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• W2076358500 abstract "Rab3A and Rab3B are highly homologous monomeric GTPases that are putative regulators of exocytosis in those tissues in which they are expressed. We have characterized and directly compared the targeting and functional properties of these isoforms in PC12 neuroendocrine cells. Rab3A and Rab3B both targeted to norepinephrine (NE)-containing large dense core vesicles (LDCVs) when stably expressed in PC12 cells, as determined by immunofluorescence and membrane fractionation. Both Rab3 isoforms also bound to recombinant rabphilin-3A in a GTP-dependent manner. The membrane association of rabphilin-3A was modestly enhanced in Rab3B-expressing PC12 cells relative to Rab3A-overexpressing cells. In addition, overexpression of Rab3A modestly inhibited Ca2+-evoked NE release, whereas Rab3B and a GTP binding mutant (Rab3B N135I) markedly stimulated the efficiency of [3H]NE secretion by PC12 cells (i.e. secretion normalized to total cell radioactivity). Expression of Rab3B and Rab3B N135I increased not only the efficiency of NE secretion but also the accumulation of [3H]NE into LDCVs (i.e. the secretory cargo available for secretion). Neither of these effects was attributable to changes in the numbers of LDCVs nor the docking of LDCVs at the plasma membrane. Our results indicate that Rab3A and Rab3B have similar membrane targeting properties and are capable of interacting with the same putative downstream effector; i.e. rabphilin-3A. However, these isoforms are functionally distinct monomeric GTPases with Rab3B stimulating a late step in Ca2+-evoked secretion when expressed in PC12 cells. Rab3A and Rab3B are highly homologous monomeric GTPases that are putative regulators of exocytosis in those tissues in which they are expressed. We have characterized and directly compared the targeting and functional properties of these isoforms in PC12 neuroendocrine cells. Rab3A and Rab3B both targeted to norepinephrine (NE)-containing large dense core vesicles (LDCVs) when stably expressed in PC12 cells, as determined by immunofluorescence and membrane fractionation. Both Rab3 isoforms also bound to recombinant rabphilin-3A in a GTP-dependent manner. The membrane association of rabphilin-3A was modestly enhanced in Rab3B-expressing PC12 cells relative to Rab3A-overexpressing cells. In addition, overexpression of Rab3A modestly inhibited Ca2+-evoked NE release, whereas Rab3B and a GTP binding mutant (Rab3B N135I) markedly stimulated the efficiency of [3H]NE secretion by PC12 cells (i.e. secretion normalized to total cell radioactivity). Expression of Rab3B and Rab3B N135I increased not only the efficiency of NE secretion but also the accumulation of [3H]NE into LDCVs (i.e. the secretory cargo available for secretion). Neither of these effects was attributable to changes in the numbers of LDCVs nor the docking of LDCVs at the plasma membrane. Our results indicate that Rab3A and Rab3B have similar membrane targeting properties and are capable of interacting with the same putative downstream effector; i.e. rabphilin-3A. However, these isoforms are functionally distinct monomeric GTPases with Rab3B stimulating a late step in Ca2+-evoked secretion when expressed in PC12 cells. INTRODUCTIONMembers of the Rab family of Ras-related monomeric GTPases are major candidates for controlling membrane docking and/or fusion in a wide variety of cell types. According to current models, the docking and fusion of donor and acceptor membranes is specified by interactions between proteins unique to transport vesicles, termed v-SNAREs, with their cognate t-SNAREs located on the intended target membrane(1.Ferro-Novick S. Jahn R. Nature. 1994; 370: 191-193Crossref PubMed Scopus (555) Google Scholar, 2.Rothman J.E. Nature. 1994; 372: 55-63Crossref PubMed Scopus (1995) Google Scholar). Rabs are not found in purified SNARE complexes(3.Söllner T. Bennett M.K. Whiteheart S.W. Scheller R.H. Rothman J.E. Cell. 1993; 75: 409-418Abstract Full Text PDF PubMed Scopus (1573) Google Scholar); however, genetic studies in yeast indicate that additional proteins, including members of the Rab and Sec1 families, serve to control SNARE assembly by proofreading the fidelity of SNARE interactions and/or by imposing an additional layer of specificity(4.S⊘gaard M. Tani K. Ye R.R. Geromanos S. Tempst P. Kirchhausen T. Rothman J.E. Söllner T. Cell. 1994; 78: 937-948Abstract Full Text PDF PubMed Scopus (441) Google Scholar, 5.Lian J-P. Stone S. Jiang Y. Lyons P. Ferro-Novick S. Nature. 1994; 372: 698-701Crossref PubMed Scopus (161) Google Scholar).The Rab family represents a large number (>30) of homologous proteins, most of which are expressed in a wide variety of tissues where they probably regulate protein traffic pathways that are common to all cells (e.g. endocytosis in the case of Rab5)(6.Bucci C. Parton R.G. Mather I.H. Stunnenberg H. Simons K. Hoflack B. Zerial M. Cell. 1992; 70: 715-728Abstract Full Text PDF PubMed Scopus (1116) Google Scholar). However, members of the Rab3 subfamily (Rab3A-Rab3D), which are 77-85% identical at the amino acid level, are tissue-specific proteins that may regulate cell type-specific secretory pathways. For example, Rab3A is a brain-specific monomeric GTPase that binds to small synaptic vesicles, from which it dissociates during neurotransmitter release(7.Fisher von Mollard G. Südhof T.C. Jahn R. Nature. 1991; 349: 79-81Crossref PubMed Scopus (331) Google Scholar). Rab3A also reportedly associates with the large secretory granules characteristic of adrenal chromaffin cells and PC12 neuroendocrine cells(8.Darchen F. Senyshyn J. Brondyk W.H. Taatjes D.J. Holz R.W. Henry J.P. Denizot J.P. Macara I.G. J. Cell Sci. 1995; 108: 1639-1649Crossref PubMed Google Scholar). Rab3B and Rab3D are primarily expressed outside of the nervous system, with Rab3B being expressed in a wide variety of epithelial tissues (9.Weber E. Berta G. Tousson A. John St., P. Green M.W. Gopalakrishnan U. Jilling T. Sorscher E.J. Elton T.S. Abrahamson D.R. Kirk K.L. J. Cell Biol. 1994; 124: 583-594Crossref Scopus (168) Google Scholar) and Rab3D enriched in adipocytes(10.Baldini G. Hohl T. Lin H.Y. Lodish H.F. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5049-5052Crossref PubMed Scopus (195) Google Scholar). Rab3C shares many properties with Rab3A including expression in brain, targeting to synaptic vesicles, and transient dissociation from synaptic vesicles during neurotransmitter release(11.Fisher von Mollard G. Stahl B. Khokhlatcev A. Südhof T.C. Jahn R. J. Biol. Chem. 1994; 269: 10971-10974PubMed Google Scholar). Rab3A and Rab3C both bind in a GTP-dependent manner to rabphilin-3A, a Ca2+ and phospholipid binding protein that is a putative downstream effector for Rab3A in neurons(12.Shirataki H. Kaibuchi K. Yamaguchi T. Wada K. Horiuchi H. Takai Y. J. Biol. Chem. 1992; 267: 10946-10949Abstract Full Text PDF PubMed Google Scholar, 13.Yamaguchi T. Shirataki H. Kishida S. Miyazaki M. Nishikawa J. Wada K. Numata S. Kaibuchi K. Takai Y. J. Biol. Chem. 1993; 268: 27164-27170Abstract Full Text PDF PubMed Google Scholar, 14.Li C. Takei K. Geppert M. Daniell L. Stenius K. Chapman E.R. Jahn R. De Camilli P. Südhof T.C. Neuron. 1994; 13: 885-898Abstract Full Text PDF PubMed Scopus (170) Google Scholar).All Rab3 isoforms have been proposed to regulate exocytosis in their respective tissues; however, in most cases direct evidence for such regulation is lacking. Moreover, there is evidence to suggest that these structurally related proteins may in fact be functionally distinct. For example, on the basis of the results of antisense RNA experiments performed on anterior pituitary cells, Lledo et al.(15.Lledo P-M. Vernier P. Vincent J-D. Mason W.T. Zorec R. Nature. 1993; 364: 540-544Crossref PubMed Scopus (179) Google Scholar) concluded that Rab3B is a positive regulator of Ca2+-evoked secretion from these cells. Conversely, Holz et al.(16.Holz R.W. Brondyk W.H. Senter R.A. Kuizon L. Macara I.G. J. Biol. Chem. 1994; 269: 10229-10234Abstract Full Text PDF PubMed Google Scholar) have reported that transient overexpression of wild type Rab3A and certain mutants inhibited Ca2+-dependent secretion from bovine chromaffin cells. A similar conclusion was drawn by Johannes et al.(17.Johannes L. Lledo P-M. Roa M. Vincent J-D. Henry J-P. Darchen F. EMBO J. 1994; 13: 2029-2037Crossref PubMed Scopus (186) Google Scholar), who examined the effects of Rab3A mutants that were microinjected or transiently expressed in PC12 neuroendocrine cells. Such data have led to the proposal that Rab3A participates in the formation of a multimeric prefusion complex that must be dissociated prior to membrane fusion and secretion. This proposal seems consistent with the phenotype exhibited by Rab3A-negative transgenic mice, which display no obvious decrement in synaptic transmission except during repetitive stimulation, i.e. when synaptic vesicle recruitment to the presynaptic membrane becomes rate-limiting(18.Geppert M. Bolshakov V.Y. Siegelbaum S.A. Takei K. De Camilli P. Hammer R.E. Südhof T.C. Nature. 1994; 369: 493-497Crossref PubMed Scopus (402) Google Scholar).The preceding results imply that Rab3 isoforms, in particular Rab3A and Rab3B, may not be functionally interchangeable molecules, despite the fact that they are highly homologous proteins (~80% amino acid identity). The goal of the present study was to test this notion by characterizing the targeting and functional properties of Rab3A and Rab3B in PC12 neuroendocrine cells. PC12 cells were utilized because they have a well characterized, regulated secretory pathway (i.e. the norepinephrine-containing LDCVs) 1The abbreviations used are: LDCVlarge dense core vesicleMEMminimal essential mediumGSTglutathione S-transferaseNEnorepinephrinePBSphosphate-buffered salineGTPγSguanosine 5′-3-O-(thio)triphosphate. and because they normally express Rab3A but not Rab3B. Our results indicate that both of these monomeric GTPases target to LDCVs when stably expressed in PC12 cells and that both are capable of binding to rabphilin-3A, a putative downstream effector for Rab3A. Moreover, our functional data confirm that Rab3A overexpression inhibits secretion, albeit modestly and to varying degrees. Conversely, we observed that wild type Rab3B and a GTP-binding mutant (i.e. Rab3B N135I) are potent stimulators of catecholamine release. Interestingly, Rab3B stimulated secretion both by increasing the efficiency of radiolabeled catecholamine release (i.e. release normalized to cell-associated radioactivity) and by increasing the uptake of exogenous catecholamine into LDCVs (i.e. increasing the cargo available for secretion). Thus, Rab3A and Rab3B are functionally distinct molecules, with Rab3B enhancing secretion by PC12 cells at multiple levels of regulation.MATERIALS AND METHODSReagents and AntibodiesRPMI 1640, poly-L-lysine (P1399), reserpine, GTPγS, and the anti-synaptophysin monoclonal antibody (Clone SVP-38) were purchased from Sigma, fetal bovine serum and equine (horse) serum from Hyclone (Logan, UT), and minimal essential medium (MEM), lipofectamine, and Opti-MEM media from Life Technologies, Inc. Hygromycin B and ionomycin were obtained from Calbiochem, [3H]norepinephrine ([3H]NE) from DuPont NEN, norepinephrine from Research Biochemical International (Natick, MA), and cell culture dishes from Costar (Cambridge, MA). PC12 cells were purchased from ATCC (catalog number ATCC CRL 1721). The generation and characterization of a Rab3B polyclonal antibody was described previously(9.Weber E. Berta G. Tousson A. John St., P. Green M.W. Gopalakrishnan U. Jilling T. Sorscher E.J. Elton T.S. Abrahamson D.R. Kirk K.L. J. Cell Biol. 1994; 124: 583-594Crossref Scopus (168) Google Scholar). The monoclonal antibodies specific for Rab3A (Cl42.2) and for Rab3A, Rab3B, and Rab3C (Cl42.1) were generously provided by Dr. R. Jahn (Department of Cell Biology, Yale University Medical School, New Haven, CT)(19.Matteoli M. Takei K. Cameron R. Hurlbut P. Johnston P.A. Südhof T.C. Jahn R. De Camilli P. J. Cell Biol. 1991; 115: 625-633Crossref PubMed Scopus (196) Google Scholar). The polyclonal rabphilin-3A antibody (14.Li C. Takei K. Geppert M. Daniell L. Stenius K. Chapman E.R. Jahn R. De Camilli P. Südhof T.C. Neuron. 1994; 13: 885-898Abstract Full Text PDF PubMed Scopus (170) Google Scholar) and the coding regions for bovine Rab3A and rat rabphilin-3A (14.Li C. Takei K. Geppert M. Daniell L. Stenius K. Chapman E.R. Jahn R. De Camilli P. Südhof T.C. Neuron. 1994; 13: 885-898Abstract Full Text PDF PubMed Scopus (170) Google Scholar) were kind gifts of Dr. T. C. Südhof (University of Texas Southwestern Medical Center, Dallas, TX). The secretogranin II polyclonal antibody was kindly provided by Dr. J. D. Neill (Department of Physiology and Biophysics, University of Alabama at Birmingham, Birmingham, AL)(20.Wei N. Kakar S.S. Neill J.D. Am. J. Physiol. 1995; 268: E145-E152PubMed Google Scholar).Cell CulturePC12 cells were cultured in RPMI 1640, 10% heat inactivated horse serum, 5% fetal bovine serum at 37°C in a humidified 5% CO2 incubator as described(21.Greene L.A. Aletta J.M. Rukenstein A. Green S.H. Methods Enzymol. 1987; 147: 207-216Crossref PubMed Scopus (245) Google Scholar). Stably transfected PC12 cells (passage numbers 1-24) and untransfected cells were cultured and plated on poly-L-lysine (50 μg/ml)-coated dishes for NE secretion assays or on glass coverslips for immunofluorescent staining. All studies were performed in the absence of nerve growth factor.cDNA Cloning, Mutagenesis, and Expression VectorThe coding region of human Rab3B was generated by reverse transcriptionpolymerase chain reaction as described(9.Weber E. Berta G. Tousson A. John St., P. Green M.W. Gopalakrishnan U. Jilling T. Sorscher E.J. Elton T.S. Abrahamson D.R. Kirk K.L. J. Cell Biol. 1994; 124: 583-594Crossref Scopus (168) Google Scholar). Sequence analysis of this clone showed two differences when compared with the human Rab3B sequence originally reported by Zahraoui et al.(22.Zahraoui A. Touchot N. Chardin P. Tavitian A. J. Biol. Chem. 1989; 264: 12394-12401Abstract Full Text PDF PubMed Google Scholar) and submitted to the GenBankTM/EMBL Data Bank (accession number M28214). These differences were as follows: AC instead of CA at nucleotide positions 25 and 26, which results in Thr versus His at amino acid position 9, and C instead of G at nucleotide position 120, which results in Phe versus Leu at amino acid position 40. To rule out the possibility of polymerase chain reaction error in the generation of our Rab3B clone, we also sequenced the human Rab3B clone originally isolated by Zahraoui et al.(22.Zahraoui A. Touchot N. Chardin P. Tavitian A. J. Biol. Chem. 1989; 264: 12394-12401Abstract Full Text PDF PubMed Google Scholar) from a human pheochromocytoma cDNA library (kindly provided by A. Zahraoui, INSERM U-248, Paris, France). The two clones are identical. These results have been confirmed by A. Zahraoui, and a corrected human Rab3B sequence has been submitted to the GenBank/EMBL Data Bank.The GTP binding mutant Rab3B N135I was generated as described(23.Kunkel T.A. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 488-492Crossref PubMed Scopus (4886) Google Scholar). Briefly, the Rab3B coding region was subcloned into M13 mp19 phage, and a point mutation was generated using the mutagenic oligonucleotide primer 5′-GTCACACTTGATCCCCACCAG-3′. The mutation was confirmed by manually sequencing the entire coding region in single-stranded M13 templates using the dideoxy chain termination method (Sequenase, U.S. Biochemical Corp.). For expression in PC12 cells, all cDNAs were ligated into pMEP4 (a gift of M.L. Tykocinski, Case Western Reserve University, Cleveland, OH), an expression vector containing the human metallothionein promoter, SV40 polyadenylation site and a hygromycin B resistance element(24.Groger R.K. Morrow D.M. Tykocinski M.L. Gene (Amst.). 1989; 81: 285-294Crossref PubMed Scopus (76) Google Scholar).Transfection of PC12 Cells5 × 106 PC12 cells were plated onto 100-mm Petri dishes and transfected using Lipofectamine and Opti-MEM media as described (25.Hawley-Nelson P. Ciccarone V. Gebeyehu G. Jessee J. Focus. 1993; 15: 73-79Google Scholar) with the following constructs: pMEP4 without insert (mock), pMEP4-Rab3B, pMEP4-Rab3B N135I, and pMEP4-Rab3A. Stably transfected colonies were selected by growth in complete media containing 320 units/ml hygromycin B. Colonies were screened for recombinant protein expression by immunofluorescence and Western blot analysis (see below). In preliminary experiments we observed that recombinant protein expression levels were generally high without adding exogenous heavy metals (i.e. Zn2+ or Cd2+) to the tissue culture media (i.e. the metallothionein promoter was constitutively active under the growth conditions used in this study). In addition, we observed that the addition of heavy metals to the growth media had nonspecific effects on protein synthesis and NE secretion in PC12 cells. 2E. Weber, T. Jilling, and K. L. Kirk, unpublished observations. For these reasons our analyses of recombinant protein targeting and function were performed in the absence of added heavy metals.Immunofluorescence and ImmunoblottingFor immunofluorescence analysis PC12 cells were grown on poly-L-lysine-coated glass coverslips for 2-4 days, rinsed briefly in PBS, fixed in 3% formaldehyde at 4°C for 1 h, and permeabilized in 0.5% saponin/PBS for 10 min. After blocking with 1% swine γ-globulin or 5% bovine serum albumin, cells were incubated with the appropriate antibodies for 30 min at 37°C. The cells were washed three times in 0.5% saponin/PBS and exposed to the secondary antibodies for 30 min at 37°C in a 1:50 dilution (swine anti-rabbit IgG conjugated to tetramethylrhodamine isothiocyanate or rabbit anti-mouse IgG conjugated to fluorescein isothiocyanate (Dako Corp., Carpinteria, CA)). Immunofluorescence images were collected using a slow scan, cooled CCD camera (Photometrics Inc., Tuscon, AR) interfaced to an inverted microscope (Zeiss IM-35; Carl Zeiss Inc., Thornwood, NY).For Western blot analysis PC12 cells were grown in poly-L-lysine-coated 100-mm Petri dishes, washed in PBS, and lysed in lysis buffer (1% Nonidet P-40, 150 mM NaCl, 20 mM Hepes (pH 7.0), 1 mM EDTA, 1% aprotinin, and 1 mM phenylmethylsulfonyl fluoride). Lysates were clarified by centrifugation at 16,000 × g for 20 min. Protein concentrations of cell lysates were determined using the micro-BCA assay kit (Pierce). Proteins were resolved on 8 or 10% SDS-polyacrylamide gels, transferred to polyvinylidene difluoride membranes, rinsed in Tris-buffered saline, and then blocked in 5% dry milk plus 0.1% Tween 20 in Tris-buffered saline (10 mM Tris-base, 150 mM NaCl, pH 7.5). Blots were incubated with primary and secondary antibodies at room temperature for 1 h in 1 M glucose, 0.5% Tween 20, 10% glycerol and 5% dry milk in Tris-buffered saline. The blots were developed by ECL (Amersham Corp.). For quantitative Western blotting the blots were analyzed by densitometry. In pilot experiments we calibrated the ECL signal by blotting serial dilutions of Rab3B-containing cell lysates and observed a linear relationship between density and protein amount.Membrane Preparation and Density Gradient Cell FractionationPC12 cell membranes and cytosol were prepared as described previously(26.Linstedt A.D. Kelly R.B. Neuron. 1991; 7: 309-317Abstract Full Text PDF PubMed Scopus (100) Google Scholar). Briefly, cells were rinsed twice in ice-cold PBS, detached by scraping in PBS supplemented with 1.0 μg/ml leupeptin, 0.5 μg/ml aprotinin, 35 μg/ml phenylmethylsulfonyl fluoride, and 1.0 μg/ml pepstatin A, and then pelleted at 1000 × g for 5 min at 4°C. Cells were resuspended in homogenization buffer (0.32 M sucrose, 10 mM Hepes, pH 7.4) containing the protease inhibitors mentioned above and homogenized in a Potter homogenizer (1 × 40 strokes). The nuclei and other cell debris were pelleted by centrifugation at 750 × g for 5 min at 4°C. To prepare cytosol and membranes, the postnuclear supernatant was centrifuged in a 90Ti rotor (Beckman Instruments Inc., Palo Alto, CA) at 45,300 rpm (127,000 × g) for 1 h at 4°C. The supernatant (i.e. cytosol) was collected, and the membrane pellet was resuspended in homogenization buffer containing protease inhibitors.For membrane fractionation, postnuclear supernatants that were prepared from cells grown on two 75-90% confluent 100-mm Petri dishes were layered onto a 0.6-1.8 M linear sucrose gradient in 10 mM Hepes, pH 7.4, with a 2.25 M sucrose pad (27.Liu Y. Schweitzer E.S. Nirenberg M.J. Pickel V.M. Evans C.J. Edwards R.H. J. Cell Biol. 1994; 127: 1419-1433Crossref PubMed Scopus (123) Google Scholar). In order to label catecholamine-containing secretory granules, two 100-mm Petri dishes were also labeled with 40 μCi of [3H]norepinephrine/dish for 1 h at 37°C and then chased for 1 h at 37°C prior to fractionation (details described below for NE release assay). All gradients contained the protease inhibitor mixture described above. Equilibrium sedimentation was achieved by centrifugation in a SW40 rotor (Beckman) at 30,000 rpm for 6 h at 4°C. Fractions were collected from the bottom of the tube (450-500 μl/fraction; 20 or 21 fractions). The protein content of each fraction from unlabeled cells was determined with the micro-BCA assay kit. The sucrose concentration of each fraction was determined by measuring the refractive index. The radioactivity of each fraction from cells preloaded with [3H]NE was measured by scintillation counting and corrected for quenching at high sucrose concentrations (as determined by counting [3H]NE standards in 0.6-1.8 M sucrose). For immunoblot analysis fractions were diluted 1:20 in water, and proteins were precipitated by incubation with 10% trichloroacetic acid and 0.1 mg/ml deoxycholate overnight at 4°C. Protein pellets were washed twice with acetone and then solubilized in 5 × SDS sample buffer prior to immunoblot analysis.Production of GST Fusion Proteins and Immunoblot Analysis of Binding ProteinsGST fusion proteins containing full-length Rab3A, Rab3B, and rabphilin-3A were expressed in bacteria using the pGEX-2T prokaryotic expression vector (Pharmacia Biotech Inc.). Transformed Escherichia coli were induced with 0.1 mM isopropyl-1-thio-β-D-galactopyranoside at 37°C for 3 h (for GST-Rab3A and GST-Rab3B) or with 50 μM isopropyl-1-thio-β-D-galactopyranoside at room temperature for 5 h (GST-rabphilin-3A). GST-fusion proteins were affinity-purified from bacterial cell lysates with glutathione-agarose, as described(28.Smith D.B. Corcoran L.M. Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. 2. John Wiley and Sons, Inc., New York1991Google Scholar). For binding experiments cells were lysed in 1% Nonidet P-40 lysis buffer (see immunoblotting protocol under “Materials and Methods”) or homogenized (see membrane preparation protocol under “Materials and Methods”) in the presence or absence of 50 μM GTPγS. Clarified cell lysates or high speed supernatants (i.e. cytosol) were incubated with GST fusion proteins (GST-3A, GST-3B, GST-rabphilin-3A) or GST alone immobilized on agarose beads for 2 h at 4°C. Beads were washed four times with lysis or homogenization buffer. Bound proteins were eluted in 5 × sample buffer and analyzed by SDS-polyacrylamide gel electrophoresis followed by immunoblotting. Equal amounts of GST and GST-fusion protein (GST-rabphilin-3A, GST-3A, GST-3B) were used in each set of experiments. Bound proteins were eluted in similar volumes of 5 × sample buffer, and equal volumes of eluates were analyzed by immunoblotting.[3H]Norepinephrine Release AssayPC12 cells were grown in 35-mm poly-L-lysine-coated Petri dishes for 2-4 days until they reached 40-70% confluency. Cells were washed in RPMI and loaded at 37°C for 1 h in RPMI, 10% horse serum, 5% fetal bovine serum containing 1-2 μCi of [3H]NE, rinsed with PBS, and then chased in complete media for an additional 1 h in a 5% CO2 humidified incubator. Release assays were performed at 37°C in 10 mM Hepes-buffered MEM (pH 7.4) or low K+/high K+ medium (10 mM Hepes, 5 or 55 mM KCl, 145 or 95 mM NaCl, 2 mM CaCl2, 10 mM glucose, pH 7.4) (29.Kongsamut S. Miller R.J. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 2243-2247Crossref PubMed Scopus (53) Google Scholar). The low K+ medium was used for assays where the dependence of secretion on extracellular Ca2+ was examined, with Ca2+ concentrations that varied from 1 μM to 1.8 mM CaCl2. PC12 cells were incubated in MEM or low K+ medium for 10 min at 37°C prior to inducing secretion with 1 μM ionomycin or 55 mM K+. Except where noted, release was assessed in time course experiments in which the media was collected and replaced every 2 min. At the end of the experiment the cells were solubilized twice in 1 ml of 0.25 N NaOH, and the lysates were carefully collected. Media cpm were determined by liquid scintillation counting and normalized to total cell cpm. Total cell cpm were determined by summing all media counts and the radioactivity remaining in the cells at the end of the experiment, the latter of which was determined by counting the cell lysates.Transmission Electron Microscopy and MorphometryPC12 cells were grown in 35-mm poly-L-lysine-coated Petri dishes for 2-4 days until they reached 40-70% confluency, rinsed twice in PBS, and incubated in MEM supplemented with 10 mM Hepes (pH 7.4) for 10 min. Monolayers were fixed in 1% buffered glutaraldehyde, postfixed in 1% buffered OsO4, dehydrated and embedded in Polybed, sectioned, and stained with uranyl acetate and lead citrate. The ultrathin sections were examined and photographed with a Hitachi-7000 transmission electron microscope. Numbers of dense core granules/cell and the numbers of granules within 100 nm of the plasma membrane were counted by six investigators in a double blind fashion.RESULTSExpression and Membrane Association of Rab3A and Rab3B in PC12 CellsIn order to directly compare the targeting and functional properties of Rab3A and Rab3B, we have generated PC12 cell lines that stably express recombinant Rab3A, Rab3B, or the Rab3B mutant, Rab3B N135I. Rab3B N135I corresponds to an oncogenic Ras mutant (H-Ras N116I) that has a substantially elevated dissociation rate for both GTP and GDP. Fig. 1A confirms for selected cell lines the expression of wild type Rab3B and Rab3B N135I protein, and the overexpression of Rab3A protein, as compared with nontransfected PC12 cells (nPC12) and mock-transfected cell lines. No expression of Rab3B protein was detected in nontransfected or in mock-transfected PC12 cells. Endogenous Rab3A and recombinant Rab3A and Rab3B all migrated as a ~25-kDa band in Western blots of total cell lysates, whereas Rab3B N135I migrated as a doublet with slightly faster mobility. Fig. 1B compares the efficiency of membrane association for Rab3A, Rab3B, and Rab3B N135I. The efficiency of Rab3A association with membranes was not substantially altered by the expression of recombinant Rab3B or Rab3B N135I. Interestingly, Rab3B N135I associated with PC12 membranes much less efficiently than either wild type Rab3B or wild type Rab3A; i.e. the great majority of mutant protein was present in the cytosol. Four out of five Rab3B N135I clones exhibited detectable but modest membrane association of the mutant protein (e.g. see clone 3B N135I 18 in Fig. 1B), whereas for one clone (3B N135I 32; see Fig. 1B) little or no Rab3B N135I protein was present on the membranes. This latter clone (mutant 32) also exhibits a different secretory phenotype than the other mutant Rab3B-expressing clones, as will be discussed below. Cytosolic Rab 3B N135I typically migrated as a doublet in which the dominant band had a higher mobility than that of wild type Rab3B, as noted above for cell lysates (Fig. 1A). Rab3B N135I also exhibits higher mobility by SDS-polyacrylamide gel electrophoresis and poorly associates with membranes when expressed in Madin-Darby canine kidney renal epithelial cells.2Rab3A and Rab3B Target to Secretory GranulesThe targeting of Rab3A and Rab3B in PC12 cells was initially characterized by immunofluorescence staining (Fig. 2). Native Rab3A in untransfected cells and mock-transfected cells, as well as recombinant Rab3A in overexpressing cells, targeted at or very near the plasma membrane; in particular, at regions of cell-cell contact. Neither expression of recombinant Rab3B (Fig. 2) nor of Rab3B N135I (data not shown) changed this pattern of Rab3A targeting. Recombinant Rab3B protein was also detected predominantly in the region of the plasma membrane. Rab3B N135I protein was not detectable in any clone by immunofluorescence when the cells were permeabilized by our standard procedures (i.e. 0.5% saponin, see “Materials and Methods”). However, if the Rab3B N135I clones were permeabilized in 0.1% saponin, faint immunoreactivity was detected near the plasma membrane (except for mutant clone 32), along with increased cytoplasmic staining.2Figure 2:Immunofluorescence analysis of the expression and targeting of Rab3A and Rab3B in PC12 cells. Left column, mock-transfected cells, middle column, PC12 cells transfected with Rab3B coding region; right column, PC12 cells transfected with Rab3A cDNA. Top row, monoclonal Rab3A-specific antibody (1:100 dilution); bottom row, polyclonal anti-Rab3B antibody (1:50 dilution). No staining was observed in parallel control experiments using isotype-matched control and preimmune IgG, respectively (data not shown). Bar, 10 μm.View Large Image Figure ViewerDownload (PPT)To further characterize the subcellular locations of Rab3A and Rab3B in PC12 cells, we fractionated selected PC12 clones by equilibrium sedimentation through sucrose density gradients (Figure 3:, Fig" @default.
• W2076358500 created "2016-06-24" @default.
• W2076358500 creator A5026417091 @default.
• W2076358500 creator A5049576480 @default.
• W2076358500 creator A5084115012 @default.
• W2076358500 date "1996-03-01" @default.
• W2076358500 modified "2023-09-26" @default.
• W2076358500 title "Distinct Functional Properties of Rab3A and Rab3B in PC12 Neuroendocrine Cells" @default.
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