor internalization indirectly (26.Pitcher J.A. Tesmer J.J.G. Freeman J.L.R. Capel W.D. Stone W.C. Lefkowitz R.J. J. Biol. Chem. 1999; 274: 34531-34534Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). Interestingly, similar findings have been obtained recently with the secretin receptor. Internalization of secretin receptors in HEK-293 cells is unaffected by expression of dynamin K44A and β-arrestin V53D but is sensitive to sucrose pretreatment (15.Walker J.K.L. Premont R.T. Barak L.S. Caron M.G. Shetzline M.A. J. Biol. Chem. 1999; 274: 31515-31523Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Presently, it is unknown through which vesicles AT1AR internalizes in HEK-293 cells. In adrenal glomerulosa and Chinese hamster ovary cells, AT1AR may internalize through clathrin-coated vesicles, although this has only been inferred from biochemical (and not morphological) experiments using hypertonic sucrose treatment or potassium depletion (27.Hunyady L. J. Am. Soc. Nephrol. 1999; 10: S47-S56PubMed Google Scholar). In vascular smooth muscle cells, however, AT1ARs have been found to internalize through noncoated pits, possibly caveolae, as well as coated pits (27.Hunyady L. J. Am. Soc. Nephrol. 1999; 10: S47-S56PubMed Google Scholar, 28.Ishizaka N. Griendling K.K. Lassègue B. Alexander R.W. Hypertension. 1998; 32: 459-466Crossref PubMed Scopus (167) Google Scholar). Thus, the internalization of AT1AR and other GPCRs (10.Vögler 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 (78) Google Scholar) appears to differ among different cell types. The identification of the budding vesicles through which AT1AR and M2 mAChR internalize in HEK-293 cells and other cell types will provide important information on the molecular mechanisms of GPCR internalization. We thank Riccarda Krudewig and Barbara Langer for expert technical assistance. We are indebted to Drs. J. P. Albanesi, L. Hein, R. J. Lefkowitz, J. T. Parsons, and S. Schmid for their gift of the various DNA plasmids and Dr. G. Rijksen for providing anti-Src antibody.

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