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• W2014211428 abstract "Some forms of G protein-coupled receptor signaling, such as activation of mitogen-activated protein kinase cascade as well as resensitization of receptors after hormone-induced desensitization, require receptor internalization via dynamin-dependent clathrin-coated pit mechanisms. Here we demonstrate that activation of β2-adrenergic receptors (β2-ARs) leads to c-Src-mediated tyrosine phosphorylation of dynamin, which is required for receptor internalization. Two tyrosine residues, Tyr231 and Tyr597, are identified as the major phosphorylation sites. Mutation of these residues to phenylalanine dramatically decreases the c-Src-mediated phosphorylation of dynamin following β2-AR stimulation. Moreover, expression of Y231F/Y597F dynamin inhibits β2-AR internalization and the isoproterenol-stimulated mitogen-activated protein kinase activation. Thus, agonist-induced, c-Src-mediated tyrosine phosphorylation of dynamin is essential for its function in clathrin mediated G protein-coupled receptor endocytosis. Some forms of G protein-coupled receptor signaling, such as activation of mitogen-activated protein kinase cascade as well as resensitization of receptors after hormone-induced desensitization, require receptor internalization via dynamin-dependent clathrin-coated pit mechanisms. Here we demonstrate that activation of β2-adrenergic receptors (β2-ARs) leads to c-Src-mediated tyrosine phosphorylation of dynamin, which is required for receptor internalization. Two tyrosine residues, Tyr231 and Tyr597, are identified as the major phosphorylation sites. Mutation of these residues to phenylalanine dramatically decreases the c-Src-mediated phosphorylation of dynamin following β2-AR stimulation. Moreover, expression of Y231F/Y597F dynamin inhibits β2-AR internalization and the isoproterenol-stimulated mitogen-activated protein kinase activation. Thus, agonist-induced, c-Src-mediated tyrosine phosphorylation of dynamin is essential for its function in clathrin mediated G protein-coupled receptor endocytosis. Agonist stimulation of cell surface receptors results in rapid attenuation of receptor responsiveness, a process termed desensitization. In the case of G protein-coupled receptors (GPCRs), 1The abbreviations used are: GPCR, G protein-coupled receptor; MAP, mitogen-activated protein; RTK, receptor tyrosine kinase; EGF, epidermal growth factor; β2-AR, β2-adrenergic receptor; GST, glutathioneS-transferase; DSP, dithiobis(succinimidylpropionate); MBP, myelin basic protein; PH, pleckstrin homology. this process is initiated by phosphorylation of agonist-occupied receptors by the G protein-coupled receptor kinase family (1Freedman N.J. Lefkowitz R.J. Rec. Prog. Hormone Res. 1996; 51: 319-351PubMed Google Scholar) and the subsequent formation of high affinity complexes with cytosolic proteins termed β-arrestins (2Gurevich V.V. Pals-Rylaarsdam R. Benovic J.L. Hosey M.M. Onorato J.J. J. Biol. Chem. 1997; 272: 28849-28852Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). β-Arrestin-bound, desensitized GPCRs then undergo β-arrestin-mediated targeting to clathrin-coated pits (3Goodman 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 (1179) Google Scholar, 4Ferguson S.S.G. Downey III, W.E. Calapietro A.M. Barak L.S. Menard L. Caron M.G. Science. 1996; 271: 363-366Crossref PubMed Scopus (853) Google Scholar), wherein they are sequestered, internalized, and ultimately either recycled to the cell surface or targeted for degradation. Interestingly, the process of GPCR internalization also plays a critical, albeit poorly understood, role in some aspects of GPCR signal transduction,e.g. MAP kinase activation (5Daaka Y. Luttrell L.M. Ahn S. Della Rocca G.J. Ferguson S.S. Caron M.C. Lefkowitz R.J. J. Biol. Chem. 1998; 273: 685-688Abstract Full Text Full Text PDF PubMed Scopus (465) Google Scholar, 6Luttrell L.M. Daaka Y. Della Rocca G.J. Lefkowitz R.J. J. Biol. Chem. 1997; 272: 31648-31656Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). The process of cell surface receptor internalization (also termed endocytosis) is dependent on the invagination and fission of clathrin-coated vesicles from the plasma membrane into the cytosol. Endocytosis of many GPCRs and receptor tyrosine kinases (RTKs) requires the GTPase activity of dynamin. Receptor stimulation leads to recruitment of cytosolic dynamin to coated pits where it induces constriction of the pits and fission of vesicles (7McClure S.J. Robinson P.J. Mol. Membr. Biol. 1996; 13: 189-215Crossref PubMed Scopus (75) Google Scholar, 8Urrutia R. Henley J.R. Cook T. McNiven M.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 377-384Crossref PubMed Scopus (256) Google Scholar). In vitro, dynamin is activated by a variety of mechanisms including binding to microtubules (9Maeda K. Nakata T. Noda Y. Sato-Yochitake R. Hirokawa N. Mol. Biol. Cell. 1992; 3: 1181-1194Crossref PubMed Scopus (92) Google Scholar), phospholipids (10Lin H.C. Barylko B. Achiriloaie M. Albanesi J.P. J. Biol. Chem. 1997; 272: 25999-26004Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar), and several proteins containing SH3 domains (11Gout I. Dhand R. Hiles I.D. Fry M.J. Panayotou G. Das P. Truong O. Totty N.F. Hsuan J. Booker G.W. Campbell I.D. Waterfield M.D. Cell. 1993; 75: 25-36Abstract Full Text PDF PubMed Scopus (485) Google Scholar). Collectively, these molecules are thought to induce a conformational change in dynamin, inducing homo-oligomerization and subsequent increase in GTPase activity (12Hinshaw J.E. Schmid S.L. Nature. 1995; 374: 190-192Crossref PubMed Scopus (665) Google Scholar). Recently, dynamin was shown to contain a GTPase effector domain that interacts with its N-terminal GTPase domain to stimulate GTP hydrolysis (13Muhlberg A.B. Warnock D.E. Schmid S.L. EMBO J. 1997; 16: 6676-6683Crossref PubMed Scopus (199) Google Scholar). GTPase defective dynamin mutants (e.g. K44A) specifically block endocytic coated vesicle formation and agonist-mediated internalization of GPCRs and RTKs (14Damke H. Baba T. Warnock D.E. Schmid S.L. J. Cell Biol. 1994; 127: 915-934Crossref PubMed Scopus (1045) Google Scholar, 15Zhang J. Ferguson S.S.G. Barak L.S. Menard L. Caron M.G. J. Biol. Chem. 1996; 271: 18302-18305Abstract Full Text Full Text PDF PubMed Scopus (398) Google Scholar). Accumulating evidence suggests that tyrosine protein phosphorylation is critical for the internalization of cell surface receptors. Exposure of cells to tyrosine kinase inhibitors profoundly attenuates cross-linking-induced internalization of B cell receptors (16Salamero J. Fougereau M. Seckinger P. Eur. J. Immunol. 1995; 25: 2757-2764Crossref PubMed Scopus (27) Google Scholar) and hormone-induced internalization of tyrosine kinase growth factor receptors (17Holen I. Stromhaug P.E. Gordon P.B. Fengsrud M. Berg T.O. Seglen P.O. J. Biol. Chem. 1995; 270: 12823-12831Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Recently, overexpression of the nonreceptor tyrosine kinase c-Src was found to cause an increase in the internalization rate constant of the epidermal growth factor (EGF) receptor following EGF treatment (18Ware M.F. Tice D.A. Parsons S.J. Lauffenburger D.A. J. Biol. Chem. 1997; 272: 30185-30190Crossref PubMed Scopus (79) Google Scholar). Although these results suggest involvement of tyrosine kinases in the process of receptor internalization, their role(s) in this process is not well defined. Therefore, we tested the hypothesis that tyrosine kinase activity might regulate endocytosis by acting on accessory molecules important for receptor internalization such as dynamin. We find not only that Src-mediated tyrosine phosphorylation of dynamin is required for β2-AR internalization but that this phosphorylation event itself is regulated by receptor stimulation. HEK293 cells were maintained and transfected precisely as described (5Daaka Y. Luttrell L.M. Ahn S. Della Rocca G.J. Ferguson S.S. Caron M.C. Lefkowitz R.J. J. Biol. Chem. 1998; 273: 685-688Abstract Full Text Full Text PDF PubMed Scopus (465) Google Scholar, 6Luttrell L.M. Daaka Y. Della Rocca G.J. Lefkowitz R.J. J. Biol. Chem. 1997; 272: 31648-31656Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). All assays were performed 40 h after transfection. HEK293 cells stably overexpressing dynamin were generated as described (19Lin F.-T. Krueger K.M. Kendall H.E. Daaka Y. Fredericks Z.L. Pitcher J.A. Lefkowitz R.J. J. Biol. Chem. 1997; 272: 31051-31057Crossref PubMed Scopus (207) Google Scholar). The β2-AR sequestration was quantified as loss of cell surface receptors determined by immunofluorescence flow cytometry as described (5Daaka Y. Luttrell L.M. Ahn S. Della Rocca G.J. Ferguson S.S. Caron M.C. Lefkowitz R.J. J. Biol. Chem. 1998; 273: 685-688Abstract Full Text Full Text PDF PubMed Scopus (465) Google Scholar). To generate single tyrosine mutants (Y231F and Y597F), tyrosine 231 or tyrosine 597 of rat dynamin I was mutated to phenylalanine by overlapping polymerase chain reaction (UAC(Y) → UUC(F)) (20Dieffenbach C.W. Dveksler G.S. PCR Primer: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1995Google Scholar). The double tyrosine mutant (Y231F/Y597F) dynamin was constructed by recombination of the two single tyrosine mutant constructs. Isolation of total cellular dynamin for analysis of tyrosine phosphorylation was performed by affinity purification with full-length GST-Grb2 fusion protein according to the method of Gout et al. (11Gout I. Dhand R. Hiles I.D. Fry M.J. Panayotou G. Das P. Truong O. Totty N.F. Hsuan J. Booker G.W. Campbell I.D. Waterfield M.D. Cell. 1993; 75: 25-36Abstract Full Text PDF PubMed Scopus (485) Google Scholar). The cDNA encoding GST-Grb2 fusion protein was generously provided by A. R. Saltiel. GST fusion proteins were prepared as glutathione-agarose conjugates as described previously (6Luttrell L.M. Daaka Y. Della Rocca G.J. Lefkowitz R.J. J. Biol. Chem. 1997; 272: 31648-31656Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar, 21Daaka Y. Pitcher J.A. Richardson M. Stoffel R.H. Robishaw J.D. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2180-2185Crossref PubMed Scopus (156) Google Scholar). Complexed proteins were dissociated from agarose beads with SDS-polyacrylamide gel electrophoresis sample buffer. For the detection of transient, agonist-promoted association of dynamin and c-Src, covalent protein cross-linking with Dithiobis(succinimidylpropionate) (DSP, Pierce) was employed. Cells were stimulated in 4.6 ml of phosphate-buffered saline containing 10 mm Hepes, pH 7.4. Stimulation was terminated by the addition of 0.4 ml of 2.5 mm DSP in Me2SO, and plates were rocked for 30 min at room temperature. Reactions were quenched by the addition of 0.1 ml of 1m Tris, pH 7.5, followed by two washes with ice-cold phosphate-buffered saline/Hepes to remove unreacted DSP. Cell pellets were lysed in RIPA buffer (5Daaka Y. Luttrell L.M. Ahn S. Della Rocca G.J. Ferguson S.S. Caron M.C. Lefkowitz R.J. J. Biol. Chem. 1998; 273: 685-688Abstract Full Text Full Text PDF PubMed Scopus (465) Google Scholar, 6Luttrell L.M. Daaka Y. Della Rocca G.J. Lefkowitz R.J. J. Biol. Chem. 1997; 272: 31648-31656Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar) prior to immunoprecipitation of c-Src-dynamin complexes. Cell lysates in RIPA buffer were incubated with the specified antibodies and protein A/G-Sepharose beads for 2 h at 4 °C. Rabbit polyclonal anti-Src antibody (Src-2, Santa Cruz Biotech. Inc.) was used for c-Src immunoprecipitation after protein cross-linking. Dynamin proteins were immunoprecipitated without cross-linking using either mouse monoclonal anti-dynamin antibody (Hudy-1; Upstate Biotech. Inc.) or rabbit polyclonal anti-dynamin I-specific antibody (kind gift of S. L. Schmid). Immunoprecipitates or GST-Grb2 fusion complexed proteins were resolved on acrylamide gels, transferred to nitrocellulose filters, immunoblotted with anti-dynamin or anti-phosphotyrosine antibodies (RC20H or PY20H, Transduction Laboratories), and visualized by enhanced chemiluminescence (Amersham Pharmacia Biotech). HEK293 cells stably expressing dynamin I were metabolically labeled with [32P]orthophosphate as described (19Lin F.-T. Krueger K.M. Kendall H.E. Daaka Y. Fredericks Z.L. Pitcher J.A. Lefkowitz R.J. J. Biol. Chem. 1997; 272: 31051-31057Crossref PubMed Scopus (207) Google Scholar). Dynamin was purified using GST-Grb2 binding as described above. Partially purified dynamin was resolved by SDS-polyacrylamide gel electrophoresis, and the dynamin band was excised from the gel and subsequently used for microsequencing after tryptic digestion (22Fredericks Z.L. Pitcher J.A. Lefkowitz R.J. J. Biol. Chem. 1996; 271: 13796-13803Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). HA-Erk2 was immunoprecipitated from HEK293 lysates using 6 μg of 12CA5 antibody. Immunoprecipitates were washed three times with RIPA buffer and twice with kinase buffer (23Hawes B.E. van Biesen T. Koch W.J. Luttrell L.M. Lefkowitz R.J. J. Biol. Chem. 1995; 270: 17148-17153Abstract Full Text Full Text PDF PubMed Scopus (413) Google Scholar).In vitro kinase reactions using myelin basic protein (MBP) as substrate were performed as described (23Hawes B.E. van Biesen T. Koch W.J. Luttrell L.M. Lefkowitz R.J. J. Biol. Chem. 1995; 270: 17148-17153Abstract Full Text Full Text PDF PubMed Scopus (413) Google Scholar). Labeled MBP was quantified using a Storm PhosphorImager (Molecular Dynamics). Spatial distribution and trafficking of receptors control their function. For example, mitogenic signaling emanating from certain GPCRs and RTKs is dependent on receptor internalization (5Daaka Y. Luttrell L.M. Ahn S. Della Rocca G.J. Ferguson S.S. Caron M.C. Lefkowitz R.J. J. Biol. Chem. 1998; 273: 685-688Abstract Full Text Full Text PDF PubMed Scopus (465) Google Scholar, 6Luttrell L.M. Daaka Y. Della Rocca G.J. Lefkowitz R.J. J. Biol. Chem. 1997; 272: 31648-31656Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar, 24Lefkowitz R.J. J. Biol. Chem. 1998; 273: 18677-18680Abstract Full Text Full Text PDF PubMed Scopus (908) Google Scholar, 25Vieira A.V. Lamaze C. Schmid S.L. Science. 1996; 274: 2086-2089Crossref PubMed Scopus (834) Google Scholar, 26Chow J.C. Condorelli G. Smith R.J. J. Biol. Chem. 1998; 273: 4672-4680Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). To determine whether tyrosine kinase activity is required for G protein-coupled receptor endocytosis, isoproterenol-induced internalization of the β2-AR was examined in HEK293 cells pretreated with tyrosine kinase inhibitors. Fig.1 A shows that pretreatment of cells with the generic tyrosine kinase inhibitor herbimycin A reduced isoproterenol-mediated internalization of the β2-AR by 60%. More dramatically, acute exposure of cells to the specific c-Src kinase inhibitor PP1 reduced the isoproterenol-induced β2-AR sequestration by approximately 80% (Fig.1 A). As shown in Fig. 1 B, expressing either a c-Src kinase inhibitor (Csk) or a kinase defective, dominant inhibitory form of c-Src (K298M) impaired the isoproterenol-mediated internalization of the β2-AR. Conversely, expression of an activated form of Src (v-Src) increased agonist-dependent sequestration of the β2-AR (Fig. 1 B). These results suggest that Src tyrosine kinase activity is required for agonist-dependent internalization of the β2-AR. A direct interaction between c-Src and dynamin in neuronal cells has been described (27Foster-Barber A. Bishop J.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4673-4677Crossref PubMed Scopus (83) Google Scholar). Because c-Src kinase activity was required for the agonist-mediated internalization of β2-AR, we examined whether dynamin is a phosphotyrosyl protein and a substrate for c-Src. Cellular dynamin was isolated using a GST-Grb2 affinity purification following agonist exposure. As shown in Fig.2 A, isoproterenol stimulation resulted in 2–2.5-fold increase in tyrosine phosphorylation of dynamin (upper immunoblot), which was maximal within 2 min of agonist exposure. These results demonstrate that β2-AR activation induces the rapid tyrosine phosphorylation of dynamin. Next, we examined whether c-Src is the kinase responsible for the tyrosine phosphorylation of dynamin following activation of the β2-AR. We expressed the c-Src inhibitors, Csk and K298M c-Src, or the constitutively active v-Src and examined their effect on isoproterenol-induced tyrosine phosphorylation of dynamin in HEK293 cells stably expressing dynamin I. Fig. 2 B shows that expression of Csk or K298M c-Src inhibited the agonist-induced tyrosine phosphorylation of dynamin, whereas expression of activated Src (v-Src) further enhanced the level of tyrosine phosphorylation of dynamin (upper immunoblot). Thus, agonist stimulation resulted in a net increase in the phosphotyrosine content of the cellular dynamin pool, which could be reversed by inhibition of Src activity. These data mirror the effects of these reagents on β2-AR sequestration (Fig. 1 B), suggesting that c-Src-regulated internalization of the β2-AR may be mediated by the c-Src-induced tyrosine phosphorylation of dynamin. Foster-Barber and Bishop (27Foster-Barber A. Bishop J.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4673-4677Crossref PubMed Scopus (83) Google Scholar) have reported that the purified SH3 domain of c-Src binds dynamin in vitro and that dynamin co-precipitates with c-Src from PC12 cell lysates. These authors did not describe tyrosine phosphorylation of dynamin or modulation of c-Src-dynamin complex formation following receptor activation. To test the hypothesis that the agonist-regulated association of dynamin and c-Src might account for the agonist-promoted increase in dynamin tyrosine phosphorylation, we examined the ability of c-Src to form a complex with dynamin following β2-AR activation. Protein cross-linking was used to stabilize potentially transient, agonist-mediated, dynamin-c-Src interactions. HEK293 cells stably expressing dynamin I were treated with or without isoproterenol for 2 min followed by covalent protein cross-linking with DSP. As shown in Fig. 2 C, treatment of cells with isoproterenol increased the amount of tyrosine phosphorylated dynamin present in c-Src immunoprecipitates. Reverse phase high pressure liquid chromatography analysis of trypsin-digested phosphodynamin isolated from isoproterenol-stimulated cells revealed the presence of two major phosphopeptides. Microsequencing and amino acid sequence analyses (19Lin F.-T. Krueger K.M. Kendall H.E. Daaka Y. Fredericks Z.L. Pitcher J.A. Lefkowitz R.J. J. Biol. Chem. 1997; 272: 31051-31057Crossref PubMed Scopus (207) Google Scholar, 22Fredericks Z.L. Pitcher J.A. Lefkowitz R.J. J. Biol. Chem. 1996; 271: 13796-13803Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar) of one peptide revealed that Tyr597 is phosphorylated (Fig.3 A). Sequence analysis of the second phosphopeptide revealed sequences just N-terminal to a GYIG motif (Tyr231) that displays high sequence homology to consensus c-Src phosphorylation sites (28Songyang Z. Carraway III, K.L. Eck M.J. Harrison S.C. Feldman R.A. Mohammadi M. Schlessinger J. Hubbard S.R. Smith D.P. Eng C. Lorenzo M.J. Ponder B.A.J. Mayer B.J. Cantley L.C. Nature. 1995; 373: 536-539Crossref PubMed Scopus (848) Google Scholar). To determine whether tyrosine phosphorylation of dynamin affected its function, each of these tyrosine residues (at positions 231 and 597) was mutated to phenylalanine singly and in combination. As shown in Fig. 3 (B and C), whereas isoproterenol treatment increased the tyrosine phosphorylation content of wild-type dynamin, the single tyrosine mutants (Y231F and Y597F), and the double tyrosine mutant (Y231F/Y597F) of dynamin exhibited diminished tyrosine phosphorylation in both isoproterenol-stimulated and nonstimulated cells. The dramatic decrease in tyrosine phosphorylation of Y231F/Y597F mutant strongly suggests that these residues represent the phosphorylated sites in vivo. In agreement with previous results (5Daaka Y. Luttrell L.M. Ahn S. Della Rocca G.J. Ferguson S.S. Caron M.C. Lefkowitz R.J. J. Biol. Chem. 1998; 273: 685-688Abstract Full Text Full Text PDF PubMed Scopus (465) Google Scholar, 15Zhang J. Ferguson S.S.G. Barak L.S. Menard L. Caron M.G. J. Biol. Chem. 1996; 271: 18302-18305Abstract Full Text Full Text PDF PubMed Scopus (398) Google Scholar), overexpression of wild-type dynamin did not enhance agonist-promoted sequestration of the β2-AR (Fig. 4 A). However, expression of Y231F or Y597F dynamin resulted in a modest reduction of agonist-induced β2-AR internalization. Expression of the Y231F/Y597F dynamin inhibited approximately 70% of the agonist-induced internalization of the β2-AR, which was equivalent to the degree of inhibition observed with GTPase-deficient dynamin K44A. These data suggest that tyrosine phosphorylation of dynamin plays an important role in its ability to support endocytosis. Recent data suggest that agonist-promoted endocytosis plays a dual regulatory role in signaling pathways emanating from GPCRs or RTKs. Activation of the Erk1/2 MAP kinase pathway by GPCRs, such as those for isoproterenol, lysophosphatidic acid, thrombin, and bombesin (5Daaka Y. Luttrell L.M. Ahn S. Della Rocca G.J. Ferguson S.S. Caron M.C. Lefkowitz R.J. J. Biol. Chem. 1998; 273: 685-688Abstract Full Text Full Text PDF PubMed Scopus (465) Google Scholar, 6Luttrell L.M. Daaka Y. Della Rocca G.J. Lefkowitz R.J. J. Biol. Chem. 1997; 272: 31648-31656Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar), as well as RTKs, such as those for EGF (25Vieira A.V. Lamaze C. Schmid S.L. Science. 1996; 274: 2086-2089Crossref PubMed Scopus (834) Google Scholar), nerve growth factor (29Riccio A. Pierchala B.A. Ciarallo C.L. Ginty D.D. Science. 1997; 277: 1097-1100Crossref PubMed Scopus (368) Google Scholar, 30Xing J. Kornhauser J.M. Xia Z.G. Thiele E.A. Greenberg M.E. Mol. Cell. Biol. 1998; 18: 1946-1955Crossref PubMed Google Scholar), and insulin-like growth factor-1 (26Chow J.C. Condorelli G. Smith R.J. J. Biol. Chem. 1998; 273: 4672-4680Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar), is dependent on receptor internalization. In the case of the β2-AR, protein kinase A-mediated phosphorylation of the receptor confers receptor-Gi coupling, with subsequent Ras-dependent Erk1/2 activation mediated by Gβγ-subunits derived from pertussis toxin-sensitive G proteins (31Daaka Y. Luttrell L.M. Lefkowitz R.J. Nature. 1997; 390: 88-91Crossref PubMed Scopus (1075) Google Scholar). In addition, expression of dominant negative mutants of β-arrestin 1 or dynamin, which attenuate agonist-mediated endocytosis, blocks β2-AR-mediated Erk1/2 activation (5Daaka Y. Luttrell L.M. Ahn S. Della Rocca G.J. Ferguson S.S. Caron M.C. Lefkowitz R.J. J. Biol. Chem. 1998; 273: 685-688Abstract Full Text Full Text PDF PubMed Scopus (465) Google Scholar). Considering the role of dynamin in endocytosis, we investigated the effects of expressing wild-type or mutant dynamin on the β2-AR-mediated activation of the Erk2 MAP kinase. Expression of the Y231F or Y597F mutated forms of dynamin reduced the isoproterenol-mediated activation of Erk2 approximately 60% (Fig. 4,B and C). However, expression of the Y231F/Y597F mutant reduced the agonist-induced Erk2 activation by 80–90%, similar to the effects observed using the K44A dynamin (Fig. 4, Band C). These data suggest that c-Src-mediated tyrosine phosphorylation of dynamin is required for Ras-mediated activation of MAP kinase by β2-AR by virtue of its involvement in receptor sequestration. Our results establish that agonist-induced tyrosine phosphorylation of dynamin is required for β2-AR internalization and internalization-dependent signaling to MAP kinase. Clathrin-coated vesicle-mediated receptor internalization is regulated by the enzymatic (GTPase) activity of dynamin. As shown in Fig.3 A, Tyr231 and Tyr597 reside in the GTPase and pleckstrin homology (PH) domains of dynamin, respectively. Intermolecular interaction between GTPase and GED domains of adjacent dynamins has been reported to regulate the GTPase activity of the enzyme (13Muhlberg A.B. Warnock D.E. Schmid S.L. EMBO J. 1997; 16: 6676-6683Crossref PubMed Scopus (199) Google Scholar). Thus, phosphorylation of Tyr231 might regulate the GTPase activity of dynamin by controlling these intermolecular interactions. Another mutation that disables the GTPase activity of dynamin (K44A) also ablates the ability of dynamin to function in internalization of receptors (14Damke H. Baba T. Warnock D.E. Schmid S.L. J. Cell Biol. 1994; 127: 915-934Crossref PubMed Scopus (1045) Google Scholar, 15Zhang J. Ferguson S.S.G. Barak L.S. Menard L. Caron M.G. J. Biol. Chem. 1996; 271: 18302-18305Abstract Full Text Full Text PDF PubMed Scopus (398) Google Scholar). PH domains in proteins such as phospholipase C-δ1 (32Lemmon M.A. Ferguson K.M. O'Brien R. Sigler P.B. Schlessinger J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10472-10476Crossref PubMed Scopus (480) Google Scholar) have been shown to mediate interactions with acidic phospholipids. Although there is as yet no direct evidence for dynamin PH domain-phospholipid interaction, acidic phospholipids such as phosphatidylinositol 4,5-bisphosphate have been shown to stimulate GTPase activity of dynamin in vitro (10Lin H.C. Barylko B. Achiriloaie M. Albanesi J.P. J. Biol. Chem. 1997; 272: 25999-26004Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar,33Lin H.C. Gilman A.G. J. Biol. Chem. 1996; 271: 27979-27982Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Additionally, G protein βγ subunits, which interact with PH domains of many proteins (34Touhara K. Inglese J. Pitcher J.A. Shaw G. Lefkowitz R.J. J Biol. Chem. 1994; 269: 10217-10220Abstract Full Text PDF PubMed Google Scholar), have been shown to control the GTPase activity of dynamin (33Lin H.C. Gilman A.G. J. Biol. Chem. 1996; 271: 27979-27982Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Tyr597 is located in one of three variable loops of the dynamin PH domain that possess positive electrostatic potential (35Ferguson K.M. Lemmon M.A. Schlessinger J. Sigler P.B. Cell. 1994; 79: 199-209Abstract Full Text PDF PubMed Scopus (243) Google Scholar). Phosphorylation of Tyr597 may alter the ability of dynamin to interact with effector molecules such as phosphatidylinositol 4,5-bisphosphate and G protein βγ subunits, resulting in modulation of dynamin GTPase activity. Although the molecular mechanism(s) whereby tyrosine phosphorylation of dynamin regulates its function remain unclear, the present results establish that the c-Src-mediated tyrosine phosphorylation of dynamin provides one mechanism by which GPCRs regulate their own internalization and MAP kinase signaling. We thank Drs. S. L. Schmid and A. R. Saltiel for reagents and Mary Holben and Donna Addison for secretarial assistance." @default.
• W2014211428 created "2016-06-24" @default.
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• W2014211428 creator A5033862949 @default.
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• W2014211428 date "1999-01-01" @default.
• W2014211428 modified "2023-10-12" @default.
• W2014211428 title "Src-mediated Tyrosine Phosphorylation of Dynamin Is Required for β2-Adrenergic Receptor Internalization and Mitogen-activated Protein Kinase Signaling" @default.
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