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W1985087493 abstract "The dynamin family of GTP-binding proteins has been implicated as playing an important role in endocytosis. InDrosophila shibire, mutations of the single dynamin gene cause blockade of endocytosis and neurotransmitter release, manifest as temperature-sensitive neuromuscular paralysis. Mammals express three dynamin genes: the neural specific dynamin I, ubiquitous dynamin II, and predominantly testicular dynamin III. Mutations of dynamin I result in a blockade of synaptic vesicle recycling and receptor-mediated endocytosis. Here, we show that dynamin II plays a key role in controlling constitutive and regulated hormone secretion from mouse pituitary corticotrope (AtT20) cells. Dynamin II is preferentially localized to the Golgi apparatus where it interacts with G-protein βγ subunit and regulates secretory vesicle release. The presence of dynamin II at the Golgi apparatus and its interaction with the βγ subunit are mediated by the pleckstrin homology domain of the GTPase. Overexpression of the pleckstrin homology domain, or a dynamin II mutant lacking the C-terminal SH3-binding domain, induces translocation of endogenous dynamin II from the Golgi apparatus to the plasma membrane and transformation of dynamin II from activity in the secretory pathway to receptor-mediated endocytosis. Thus, dynamin II regulates secretory vesicle formation from the Golgi apparatus and hormone release from mammalian neuroendocrine cells. The dynamin family of GTP-binding proteins has been implicated as playing an important role in endocytosis. InDrosophila shibire, mutations of the single dynamin gene cause blockade of endocytosis and neurotransmitter release, manifest as temperature-sensitive neuromuscular paralysis. Mammals express three dynamin genes: the neural specific dynamin I, ubiquitous dynamin II, and predominantly testicular dynamin III. Mutations of dynamin I result in a blockade of synaptic vesicle recycling and receptor-mediated endocytosis. Here, we show that dynamin II plays a key role in controlling constitutive and regulated hormone secretion from mouse pituitary corticotrope (AtT20) cells. Dynamin II is preferentially localized to the Golgi apparatus where it interacts with G-protein βγ subunit and regulates secretory vesicle release. The presence of dynamin II at the Golgi apparatus and its interaction with the βγ subunit are mediated by the pleckstrin homology domain of the GTPase. Overexpression of the pleckstrin homology domain, or a dynamin II mutant lacking the C-terminal SH3-binding domain, induces translocation of endogenous dynamin II from the Golgi apparatus to the plasma membrane and transformation of dynamin II from activity in the secretory pathway to receptor-mediated endocytosis. Thus, dynamin II regulates secretory vesicle formation from the Golgi apparatus and hormone release from mammalian neuroendocrine cells. pleckstrin homology Src homology domain 3 polymerase chain reaction phosphate-buffered saline mitogen-activated protein kinase polyacrylamide gel electrophoresis tetramethylrhodamine isothiocyanate trans-Golgi network. Dynamin is a polypeptide with a modular structure comprising a GTP-binding domain in the N-terminal third, a middle domain of unknown function, a pleckstrin homology (PH)1 domain and a C-terminal proline-rich or Src homology 3- (SH3-) binding domain (for reviews, see Refs. 1McNiven M.A. Cao I. Pitts K.R. Yoon I. Trends Biochem. Sci. 2000; 25: 115-120Abstract Full Text Full Text PDF PubMed Scopus (307) Google Scholar, 2van der Bliek A.M. Trends Cell Biol. 1999; 9: 96-102Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar, 3Kelly R.B. Nat. Cell. Biol. 1999; 1: E8-E9Crossref PubMed Scopus (19) Google Scholar, 4Urrutia R. Henley J.R. Cook T. McNiven M.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 377-384Crossref PubMed Scopus (253) Google Scholar, 5Warnock D.E. Schmid S.L. Bioessays. 1996; 18: 885-893Crossref PubMed Scopus (137) Google Scholar, 6De Camilli P. Takei K. Neuron. 1996; 16: 481-486Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar, 7Vallee R.B. Okamoto P.M. Trends Cell Biol. 1995; 5: 43-47Abstract Full Text PDF PubMed Scopus (50) Google Scholar, 8Liu J.-P. Robinson P.J. Endocrine Rev. 1995; 16: 590-607PubMed Google Scholar). Mammals have at least three dynamin genes which code for dynamin I, II, and III. Although the homology between dynamin I and II proteins is 79% and between dynamin I and III proteins is 89%, significant variation occurs at the C-terminal regions. In addition, dynamin I is specifically expressed in neural tissues, whereas dynamin II is ubiquitous and dynamin III is predominantly testicular. These developmentally divergent dynamins may thus represent a large protein family apparently performing a range of functions in association with distinctive sites in mammals (1McNiven M.A. Cao I. Pitts K.R. Yoon I. Trends Biochem. Sci. 2000; 25: 115-120Abstract Full Text Full Text PDF PubMed Scopus (307) Google Scholar, 2van der Bliek A.M. Trends Cell Biol. 1999; 9: 96-102Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar).While dynamin I has been implicated as playing an important role in mediating synaptic vesicle recycling, little is known of the physiological function of dynamin II. Since dynamin I complexes a large ring-like structure surrounding the necks of clathrin-coated endocytic pits on the cytoplasmic surface of presynaptic plasma membrane during synaptic vesicle recycling (3Kelly R.B. Nat. Cell. Biol. 1999; 1: E8-E9Crossref PubMed Scopus (19) Google Scholar, 6De Camilli P. Takei K. Neuron. 1996; 16: 481-486Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar), it is thought that dynamin II plays a similar role to that of dynamin I in receptor-mediated endocytosis in non-neuronal cells (4Urrutia R. Henley J.R. Cook T. McNiven M.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 377-384Crossref PubMed Scopus (253) Google Scholar, 5Warnock D.E. Schmid S.L. Bioessays. 1996; 18: 885-893Crossref PubMed Scopus (137) Google Scholar, 6De Camilli P. Takei K. Neuron. 1996; 16: 481-486Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar, 7Vallee R.B. Okamoto P.M. Trends Cell Biol. 1995; 5: 43-47Abstract Full Text PDF PubMed Scopus (50) Google Scholar, 8Liu J.-P. Robinson P.J. Endocrine Rev. 1995; 16: 590-607PubMed Google Scholar). In support of this hypothesis are the findings that the GTPase activities of both dynamin I and II, and of their SH3-binding domain truncation mutants, are similarly stimulatedin vitro by phospholipids, grb2, and microtubules (9Lin 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), suggesting that the modes of interaction of the PH domains from dynamin I and II with phosphatidylinositol (4,5)-bisphosphate and of the SH3-binding domain with microtubules and grb2 are similar in vitro (9Lin 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). However, it is not yet established why dynamin I and II are concomitantly present in neurons (4Urrutia R. Henley J.R. Cook T. McNiven M.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 377-384Crossref PubMed Scopus (253) Google Scholar), and why rapid endocytosis in adrenal chromaffin PC12 cells is significantly inhibited by the microinjected PH domain of dynamin I but not by the PH domain of dynamin II (10Artalejo C.A. Lemmon M.A. Schlessinger J. Palfrey H.C. EMBO J. 1997; 16: 1565-1574Crossref PubMed Scopus (70) Google Scholar). Given the previous reports that dynamin II associates with the trans-Golgi network in human hepatic (HepG2) cells (11Maier O. Knoblich M. Westermann P. Biochem. Biophys. Res. Commun. 1996; 223: 229-233Crossref PubMed Scopus (49) Google Scholar), and that dynamin I and/or II immunoreactivity is present in the Golgi apparatus of human foreskin melanocytes and fibroblasts (12Henley J.R. McNiven M.A. J. Cell Biol. 1996; 133: 761-775Crossref PubMed Scopus (107) Google Scholar), it is conceivable that dynamin II might have a different function in membrane trafficking from the endocytic role of dynamin I in mammalian cells (4Urrutia R. Henley J.R. Cook T. McNiven M.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 377-384Crossref PubMed Scopus (253) Google Scholar). To establish a biological function of dynamin II, the present study was undertaken to explore the activity of dynamin II in mouse pituitary corticotropes (AtT20 cells). We found that dynamin II plays a key role in controlling both constitutive and regulated hormone secretions at the Golgi apparatus of these neuroendocrine cells.DISCUSSIONThe present study defines for the first time dynamin II as a Golgi membrane trafficking protein required for both constitutive and regulated hormone secretion from neuroendocrine cells. A striking consequence of loss of dynamin II function in neuroendocrine cells is lowered hormone secretion with both basal and CRH-stimulated secretion of β-endorphin being affected. A similar phenotype is also found when antisense mRNA is used to block the synthesis of endogenous dynamin II. By contrast, an increase in β-endorphin secretion is observed when wild-type dynamin II is overexpressed. Thus, the extent of both constitutive and regulated hormone secretions from these neuroendocrine cells may be limited by the expression levels of endogenous dynamin II under physiological conditions. This finding is in line with previous findings that dynamin II is associated with the Golgi vesicular buddingin vitro (23Jones S.M. Howell K.E. Henley J.R. Cao H. McNiven M.A. Science. 1998; 279: 573-577Crossref PubMed Scopus (271) Google Scholar) and in cultured rat epithelial cells (24Cao H. Garcia F. McNiven M.A. Mol. Biol. Cell. 1998; 9: 2595-2609Crossref PubMed Scopus (340) Google Scholar), and that microinjection of dynamin II does not mimic that of dynamin I on rapid endocytosis in adrenal chromaffin cells (10Artalejo C.A. Lemmon M.A. Schlessinger J. Palfrey H.C. EMBO J. 1997; 16: 1565-1574Crossref PubMed Scopus (70) Google Scholar). Since dynamin II has also been implicated in caveolae endocytosis in hepatocytes (25Henley J.R. Krueger E.W. Oswald B.J. McNiven M.A. J. Cell Biol. 1998; 141: 85-99Crossref PubMed Scopus (617) Google Scholar) and receptor endocytosis in adipocytes (26Volchuk A. Narine S. Foster L.J. Grabs D. De Camilli P. Klip A. J. Biol. Chem. 1998; 273: 8169-8176Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar), HeLa cells and Madin-Darby canine kidney cells without affecting exocytotic pathways (27Altschuler Y. Barbas S.M. Terlecky L.J. Tang K. Hardy S. Mostov K.E. Schmid S.L. J. Cell Biol. 1998; 143: 1871-1881Crossref PubMed Scopus (186) Google Scholar, 28Kasai K. Shin H.W. Shinotsuka C. Murakami K. Nakayama K. J. Biochem. (Tokyo). 1999; 125: 780-789Crossref PubMed Scopus (44) Google Scholar), it is possible that the cellular functions of dynamin II are largely determined by its specifically targeted subcellular localization in a cell specific fashion. Subcellular localization studies in neuroendocrine cells suggest the presence of dynamin II at the Golgi apparatus, consistent with dynamin II-regulated hormone secretion. Furthermore, overexpression of the wild type or dynamin II mutant regulates de novo production of secretory vesicles from purified Golgi apparatus and semi-permeabilized cells. Thus, dynamin II may be a major form of the dynamin family playing an obligatory role in secretory vesicle biogenesis at the Golgi apparatus during hormone secretion from neuroendocrine cells, consistent with the most recent findings that dynamin II regulates post-Golgi transport of a plasma-membrane protein (29Kreitzer G. Marmorstein A. Okamoto P. Vallee R. Rodriguez-Boulan E. Nat. Cell Biol. 2000; 2: 125-127Crossref PubMed Scopus (200) Google Scholar).In addition, structure-function analysis suggests a crucial role for the PH domain in targeting dynamin II to the Golgi. The finding that overexpressed dynamin II PH domain induces dissociation of endogenous dynamin II from the Golgi and inhibition of hormone secretion strongly suggests that the PH domain of dynamin II is involved in interacting with Golgi membrane docking molecule(s) of dynamin II. Given that PH domains from diverse proteins are capable of binding to the phosphate groups of acidic phospholipids (such as phosphatidylinositol (4,5)-bisphosphate) (30Lemmon M.A. Ferguson K.M. Schlessinger J. Cell. 1996; 85: 621-624Abstract Full Text Full Text PDF PubMed Scopus (428) Google Scholar), it is possible that dynamin II interacts with these lipid molecules but is specifically targeted at the Golgi apparatus by protein-protein interactions involving its PH domain. In an attempt to search for dynamin II interactive proteins, we detected no interaction between dynamin II and γ-adaptin by immunoprecipitation, although previous studies have shown that dynamin I interacts with α-adaptin (31Wang L.H. Sudhof T.C. Anderson R.G. J. Biol. Chem. 1995; 270: 10079-10083Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar) and dynamin II mediates clathrin-coated vesicle budding from the TGN (23Jones S.M. Howell K.E. Henley J.R. Cao H. McNiven M.A. Science. 1998; 279: 573-577Crossref PubMed Scopus (271) Google Scholar). In contrast, the interaction between dynamin II and the βγ subunit of G-proteins via the dynamin II PH domain is specific, since the small GTP-binding protein Rab6 does not coimmunoprecipitate with G-protein β subunit, nor does dynamin II with β-COP (a Golgi coatamer protein also possessing a WD40 repeat). Previous studies have also shown that purified βγ subunits bind to purified dynamin (32Liu J.-P. Yajima Y. Li H. Ackland S. Akita Y. Stewart J. Kawashima S. Mol. Cell. Endocrinol. 1997; 132: 61-71Crossref PubMed Scopus (20) Google Scholar) and stimulate secretory vesicle formation (33Barr F.A. Leyte A. Mollner S. Pfeuffer T. Tooze S.A. Huttner W.B. FEBS Lett. 1991; 294: 239-243Crossref PubMed Scopus (74) Google Scholar).In addition to the PH domain, our data also support a role for the SH3-binding domain in dynamin II-mediated secretory vesicle production at the Golgi apparatus. Overexpression of dynamin II lacking the SH3-binding domain not only induces dislocation of endogenous dynamin II from the Golgi but also produces inhibition of hormone secretion. The translocation of endogenous dynamin II from the Golgi to the plasma membrane suggests an involvement of the SH3-binding domain in dynamin II association with secretory vesicle membranes (Fig.10). Thus, uncoupling of the SH3-binding domain renders dynamin II ineffective in mediating secretory vesicle biogenesis, an effect paralleling previous finding for dynamin I that disruption of the SH3-binding domain impairs synaptic vesicle recycling (34Shupliakov O. Low P. Grabs D. Gad H. Chen H. David C. Takei K. De Camilli P. Brodin L. Science. 1997; 276: 259-263Crossref PubMed Scopus (397) Google Scholar).It is also noteworthy that the SH3-binding domain deletion mutant is more potent than the PH domain mutant in causing inhibition of hormone release (Fig. 7 A) and stimulation of receptor-mediated endocytosis (Fig. 7 B). This difference in potency may reflect a difference between the mutants in competing with endogenous dynamin II at the Golgi. Since the region immediately C-terminal to the PH domain of β-adrenergic receptor kinase is also involved in binding to the βγ subunit (35Inglese J. Luttrell L.M. Iniguez-Lluhi J.A. Touhara K. Koch W.J. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3637-3641Crossref PubMed Scopus (71) Google Scholar), it is possible that other regions outside the PH domain of dynamin II are also involved in targeting the protein to the Golgi, and thus that the SH3-binding domain deletion mutant is more effective than the PH domain-only mutant in causing translocation. Once on the plasma membrane, the misallocated dynamin II is capable of mediating clathrin-dependent endocytosis, suggesting that dynamin II is structurally competent to participate in both secretory and endocytic vesicular trafficking, and that its physiological role in the secretory pathway is determined by its localization to the Golgi.Collectively, these data show that dynamin II GTPase is a dominant regulator of both constitutive and regulated hormone secretions from neuroendocrine cells, operating as a polymer at the Golgi controlling secretory vesicle biogenesis. During this process, dynamin II interacts with βγ subunit of G-proteins via a region containing the PH domain; disruption of the interaction causes translocation of dynamin II from the Golgi to plasma membrane. The parallel increases and decreases in dynamin II expression with constitutive and regulated hormone secretion are consistent with expressed levels of dynamin II playing an important physiological role in regulating the capacity of cell secretion in response to environmental stimuli. Molecular targeting to particular dynamin isoforms may therefore provide a potential therapeutic means for controlling Golgi protein trafficking and hormone secretion under particular circumstances (36Shields D. Arvan P. Curr. Opin. Cell. Biol. 1999; 11: 489-494Crossref PubMed Scopus (20) Google Scholar). Dynamin is a polypeptide with a modular structure comprising a GTP-binding domain in the N-terminal third, a middle domain of unknown function, a pleckstrin homology (PH)1 domain and a C-terminal proline-rich or Src homology 3- (SH3-) binding domain (for reviews, see Refs. 1McNiven M.A. Cao I. Pitts K.R. Yoon I. Trends Biochem. Sci. 2000; 25: 115-120Abstract Full Text Full Text PDF PubMed Scopus (307) Google Scholar, 2van der Bliek A.M. Trends Cell Biol. 1999; 9: 96-102Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar, 3Kelly R.B. Nat. Cell. Biol. 1999; 1: E8-E9Crossref PubMed Scopus (19) Google Scholar, 4Urrutia R. Henley J.R. Cook T. McNiven M.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 377-384Crossref PubMed Scopus (253) Google Scholar, 5Warnock D.E. Schmid S.L. Bioessays. 1996; 18: 885-893Crossref PubMed Scopus (137) Google Scholar, 6De Camilli P. Takei K. Neuron. 1996; 16: 481-486Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar, 7Vallee R.B. Okamoto P.M. Trends Cell Biol. 1995; 5: 43-47Abstract Full Text PDF PubMed Scopus (50) Google Scholar, 8Liu J.-P. Robinson P.J. Endocrine Rev. 1995; 16: 590-607PubMed Google Scholar). Mammals have at least three dynamin genes which code for dynamin I, II, and III. Although the homology between dynamin I and II proteins is 79% and between dynamin I and III proteins is 89%, significant variation occurs at the C-terminal regions. In addition, dynamin I is specifically expressed in neural tissues, whereas dynamin II is ubiquitous and dynamin III is predominantly testicular. These developmentally divergent dynamins may thus represent a large protein family apparently performing a range of functions in association with distinctive sites in mammals (1McNiven M.A. Cao I. Pitts K.R. Yoon I. Trends Biochem. Sci. 2000; 25: 115-120Abstract Full Text Full Text PDF PubMed Scopus (307) Google Scholar, 2van der Bliek A.M. Trends Cell Biol. 1999; 9: 96-102Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar). While dynamin I has been implicated as playing an important role in mediating synaptic vesicle recycling, little is known of the physiological function of dynamin II. Since dynamin I complexes a large ring-like structure surrounding the necks of clathrin-coated endocytic pits on the cytoplasmic surface of presynaptic plasma membrane during synaptic vesicle recycling (3Kelly R.B. Nat. Cell. Biol. 1999; 1: E8-E9Crossref PubMed Scopus (19) Google Scholar, 6De Camilli P. Takei K. Neuron. 1996; 16: 481-486Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar), it is thought that dynamin II plays a similar role to that of dynamin I in receptor-mediated endocytosis in non-neuronal cells (4Urrutia R. Henley J.R. Cook T. McNiven M.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 377-384Crossref PubMed Scopus (253) Google Scholar, 5Warnock D.E. Schmid S.L. Bioessays. 1996; 18: 885-893Crossref PubMed Scopus (137) Google Scholar, 6De Camilli P. Takei K. Neuron. 1996; 16: 481-486Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar, 7Vallee R.B. Okamoto P.M. Trends Cell Biol. 1995; 5: 43-47Abstract Full Text PDF PubMed Scopus (50) Google Scholar, 8Liu J.-P. Robinson P.J. Endocrine Rev. 1995; 16: 590-607PubMed Google Scholar). In support of this hypothesis are the findings that the GTPase activities of both dynamin I and II, and of their SH3-binding domain truncation mutants, are similarly stimulatedin vitro by phospholipids, grb2, and microtubules (9Lin 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), suggesting that the modes of interaction of the PH domains from dynamin I and II with phosphatidylinositol (4,5)-bisphosphate and of the SH3-binding domain with microtubules and grb2 are similar in vitro (9Lin 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). However, it is not yet established why dynamin I and II are concomitantly present in neurons (4Urrutia R. Henley J.R. Cook T. McNiven M.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 377-384Crossref PubMed Scopus (253) Google Scholar), and why rapid endocytosis in adrenal chromaffin PC12 cells is significantly inhibited by the microinjected PH domain of dynamin I but not by the PH domain of dynamin II (10Artalejo C.A. Lemmon M.A. Schlessinger J. Palfrey H.C. EMBO J. 1997; 16: 1565-1574Crossref PubMed Scopus (70) Google Scholar). Given the previous reports that dynamin II associates with the trans-Golgi network in human hepatic (HepG2) cells (11Maier O. Knoblich M. Westermann P. Biochem. Biophys. Res. Commun. 1996; 223: 229-233Crossref PubMed Scopus (49) Google Scholar), and that dynamin I and/or II immunoreactivity is present in the Golgi apparatus of human foreskin melanocytes and fibroblasts (12Henley J.R. McNiven M.A. J. Cell Biol. 1996; 133: 761-775Crossref PubMed Scopus (107) Google Scholar), it is conceivable that dynamin II might have a different function in membrane trafficking from the endocytic role of dynamin I in mammalian cells (4Urrutia R. Henley J.R. Cook T. McNiven M.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 377-384Crossref PubMed Scopus (253) Google Scholar). To establish a biological function of dynamin II, the present study was undertaken to explore the activity of dynamin II in mouse pituitary corticotropes (AtT20 cells). We found that dynamin II plays a key role in controlling both constitutive and regulated hormone secretions at the Golgi apparatus of these neuroendocrine cells. DISCUSSIONThe present study defines for the first time dynamin II as a Golgi membrane trafficking protein required for both constitutive and regulated hormone secretion from neuroendocrine cells. A striking consequence of loss of dynamin II function in neuroendocrine cells is lowered hormone secretion with both basal and CRH-stimulated secretion of β-endorphin being affected. A similar phenotype is also found when antisense mRNA is used to block the synthesis of endogenous dynamin II. By contrast, an increase in β-endorphin secretion is observed when wild-type dynamin II is overexpressed. Thus, the extent of both constitutive and regulated hormone secretions from these neuroendocrine cells may be limited by the expression levels of endogenous dynamin II under physiological conditions. This finding is in line with previous findings that dynamin II is associated with the Golgi vesicular buddingin vitro (23Jones S.M. Howell K.E. Henley J.R. Cao H. McNiven M.A. Science. 1998; 279: 573-577Crossref PubMed Scopus (271) Google Scholar) and in cultured rat epithelial cells (24Cao H. Garcia F. McNiven M.A. Mol. Biol. Cell. 1998; 9: 2595-2609Crossref PubMed Scopus (340) Google Scholar), and that microinjection of dynamin II does not mimic that of dynamin I on rapid endocytosis in adrenal chromaffin cells (10Artalejo C.A. Lemmon M.A. Schlessinger J. Palfrey H.C. EMBO J. 1997; 16: 1565-1574Crossref PubMed Scopus (70) Google Scholar). Since dynamin II has also been implicated in caveolae endocytosis in hepatocytes (25Henley J.R. Krueger E.W. Oswald B.J. McNiven M.A. J. Cell Biol. 1998; 141: 85-99Crossref PubMed Scopus (617) Google Scholar) and receptor endocytosis in adipocytes (26Volchuk A. Narine S. Foster L.J. Grabs D. De Camilli P. Klip A. J. Biol. Chem. 1998; 273: 8169-8176Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar), HeLa cells and Madin-Darby canine kidney cells without affecting exocytotic pathways (27Altschuler Y. Barbas S.M. Terlecky L.J. Tang K. Hardy S. Mostov K.E. Schmid S.L. J. Cell Biol. 1998; 143: 1871-1881Crossref PubMed Scopus (186) Google Scholar, 28Kasai K. Shin H.W. Shinotsuka C. Murakami K. Nakayama K. J. Biochem. (Tokyo). 1999; 125: 780-789Crossref PubMed Scopus (44) Google Scholar), it is possible that the cellular functions of dynamin II are largely determined by its specifically targeted subcellular localization in a cell specific fashion. Subcellular localization studies in neuroendocrine cells suggest the presence of dynamin II at the Golgi apparatus, consistent with dynamin II-regulated hormone secretion. Furthermore, overexpression of the wild type or dynamin II mutant regulates de novo production of secretory vesicles from purified Golgi apparatus and semi-permeabilized cells. Thus, dynamin II may be a major form of the dynamin family playing an obligatory role in secretory vesicle biogenesis at the Golgi apparatus during hormone secretion from neuroendocrine cells, consistent with the most recent findings that dynamin II regulates post-Golgi transport of a plasma-membrane protein (29Kreitzer G. Marmorstein A. Okamoto P. Vallee R. Rodriguez-Boulan E. Nat. Cell Biol. 2000; 2: 125-127Crossref PubMed Scopus (200) Google Scholar).In addition, structure-function analysis suggests a crucial role for the PH domain in targeting dynamin II to the Golgi. The finding that overexpressed dynamin II PH domain induces dissociation of endogenous dynamin II from the Golgi and inhibition of hormone secretion strongly suggests that the PH domain of dynamin II is involved in interacting with Golgi membrane docking molecule(s) of dynamin II. Given that PH domains from diverse proteins are capable of binding to the phosphate groups of acidic phospholipids (such as phosphatidylinositol (4,5)-bisphosphate) (30Lemmon M.A. Ferguson K.M. Schlessinger J. Cell. 1996; 85: 621-624Abstract Full Text Full Text PDF PubMed Scopus (428) Google Scholar), it is possible that dynamin II interacts with these lipid molecules but is specifically targeted at the Golgi apparatus by protein-protein interactions involving its PH domain. In an attempt to search for dynamin II interactive proteins, we detected no interaction between dynamin II and γ-adaptin by immunoprecipitation, although previous studies have shown that dynamin I interacts with α-adaptin (31Wang L.H. Sudhof T.C. Anderson R.G. J. Biol. Chem. 1995; 270: 10079-10083Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar) and dynamin II mediates clathrin-coated vesicle budding from the TGN (23Jones S.M. Howell K.E. Henley J.R. Cao H. McNiven M.A. Science. 1998; 279: 573-577Crossref PubMed Scopus (271) Google Scholar). In contrast, the interaction between dynamin II and the βγ subunit of G-proteins via the dynamin II PH domain is specific, since the small GTP-binding protein Rab6 does not coimmunoprecipitate with G-protein β subunit, nor does dynamin II with β-COP (a Golgi coatamer protein also possessing a WD40 repeat). Previous studies have also shown that purified βγ subunits bind to purified dynamin (32Liu J.-P. Yajima Y. Li H. Ackland S. Akita Y. Stewart J. Kawashima S. Mol. Cell. Endocrinol. 1997; 132: 61-71Crossref PubMed Scopus (20) Google Scholar) and stimulate secretory vesicle formation (33Barr F.A. Leyte A. Mollner S. Pfeuffer T. Tooze S.A. Huttner W.B. FEBS Lett. 1991; 294: 239-243Crossref PubMed Scopus (74) Google Scholar).In addition to the PH domain, our data also support a role for the SH3-binding domain in dynamin II-mediated secretory vesicle production at the Golgi apparatus. Overexpression of dynamin II lacking the SH3-binding domain not only induces dislocation of endogenous dynamin II from the Golgi but also produces inhibition of hormone secretion. The translocation of endogenous dynamin II from the Golgi to the plasma membrane suggests an involvement of the SH3-binding domain in dynamin II association with secretory vesicle membranes (Fig.10). Thus, uncoupling of the SH3-binding domain renders dynamin II ineffective in mediating secretory vesicle biogenesis, an effect paralleling previous finding for dynamin I that disruption of the SH3-binding domain impairs synaptic vesicle recycling (34Shupliakov O. Low P. Grabs D. Gad H. Chen H. David C. Takei K. De Camilli P. Brodin L. Science. 1997; 276: 259-263Crossref PubMed Scopus (397) Google Scholar).It is also noteworthy that the SH3-binding domain deletion mutant is more potent than the PH domain mutant in causing inhibition of hormone release (Fig. 7 A) and stimulation of receptor-mediated endocytosis (Fig. 7 B). This difference in potency may reflect a difference between the mutants in competing with endogenous dynamin II at the Golgi. Since the region immediately C-terminal to the PH domain of β-adrenergic receptor kinase is also involved in binding to the βγ subunit (35Inglese J. Luttrell L.M. Iniguez-Lluhi J.A. Touhara K. Koch W.J. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3637-3641Crossref PubMed Scopus (71) Google Scholar), it is possible that other regions outside the PH domain of dynamin II are also involved in targeting the protein to the Golgi, and thus that the SH3-binding domain deletion mutant is more effective than the PH domain-only mutant in causing translocation. Once on the plasma membrane, the misallocated dynamin II is capable of mediating clathrin-dependent endocytosis, suggesting that dynamin II is structurally competent to participate in both secretory and endocytic vesicular trafficking, and that its physiological role in the secretory pathway is determined by its localization to the Golgi.Collectively, these data show that dynamin II GTPase is a dominant regulator of both constitutive and regulated hormone secretions from neuroendocrine cells, operating as a polymer at the Golgi controlling secretory vesicle biogenesis. During this process, dynamin II interacts with βγ subunit of G-proteins via a region containing the PH domain; disruption of the interaction causes translocation of dynamin II from the Golgi to plasma membrane. The parallel increases and decreases in dynamin II expression with constitutive and regulated hormone secretion are consistent with expressed levels of dynamin II playing an important physiological role in regulating the capacity of cell secretion in response to environmental stimuli. Molecular targeting to particular dynamin isoforms may therefore provide a potential therapeutic means for controlling Golgi protein trafficking and hormone secretion under particular circumstances (36Shields D. Arvan P. Curr. Opin. Cell. Biol. 1999; 11: 489-494Crossref PubMed Scopus (20) Google Scholar). The present study defines for the first time dynamin II as a Golgi membrane trafficking protein required for both constitutive and regulated hormone secretion from neuroendocrine cells. A striking consequence of loss of dynamin II function in neuroendocrine cells is lowered hormone secretion with both basal and CRH-stimulated secretion of β-endorphin being affected. A similar phenotype is also found when antisense mRNA is used to block the synthesis of endogenous dynamin II. By contrast, an increase in β-endorphin secretion is observed when wild-type dynamin II is overexpressed. Thus, the extent of both constitutive and regulated hormone secretions from these neuroendocrine cells may be limited by the expression levels of endogenous dynamin II under physiological conditions. This finding is in line with previous findings that dynamin II is associated with the Golgi vesicular buddingin vitro (23Jones S.M. Howell K.E. Henley J.R. Cao H. McNiven M.A. Science. 1998; 279: 573-577Crossref PubMed Scopus (271) Google Scholar) and in cultured rat epithelial cells (24Cao H. Garcia F. McNiven M.A. Mol. Biol. Cell. 1998; 9: 2595-2609Crossref PubMed Scopus (340) Google Scholar), and that microinjection of dynamin II does not mimic that of dynamin I on rapid endocytosis in adrenal chromaffin cells (10Artalejo C.A. Lemmon M.A. Schlessinger J. Palfrey H.C. EMBO J. 1997; 16: 1565-1574Crossref PubMed Scopus (70) Google Scholar). Since dynamin II has also been implicated in caveolae endocytosis in hepatocytes (25Henley J.R. Krueger E.W. Oswald B.J. McNiven M.A. J. Cell Biol. 1998; 141: 85-99Crossref PubMed Scopus (617) Google Scholar) and receptor endocytosis in adipocytes (26Volchuk A. Narine S. Foster L.J. Grabs D. De Camilli P. Klip A. J. Biol. Chem. 1998; 273: 8169-8176Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar), HeLa cells and Madin-Darby canine kidney cells without affecting exocytotic pathways (27Altschuler Y. Barbas S.M. Terlecky L.J. Tang K. Hardy S. Mostov K.E. Schmid S.L. J. Cell Biol. 1998; 143: 1871-1881Crossref PubMed Scopus (186) Google Scholar, 28Kasai K. Shin H.W. Shinotsuka C. Murakami K. Nakayama K. J. Biochem. (Tokyo). 1999; 125: 780-789Crossref PubMed Scopus (44) Google Scholar), it is possible that the cellular functions of dynamin II are largely determined by its specifically targeted subcellular localization in a cell specific fashion. Subcellular localization studies in neuroendocrine cells suggest the presence of dynamin II at the Golgi apparatus, consistent with dynamin II-regulated hormone secretion. Furthermore, overexpression of the wild type or dynamin II mutant regulates de novo production of secretory vesicles from purified Golgi apparatus and semi-permeabilized cells. Thus, dynamin II may be a major form of the dynamin family playing an obligatory role in secretory vesicle biogenesis at the Golgi apparatus during hormone secretion from neuroendocrine cells, consistent with the most recent findings that dynamin II regulates post-Golgi transport of a plasma-membrane protein (29Kreitzer G. Marmorstein A. Okamoto P. Vallee R. Rodriguez-Boulan E. Nat. Cell Biol. 2000; 2: 125-127Crossref PubMed Scopus (200) Google Scholar). In addition, structure-function analysis suggests a crucial role for the PH domain in targeting dynamin II to the Golgi. The finding that overexpressed dynamin II PH domain induces dissociation of endogenous dynamin II from the Golgi and inhibition of hormone secretion strongly suggests that the PH domain of dynamin II is involved in interacting with Golgi membrane docking molecule(s) of dynamin II. Given that PH domains from diverse proteins are capable of binding to the phosphate groups of acidic phospholipids (such as phosphatidylinositol (4,5)-bisphosphate) (30Lemmon M.A. Ferguson K.M. Schlessinger J. Cell. 1996; 85: 621-624Abstract Full Text Full Text PDF PubMed Scopus (428) Google Scholar), it is possible that dynamin II interacts with these lipid molecules but is specifically targeted at the Golgi apparatus by protein-protein interactions involving its PH domain. In an attempt to search for dynamin II interactive proteins, we detected no interaction between dynamin II and γ-adaptin by immunoprecipitation, although previous studies have shown that dynamin I interacts with α-adaptin (31Wang L.H. Sudhof T.C. Anderson R.G. J. Biol. Chem. 1995; 270: 10079-10083Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar) and dynamin II mediates clathrin-coated vesicle budding from the TGN (23Jones S.M. Howell K.E. Henley J.R. Cao H. McNiven M.A. Science. 1998; 279: 573-577Crossref PubMed Scopus (271) Google Scholar). In contrast, the interaction between dynamin II and the βγ subunit of G-proteins via the dynamin II PH domain is specific, since the small GTP-binding protein Rab6 does not coimmunoprecipitate with G-protein β subunit, nor does dynamin II with β-COP (a Golgi coatamer protein also possessing a WD40 repeat). Previous studies have also shown that purified βγ subunits bind to purified dynamin (32Liu J.-P. Yajima Y. Li H. Ackland S. Akita Y. Stewart J. Kawashima S. Mol. Cell. Endocrinol. 1997; 132: 61-71Crossref PubMed Scopus (20) Google Scholar) and stimulate secretory vesicle formation (33Barr F.A. Leyte A. Mollner S. Pfeuffer T. Tooze S.A. Huttner W.B. FEBS Lett. 1991; 294: 239-243Crossref PubMed Scopus (74) Google Scholar). In addition to the PH domain, our data also support a role for the SH3-binding domain in dynamin II-mediated secretory vesicle production at the Golgi apparatus. Overexpression of dynamin II lacking the SH3-binding domain not only induces dislocation of endogenous dynamin II from the Golgi but also produces inhibition of hormone secretion. The translocation of endogenous dynamin II from the Golgi to the plasma membrane suggests an involvement of the SH3-binding domain in dynamin II association with secretory vesicle membranes (Fig.10). Thus, uncoupling of the SH3-binding domain renders dynamin II ineffective in mediating secretory vesicle biogenesis, an effect paralleling previous finding for dynamin I that disruption of the SH3-binding domain impairs synaptic vesicle recycling (34Shupliakov O. Low P. Grabs D. Gad H. Chen H. David C. Takei K. De Camilli P. Brodin L. Science. 1997; 276: 259-263Crossref PubMed Scopus (397) Google Scholar). It is also noteworthy that the SH3-binding domain deletion mutant is more potent than the PH domain mutant in causing inhibition of hormone release (Fig. 7 A) and stimulation of receptor-mediated endocytosis (Fig. 7 B). This difference in potency may reflect a difference between the mutants in competing with endogenous dynamin II at the Golgi. Since the region immediately C-terminal to the PH domain of β-adrenergic receptor kinase is also involved in binding to the βγ subunit (35Inglese J. Luttrell L.M. Iniguez-Lluhi J.A. Touhara K. Koch W.J. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3637-3641Crossref PubMed Scopus (71) Google Scholar), it is possible that other regions outside the PH domain of dynamin II are also involved in targeting the protein to the Golgi, and thus that the SH3-binding domain deletion mutant is more effective than the PH domain-only mutant in causing translocation. Once on the plasma membrane, the misallocated dynamin II is capable of mediating clathrin-dependent endocytosis, suggesting that dynamin II is structurally competent to participate in both secretory and endocytic vesicular trafficking, and that its physiological role in the secretory pathway is determined by its localization to the Golgi. Collectively, these data show that dynamin II GTPase is a dominant regulator of both constitutive and regulated hormone secretions from neuroendocrine cells, operating as a polymer at the Golgi controlling secretory vesicle biogenesis. During this process, dynamin II interacts with βγ subunit of G-proteins via a region containing the PH domain; disruption of the interaction causes translocation of dynamin II from the Golgi to plasma membrane. The parallel increases and decreases in dynamin II expression with constitutive and regulated hormone secretion are consistent with expressed levels of dynamin II playing an important physiological role in regulating the capacity of cell secretion in response to environmental stimuli. Molecular targeting to particular dynamin isoforms may therefore provide a potential therapeutic means for controlling Golgi protein trafficking and hormone secretion under particular circumstances (36Shields D. Arvan P. Curr. Opin. Cell. Biol. 1999; 11: 489-494Crossref PubMed Scopus (20) Google Scholar). We thank S. Kawashima, P. J. Robinson, and T. C. Sudhof for materials and advice, D. Autelitano and R. Dilley for methodological assistance, and S. L. Sabol and K. Sheppard for supplying the AtT20 cells." @default.
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W1985087493 title "Dynamin II Regulates Hormone Secretion in Neuroendocrine Cells" @default.
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