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

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; PubMed Scopus Google Scholar, B. Albanesi J.P. J. Biol. Chem. 1998; PubMed Scopus Google Scholar, M.A. J. Biol. Chem. 1998; PubMed Scopus Google Scholar). three dynamin isoforms contain a that is to of to dynamin not only strongly the GTPase activity of dynamin but also to dynamin to the plasma dynamin the 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; PubMed Scopus Google Scholar, B. Albanesi J.P. J. Biol. Chem. 1998; PubMed Scopus Google Scholar, M.A. J. Biol. Chem. 1998; PubMed Scopus Google Scholar). Expression of the dynamin which is not stimulatable by receptor internalization in clathrin-coated (6.Achiriloaie M. Barylko B. Albanesi J.P. Mol. Cell. Biol. 1999; 19: 1410-1415Crossref PubMed Scopus (143) Google of indicate that most including and muscarinic acetylcholine receptors in HEK-293 internalize in clathrin-coated in a This is the of the dominant-negative inhibitor of K44A dynamin P. J. Biol. Chem. 1998; PubMed Scopus Google Scholar, B. M. M. J. Biol. Chem. 1999; PubMed Scopus Google Scholar, Gurevich V.V. Benovic J.L. J. Biol. Chem. PubMed Scopus Google Scholar, J. J. Biol. Chem. 1996; PubMed Scopus Google Scholar). In contrast, M2 mAChRs internalize in a and irrespective of expression of K44A dynamin in HEK-293 cells P. J. Biol. Chem. 1998; PubMed Scopus Google Scholar, Gurevich V.V. Benovic J.L. J. Biol. Chem. PubMed Scopus Google Scholar). internalization of angiotensin AT1A receptors J. J. Biol. Chem. 1996; PubMed Scopus Google receptors M. J. Cell Biol. 1999; PubMed Scopus Google and receptors M.A. J. Biol. Chem. 1999; PubMed Scopus Google is also to expression of K44A This has led to the proposal that internalization of these GPCRs is dynamin-independent. in of the that the binding of GTP to dynamin probably binding to all three binding motifs in the GTP-binding we that a dynamin all three GTP-binding motifs be a dynamin to internalization of a is dynamin-dependent. we that internalization of M2 mAChR and AT1AR is strongly by expression of N272 dynamin, which lacks the GTP-binding expression of K535M dynamin, which lacks GTPase internalization of these the the dynamin essential in the internalization of a has been by K44A dynamin as dominant-negative internalization of most GPCRs is by expression of K44A dynamin, some GPCRs like the M2 and internalize irrespective of K44A dynamin that internalization of these GPCRs is P. J. Biol. Chem. 1998; PubMed Scopus Google Scholar, Gurevich V.V. Benovic J.L. J. Biol. Chem. PubMed Scopus Google Scholar, J. J. Biol. Chem. 1996; PubMed Scopus Google Scholar, M. J. Cell Biol. 1999; PubMed Scopus Google Scholar, M.A. J. Biol. Chem. 1999; PubMed Scopus Google Scholar). report that, contrary to what is postulated, internalization of M2 mAChR and AT1AR is dynamin-dependent. of the dominant-negative dynamin N272 and K535M strongly M2 mAChR and AT1AR internalization in HEK-293 cells. findings that N272 and K535M dynamin are dominant-negative dynamin K44A In this be to J.S. Burgess C.C. Obar R.A. Vallee R.B. J. Cell Biol. 1993; 122: 565-578Crossref PubMed Scopus (394) Google H. Schmid S.L. J. Cell Biol. PubMed Scopus Google and internalization of Cell 1998; PubMed Scopus Google are by expression of N272 or K535M dynamin these are not by K44A dynamin and are to be dynamin-independent. is that N272 dynamin, which lacks all three GTP-binding inhibits internalization of M1 and M2 K44A dynamin, which lacks only the first GTP-binding only internalization. is that K44A dynamin essential of the M1 but not of the M2 mAChR internalization to the that K44A dynamin is to with dynamin Schmid S.L. J. Biol. Chem. 1996; PubMed Scopus Google Scholar). dynamin and interaction of dynamin with other proteins requires the of the dynamin, which the dynamin isoforms B. der Bliek A.M. J. Biol. Chem. 1999; PubMed Scopus Google internalization dynamin a internalization of K44A K44A dynamin is to the of M2 and from the plasma but is not to internalization of M1 mAChRs in clathrin-coated the we that mAChR and AT1AR internalization in HEK-293 cells is strongly by expression of K535M dynamin, a dynamin mutant, which lacks the putative binding (6.Achiriloaie M. Barylko B. Albanesi J.P. Mol. Cell. Biol. 1999; 19: 1410-1415Crossref PubMed Scopus (143) Google Scholar). is which of the vesicle process binding to dynamin is has been that, of dynamin to the clathrin-coated interaction with the plasma is by the binding of with in the plasma B. Albanesi J.P. J. Biol. Chem. 1998; PubMed Scopus Google Scholar). In binding of the dynamin the of the clathrin-coated pit and GTPase activity B. Albanesi J.P. J. Biol. Chem. 1998; PubMed Scopus Google Scholar, M.A. J. Biol. Chem. 1998; PubMed Scopus Google Scholar). is that in the of dynamin to interaction of dynamin with proteins with (6.Achiriloaie M. Barylko B. Albanesi J.P. Mol. Cell. Biol. 1999; 19: 1410-1415Crossref PubMed Scopus (143) Google Scholar). of the the of in internalization. of the binding of be important for the of with the of internalization of receptors in HEK-293 cells S. S. J. Biol. Chem. 1999; PubMed Scopus Google Scholar, S. H. 1999; PubMed Scopus Google we that internalization of M1 mAChRs is strongly reduced by of c-Src activity and by of Y231F,Y597F dynamin, which cannot be phosphorylated by M1 mAChRs internalize in clathrin-coated in a we that, in to internalization of M1 mAChRs of receptor in the clathrin-coated pit and of c-Src by c-Src then dynamin, a process that is for M1 mAChR internalization in HEK-293 cells. tyrosine phosphorylation activates dynamin or of dynamin by other to be In contrast, internalization of M2 mAChRs not to be by expression of kinase-defective K298M c-Src or treatment of the cells with the c-Src inhibitor Thus, c-Src not a in M2 mAChR internalization. findings are by that M2 mAChR internalization in HEK-293 cells is B. M. M. J. Biol. Chem. 1999; PubMed Scopus Google Scholar, Gurevich V.V. Benovic J.L. J. Biol. Chem. PubMed Scopus Google Scholar). treatment of the cells with the generic tyrosine or of Y231F,Y597F dynamin M2 mAChR internalization. This that M2 mAChR internalization is regulated to a by phosphorylation of dynamin by tyrosine kinases other the of the and findings B. M. M. J. Biol. Chem. 1999; PubMed Scopus Google we that M2 mAChR internalization in HEK-293 cells is catalyzed by a dynamin that from the dynamin involved in M1 mAChR internalization. to be the internalization of M2 mAChR in HEK-293 cells. have that of HEK-293 cells with M2 mAChR internalization in HEK-293 cells. H. and J. expression of a dominant-negative clathrin or β-arrestin which inhibits internalization of M1 but not M2 mAChR internalization B. M. M. J. Biol. Chem. 1999; PubMed Scopus Google Scholar). Similarly, expression of the domain, which the of dynamin to clathrin-coated inhibits M1 but not M2mAChR internalization in HEK-293 the of these we that M2 mAChR internalization in HEK-293 cells is and that the of receptor internalization is not for internalization. In this is important to that treatment of HEK-293 cells not only vesicle the plasma but also other including kinase J. Biol. Chem. 1999; PubMed Scopus Google which inhibit receptor internalization J. Biol. Chem. 1999; PubMed Scopus Google Scholar). findings have been with the of receptors in HEK-293 cells is by expression of dynamin K44A and β-arrestin but is to M.A. J. Biol. Chem. 1999; PubMed Scopus Google Scholar). is which AT1AR in HEK-293 cells. In and AT1AR internalize clathrin-coated vesicles, this has only been from not treatment or J. 1999; 10: Google Scholar). In have been to internalize as well as J. 1999; 10: Google Scholar, B. 1998; PubMed Scopus Google Scholar). Thus, the internalization of AT1AR and other GPCRs P. J. Biol. Chem. 1998; PubMed Scopus Google to The of the which AT1AR and M2 mAChR internalize in HEK-293 cells and other important the of 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; PubMed Scopus Google Scholar, B. Albanesi J.P. J. Biol. Chem. 1998; PubMed Scopus Google Scholar, M.A. J. Biol. Chem. 1998; PubMed Scopus Google Scholar). three dynamin isoforms contain a that is to of to dynamin not only strongly the GTPase activity of dynamin but also to dynamin to the plasma dynamin the 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; PubMed Scopus Google Scholar, B. Albanesi J.P. J. Biol. Chem. 1998; PubMed Scopus Google Scholar, M.A. J. Biol. Chem. 1998; PubMed Scopus Google Scholar). Expression of the dynamin which is not stimulatable by receptor internalization in clathrin-coated (6.Achiriloaie M. Barylko B. Albanesi J.P. Mol. Cell. Biol. 1999; 19: 1410-1415Crossref PubMed Scopus (143) Google Scholar). A of indicate that most including and muscarinic acetylcholine receptors in HEK-293 internalize in clathrin-coated in a This is the of the dominant-negative inhibitor of K44A dynamin P. J. Biol. Chem. 1998; PubMed Scopus Google Scholar, B. M. M. J. Biol. Chem. 1999; PubMed Scopus Google Scholar, Gurevich V.V. Benovic J.L. J. Biol. Chem. PubMed Scopus Google Scholar, J. J. Biol. Chem. 1996; PubMed Scopus Google Scholar). In contrast, M2 mAChRs internalize in a and irrespective of expression of K44A dynamin in HEK-293 cells P. J. Biol. Chem. 1998; PubMed Scopus Google Scholar, Gurevich V.V. Benovic J.L. J. Biol. Chem. PubMed Scopus Google Scholar). internalization of angiotensin AT1A receptors J. J. Biol. Chem. 1996; PubMed Scopus Google receptors M. J. Cell Biol. 1999; PubMed Scopus Google and receptors M.A. J. Biol. Chem. 1999; PubMed Scopus Google is also to expression of K44A This has led to the proposal that internalization of these GPCRs is dynamin-independent. in of the that the binding of GTP to dynamin probably binding to all three binding motifs in the GTP-binding we that a dynamin all three GTP-binding motifs be a dynamin to internalization of a is dynamin-dependent. we that internalization of M2 mAChR and AT1AR is strongly by expression of N272 dynamin, which lacks the GTP-binding expression of K535M dynamin, which lacks GTPase internalization of these the the dynamin essential in the internalization of a has been by K44A dynamin as dominant-negative internalization of most GPCRs is by expression of K44A dynamin, some GPCRs like the M2 and internalize irrespective of K44A dynamin that internalization of these GPCRs is P. J. Biol. Chem. 1998; PubMed Scopus Google Scholar, Gurevich V.V. Benovic J.L. J. Biol. Chem. PubMed Scopus Google Scholar, J. J. Biol. Chem. 1996; PubMed Scopus Google Scholar, M. J. Cell Biol. 1999; PubMed Scopus Google Scholar, M.A. J. Biol. Chem. 1999; PubMed Scopus Google Scholar). report that, contrary to what is postulated, internalization of M2 mAChR and AT1AR is dynamin-dependent. of the dominant-negative dynamin N272 and K535M strongly M2 mAChR and AT1AR internalization in HEK-293 cells. findings that N272 and K535M dynamin are dominant-negative dynamin K44A In this be to J.S. Burgess C.C. Obar R.A. Vallee R.B. J. Cell Biol. 1993; 122: 565-578Crossref PubMed Scopus (394) Google H. Schmid S.L. J. Cell Biol. PubMed Scopus Google and internalization of Cell 1998; PubMed Scopus Google are by expression of N272 or K535M dynamin these are not by K44A dynamin and are to be dynamin-independent. is that N272 dynamin, which lacks all three GTP-binding inhibits internalization of M1 and M2 K44A dynamin, which lacks only the first GTP-binding only internalization. is that K44A dynamin essential of the M1 but not of the M2 mAChR internalization to the that K44A dynamin is to with dynamin Schmid S.L. J. Biol. Chem. 1996; PubMed Scopus Google Scholar). dynamin and interaction of dynamin with other proteins requires the of the dynamin, which the dynamin isoforms B. der Bliek A.M. J. Biol. Chem. 1999; PubMed Scopus Google internalization dynamin a internalization of K44A K44A dynamin is to the of M2 and from the plasma but is not to internalization of M1 mAChRs in clathrin-coated the we that mAChR and AT1AR internalization in HEK-293 cells is strongly by expression of K535M dynamin, a dynamin mutant, which lacks the putative binding (6.Achiriloaie M. Barylko B. Albanesi J.P. Mol. Cell. Biol. 1999; 19: 1410-1415Crossref PubMed Scopus (143) Google Scholar). is which of the vesicle process binding to dynamin is has been that, of dynamin to the clathrin-coated interaction with the plasma is by the binding of with in the plasma B. Albanesi J.P. J. Biol. Chem. 1998; PubMed Scopus Google Scholar). In binding of the dynamin the of the clathrin-coated pit and GTPase activity B. Albanesi J.P. J. Biol. Chem. 1998; PubMed Scopus Google Scholar, M.A. J. Biol. Chem. 1998; PubMed Scopus Google Scholar). is that in the of dynamin to interaction of dynamin with proteins with (6.Achiriloaie M. Barylko B. Albanesi J.P. Mol. Cell. Biol. 1999; 19: 1410-1415Crossref PubMed Scopus (143) Google Scholar). of the the of in internalization. of the binding of be important for the of with the of internalization of receptors in HEK-293 cells S. S. J. Biol. Chem. 1999; PubMed Scopus Google Scholar, S. H. 1999; PubMed Scopus Google we that internalization of M1 mAChRs is strongly reduced by of c-Src activity and by of Y231F,Y597F dynamin, which cannot be phosphorylated by M1 mAChRs internalize in clathrin-coated in a we that, in to internalization of M1 mAChRs of receptor in the clathrin-coated pit and of c-Src by c-Src then dynamin, a process that is for M1 mAChR internalization in HEK-293 cells. tyrosine phosphorylation activates dynamin or of dynamin by other to be In contrast, internalization of M2 mAChRs not to be by expression of kinase-defective K298M c-Src or treatment of the cells with the c-Src inhibitor Thus, c-Src not a in M2 mAChR internalization. findings are by that M2 mAChR internalization in HEK-293 cells is B. M. M. J. Biol. Chem. 1999; PubMed Scopus Google Scholar, Gurevich V.V. Benovic J.L. J. Biol. Chem. PubMed Scopus Google Scholar). treatment of the cells with the generic tyrosine or of Y231F,Y597F dynamin M2 mAChR internalization. This that M2 mAChR internalization is regulated to a by phosphorylation of dynamin by tyrosine kinases other the of the and findings B. M. M. J. Biol. Chem. 1999; PubMed Scopus Google we that M2 mAChR internalization in HEK-293 cells is catalyzed by a dynamin that from the dynamin involved in M1 mAChR internalization. to be the internalization of M2 mAChR in HEK-293 cells. have that of HEK-293 cells with M2 mAChR internalization in HEK-293 cells. H. and J. expression of a dominant-negative clathrin or β-arrestin which inhibits internalization of M1 but not M2 mAChR internalization B. M. M. J. Biol. Chem. 1999; PubMed Scopus Google Scholar). Similarly, expression of the domain, which the of dynamin to clathrin-coated inhibits M1 but not M2mAChR internalization in HEK-293 the of these we that M2 mAChR internalization in HEK-293 cells is and that the of receptor internalization is not for internalization. In this is important to that treatment of HEK-293 cells not only vesicle the plasma but also other including kinase J. Biol. Chem. 1999; PubMed Scopus Google which inhibit receptor internalization J. Biol. Chem. 1999; PubMed Scopus Google Scholar). findings have been with the of receptors in HEK-293 cells is by expression of dynamin K44A and β-arrestin but is to M.A. J. Biol. Chem. 1999; PubMed Scopus Google Scholar). is which AT1AR in HEK-293 cells. In and AT1AR internalize clathrin-coated vesicles, this has only been from not treatment or J. 1999; 10: Google Scholar). In have been to internalize as well as J. 1999; 10: Google Scholar, B. 1998; PubMed Scopus Google Scholar). Thus, the internalization of AT1AR and other GPCRs P. J. Biol. Chem. 1998; PubMed Scopus Google to The of the which AT1AR and M2 mAChR internalize in HEK-293 cells and other important the of internalization. In the the dynamin essential in the internalization of a has been by K44A dynamin as dominant-negative internalization of most GPCRs is by expression of K44A dynamin, some GPCRs like the M2 and internalize irrespective of K44A dynamin that internalization of these GPCRs is P. J. Biol. Chem. 1998; PubMed Scopus Google Scholar, Gurevich V.V. Benovic J.L. J. Biol. Chem. PubMed Scopus Google Scholar, J. J. Biol. Chem. 1996; PubMed Scopus Google Scholar, M. J. Cell Biol. 1999; PubMed Scopus Google Scholar, M.A. J. Biol. Chem. 1999; PubMed Scopus Google Scholar). report that, contrary to what is postulated, internalization of M2 mAChR and AT1AR is dynamin-dependent. of the dominant-negative dynamin N272 and K535M strongly M2 mAChR and AT1AR internalization in HEK-293 cells. findings that N272 and K535M dynamin are dominant-negative dynamin K44A In this be to J.S. Burgess C.C. Obar R.A. Vallee R.B. J. Cell Biol. 1993; 122: 565-578Crossref PubMed Scopus (394) Google H. Schmid S.L. J. Cell Biol. PubMed Scopus Google and internalization of Cell 1998; PubMed Scopus Google are by expression of N272 or K535M dynamin these are not by K44A dynamin and are to be dynamin-independent. is that N272 dynamin, which lacks all three GTP-binding inhibits internalization of M1 and M2 K44A dynamin, which lacks only the first GTP-binding only internalization. is that K44A dynamin essential of the M1 but not of the M2 mAChR internalization to the that K44A dynamin is to with dynamin Schmid S.L. J. Biol. Chem. 1996; PubMed Scopus Google Scholar). dynamin and interaction of dynamin with other proteins requires the of the dynamin, which the dynamin isoforms B. der Bliek A.M. J. Biol. Chem. 1999; PubMed Scopus Google internalization dynamin a internalization of K44A K44A dynamin is to the of M2 and from the plasma but is not to internalization of M1 mAChRs in clathrin-coated In the we that mAChR and AT1AR internalization in HEK-293 cells is strongly by expression of K535M dynamin, a dynamin mutant, which lacks the putative binding (6.Achiriloaie M. Barylko B. Albanesi J.P. Mol. Cell. Biol. 1999; 19: 1410-1415Crossref PubMed Scopus (143) Google Scholar). is which of the vesicle process binding to dynamin is has been that, of dynamin to the clathrin-coated interaction with the plasma is by the binding of with in the plasma B. Albanesi J.P. J. Biol. Chem. 1998; PubMed Scopus Google Scholar). In binding of the dynamin the of the clathrin-coated pit and GTPase activity B. Albanesi J.P. J. Biol. Chem. 1998; PubMed Scopus Google Scholar, M.A. J. Biol. Chem. 1998; PubMed Scopus Google Scholar). is that in the of dynamin to interaction of dynamin with proteins with (6.Achiriloaie M. Barylko B. Albanesi J.P. Mol. Cell. Biol. 1999; 19: 1410-1415Crossref PubMed Scopus (143) Google Scholar). of the the of in internalization. of the binding of be important for the of internalization. In with the of internalization of receptors in HEK-293 cells S. S. J. Biol. Chem. 1999; PubMed Scopus Google Scholar, S. H. 1999; PubMed Scopus Google we that internalization of M1 mAChRs is strongly reduced by of c-Src activity and by of Y231F,Y597F dynamin, which cannot be phosphorylated by M1 mAChRs internalize in clathrin-coated in a we that, in to internalization of M1 mAChRs of receptor in the clathrin-coated pit and of c-Src by c-Src then dynamin, a process that is for M1 mAChR internalization in HEK-293 cells. tyrosine phosphorylation activates dynamin or of dynamin by other to be In contrast, internalization of M2 mAChRs not to be by expression of kinase-defective K298M c-Src or treatment of the cells with the c-Src inhibitor Thus, c-Src not a in M2 mAChR internalization. findings are by that M2 mAChR internalization in HEK-293 cells is B. M. M. J. Biol. Chem. 1999; PubMed Scopus Google Scholar, Gurevich V.V. Benovic J.L. J. Biol. Chem. PubMed Scopus Google Scholar). treatment of the cells with the generic tyrosine or of Y231F,Y597F dynamin M2 mAChR internalization. This that M2 mAChR internalization is regulated to a by phosphorylation of dynamin by tyrosine kinases other the of the and findings B. M. M. J. Biol. Chem. 1999; PubMed Scopus Google we that M2 mAChR internalization in HEK-293 cells is catalyzed by a dynamin that from the dynamin involved in M1 mAChR internalization. to be the internalization of M2 mAChR in HEK-293 cells. have that of HEK-293 cells with M2 mAChR internalization in HEK-293 cells. H. and J. expression of a dominant-negative clathrin or β-arrestin which inhibits internalization of M1 but not M2 mAChR internalization B. M. M. J. Biol. Chem. 1999; PubMed Scopus Google Scholar). Similarly, expression of the domain, which the of dynamin to clathrin-coated inhibits M1 but not M2mAChR internalization in HEK-293 the of these we that M2 mAChR internalization in HEK-293 cells is and that the of receptor internalization is not for internalization. In this is important to that treatment of HEK-293 cells not only vesicle the plasma but also other including kinase J. Biol. Chem. 1999; PubMed Scopus Google which inhibit receptor internalization J. Biol. Chem. 1999; PubMed Scopus Google Scholar). findings have been with the of receptors in HEK-293 cells is by expression of dynamin K44A and β-arrestin but is to M.A. J. Biol. Chem. 1999; PubMed Scopus Google Scholar). is which AT1AR in HEK-293 cells. In and AT1AR internalize clathrin-coated vesicles, this has only been from not treatment or J. 1999; 10: Google Scholar). In have been to internalize as well as J. 1999; 10: Google Scholar, B. 1998; PubMed Scopus Google Scholar). Thus, the internalization of AT1AR and other GPCRs P. J. Biol. Chem. 1998; PubMed Scopus Google to The of the which AT1AR and M2 mAChR internalize in HEK-293 cells and other important the of internalization. and for are to J. P. J. J. and S. Schmid for of the and for