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Serotonin Transamidates Rab4 and Facilitates Its Binding to the C Terminus of Serotonin Transporter

2008; Elsevier BV; Volume: 283; Issue: 14 Linguagem: Inglês

10.1074/jbc.m706367200

ISSN

1083-351X

Autores

Billow A. Ahmed, Brandon C. Jeffus, Syed I. A. Bukhari, Justin T. Harney, Reşat Ünal, Vladimir Lupashin, Peter van der Sluijs, Fusun Kilic,

Tópico(s)

Pancreatic function and diabetes

Resumo

The serotonin transporter (SERT) on the plasma membrane is the major mechanism for the clearance of plasma serotonin (5-hydroxytryptamine (5HT)). The uptake rates of cells depend on the density of SERT molecules on the plasma membrane. Interestingly, the number of SERT molecules on the platelet surface is down-regulated when plasma 5HT ([5HT]ex) is elevated. It is well reported that stimulation of cells with high [5HT]ex induces transamidation of a small GTPase, Rab4. Modification with 5HT stabilizes Rab4 in its active, GTP-bound form, Rab4-GTP. Although investigating the mechanism by which elevated plasma 5HT level down-regulates the density of SERT molecules on the plasma membrane, we studied Rab4 and SERT in heterologous and platelet expression systems. Our data demonstrate that, in response to elevated [5HT]ex, Rab4-GTP co-localizes with and binds to SERT. The association of SERT with Rab4-GTP depends on: (i) 5HT modification and (ii) the GTP-binding ability of Rab4. Their association retains transporter molecules intracellularly. Furthermore, we mapped the Rab4-SERT association domain to amino acids 616-624 in the cytoplasmic tail of SERT. This finding provides an explanation for the role of the C terminus in the localization and trafficking of SERT via Rab4 in a plasma 5HT-dependent manner. Therefore, we propose that elevated [5HT]ex"paralyzes" the translocation of SERT from intracellular locations to the plasma membrane by controlling transamidation and Rab4-GTP formation. The serotonin transporter (SERT) on the plasma membrane is the major mechanism for the clearance of plasma serotonin (5-hydroxytryptamine (5HT)). The uptake rates of cells depend on the density of SERT molecules on the plasma membrane. Interestingly, the number of SERT molecules on the platelet surface is down-regulated when plasma 5HT ([5HT]ex) is elevated. It is well reported that stimulation of cells with high [5HT]ex induces transamidation of a small GTPase, Rab4. Modification with 5HT stabilizes Rab4 in its active, GTP-bound form, Rab4-GTP. Although investigating the mechanism by which elevated plasma 5HT level down-regulates the density of SERT molecules on the plasma membrane, we studied Rab4 and SERT in heterologous and platelet expression systems. Our data demonstrate that, in response to elevated [5HT]ex, Rab4-GTP co-localizes with and binds to SERT. The association of SERT with Rab4-GTP depends on: (i) 5HT modification and (ii) the GTP-binding ability of Rab4. Their association retains transporter molecules intracellularly. Furthermore, we mapped the Rab4-SERT association domain to amino acids 616-624 in the cytoplasmic tail of SERT. This finding provides an explanation for the role of the C terminus in the localization and trafficking of SERT via Rab4 in a plasma 5HT-dependent manner. Therefore, we propose that elevated [5HT]ex"paralyzes" the translocation of SERT from intracellular locations to the plasma membrane by controlling transamidation and Rab4-GTP formation. Withdrawal: Serotonin transamidates Rab4 and facilitates its binding to the C terminus of serotonin transporterJournal of Biological ChemistryVol. 294Issue 24PreviewVOLUME 283 (2008) PAGES 9388–9398 Full-Text PDF Open AccessExpression of Concern: Serotonin transamidates Rab4 and facilitates its binding to the C terminus of serotonin transporter.Journal of Biological ChemistryVol. 294Issue 13PreviewVOLUME 283 (2008) PAGES 9388–9398 Full-Text PDF Open Access The serotonin transporter (SERT) 2The abbreviations used are: SERTserotonin transporterAbantibodyhSERThuman serotonin transporterhDAThuman dopamine transporterhNEThuman norepinephrine transporterDATdopamine transporterNETnorepinephrine transporter5HTserotoninSSRIselective serotonin reuptake inhibitorsprotein kinase Cprotein kinase CYFPyellow fluorescent proteinDMEMDulbecco's modified eagle's mediumPBSphosphate buffered salineCHOChinese hamster ovaryIFimmunofluorescenceDTTdithiolthreitolTBSTris-buffered salinePIMprotease inhibitor mixtureWGAwheat germ agglutininWBWestern blotting. is a member of the Cl-- and Na+-dependent monoamine transporter family, which also includes the dopamine transporter (DAT) and the norepinephrine transporter. SERT is a 630-amino acid plasma membrane-bound glycoprotein. Hydropathy analysis predicts that SERT contains 12 transmembrane domains and that both the N and C termini are exposed to the cytoplasm. The primary function of SERT in the central nervous system involves the regulation of serotonergic signaling via transport of serotonin (5-hydroxytryptamine (5HT)) molecules from the synaptic cleft into the pre-synaptic terminal for re-utilization. SERT is also expressed in non-neuronal cells, including platelets, placental, intestinal and adrenal cell lines, but the exact function of SERT in these cell lines is still under investigation (1Blakely R.D. Berson H.E. Fremeau Jr., R.T. Caron M.G. Peek M.M. Prince H.K. Bradley C.C. Nature. 1991; 354: 66-70Crossref PubMed Scopus (686) Google Scholar, 2Hoffman B.J. Mezey E. Brownstein M.J. Science. 1991; 254: 579-580Crossref PubMed Scopus (507) Google Scholar, 3Ramamoorthy S. Leibach F.H. Mahesh V.B. Ganapathy V. 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The C- and N-terminal regions of monoamine transporter proteins have just recently garnered increased attention for their importance in transport function and localization. Significant work has been accomplished in identifying the importance of the C-terminal region of DAT and norepinephrine transporter in transporter function, expression, and localization (6Binda F. Lute B.J. Dipace C. Blakely R.D. Galli A. Neuropharmacology. 2006; 50: 354-361Crossref PubMed Scopus (13) Google Scholar, 7Bjerggaard C. Fog J.U. Hastrup H. Madsen K. Loland C.J. Javitch J.A. Gether U. J. Neurosci. 2004; 24: 7024-7036Crossref PubMed Scopus (77) Google Scholar, 8Distelmaier F. Wiedemann P. Bruss M. Bonisch H. J. Neurochem. 2004; 91: 537-546Crossref PubMed Scopus (20) Google Scholar, 9Bauman P.A. Blakely R.D. Arch. Biochem. Biophys. 2002; 404: 80-91Crossref PubMed Scopus (49) Google Scholar). The proteins interacting with the N terminus of SERT are syntaxin 1A (10Quick M.W. Int. J. Dev. Neurosci. 2002; 20: 219-224Crossref PubMed Scopus (48) Google Scholar, 11Quick M.W. Neuron. 2003; 40: 537-549Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar) and secretory carrier membrane protein 2 (12Muller H.K. Wiborg O. Haase J. J. Biol. Chem. 2006; 281: 28901-28909Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). SERT also complexes with Hic-5 (13Carneiro A.M. Blakely R.D. J. Biol. Chem. 2006; 281: 24769-24780Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar) and α-synuclein (14Wersinger C. Rusnak M. Sidhu A. Eur. J. Neurosci. 2006; 24: 55-64Crossref PubMed Scopus (71) Google Scholar), but the functional significance of these interactions are not known. PICK1 (15Torres G.E. Yao W.D. Mohn A.R. Quan H. Kim K.M. Levey A.I. Staudinger J. Caron M.G. Neuron. 2001; 30: 121-134Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar), MacMARCKS (16Jess U. El Far O. Kirsch J. Betz H. Biochem. Biophys. Res. Commun. 2002; 294: 272-279Crossref PubMed Scopus (40) Google Scholar), the actin cytoskeleton (49Mochizuki H. Amano T. Seki T. Matsubayashi H. Mitsuhata C. Morita K. Kitayama S. Dohi T. Mishima H.K. Sakai N. Neurochem. Int. 2005; 46: 93-105Crossref PubMed Scopus (23) Google Scholar), neuronal nitric-oxide synthase, and Sec23A and Sec24C (17Chanrion B. Mannoury la Cour C. Bertaso F. Lerner-Natoli M. Freissmuth M. Millan M.J. Bockaert J. Marin P. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 8119-8124Crossref PubMed Scopus (144) Google Scholar) associate with the C terminus of SERT. It has been suggested that the final 20 amino acids from the C-terminal of SERT are critical for the functional expression of the transporter (18Larsen M.B. Fjorback A.W. Wiborg O. Biochemistry. 2006; 45: 1331-1337Crossref PubMed Scopus (29) Google Scholar). However, the role of this region on the protein-protein interactions of SERT during protein folding or plasma membrane localization processes is incompletely understood. SERT is an important target for many therapeutic agents that enhance serotonergic signaling (e.g. for treatment of affective neuropsychiatric disorders) and is involved in the mechanism of some drugs of abuse (e.g. cocaine and 3,4-methylenedioxy-N-methamphetamine (also known as ecstasy)) (19Kilic F. Murphy D. Rudnick G. Mol. Pharmacol. 2003; 64: 4-12Crossref Scopus (126) Google Scholar, 20Murphy D.L. Andrews A.M. Wichems C.H. Li Q. Tohda M. Greenberg B. J. Clin. Psychiatry. 1998; 59: 4-12PubMed Google Scholar). These compounds effect recycling and internalization and thereby the density of SERT on the plasma membrane (21Ramamoorthy S. Blakely R.D. Science. 1999; 285: 763-766Crossref PubMed Scopus (313) Google Scholar). The role of substrates in the trafficking of γ-aminobutyric acid transporter (22Wang D. Quick M.W. J. Biol. Chem. 2005; 280: 18703-18799Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 23Farhan H. Reiterer V. Korkhov V.M. Schmid J.A. Freissmuth M. Sitte H.H. J. Biol. Chem. 2007; 282: 7679-7689Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar), DAT (24Holton K.L. Loder M.K. Melikian H.E. Nat. Neurosci. 2005; 8: 881-888Crossref PubMed Scopus (114) Google Scholar), and the excitatory amino acid transporter 1 is to regulate the expression of the transporter on the cell membrane, i.e. glutamate increases the surface expression of excitatory amino acid transporter 1 (25Duan S. Anderson C.M. Stein B.A. Swanson R.A. J. Neurosci. 1999; 19: 10193-10200Crossref PubMed Google Scholar). Activation of protein kinase C decreases the number of SERT molecules on the plasma membrane; however, this effect of protein kinase C is blocked in the presence of [5HT]ex at high levels (21Ramamoorthy S. Blakely R.D. Science. 1999; 285: 763-766Crossref PubMed Scopus (313) Google Scholar). Importantly, Whitworth et al., (26Whitworth T.L. Herndon L.C. Quick M.W. J. Neurosci. 2003; 22: RC192Crossref Google Scholar) demonstrated that 5HT-mediated signals have a role in regulating the number of transporters at or near the synapse by changing the subcellular redistribution of SERT in neurons and glia. These studies confirm that the functional efficiency of SERT is regulated by its membrane-trafficking pathways, i.e. internalization and recycling of the transporter (27Ramamoorthy S. Baumen A.L. Moore K.R. Han H. Yang-Fen T. Chang A.S. Ganapathy V. Blakely R.D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2542-2546Crossref PubMed Scopus (782) Google Scholar, 28Wang X. Baumann M.H. Xu H. Morales M. Rothman R.B. J. Pharmacol. Exp. Ther. 2005; 314: 1002-1012Crossref PubMed Scopus (49) Google Scholar, 29Muller H.K. Wiborg O. Haase J. J. Biol. Chem. 2006; 281: 28901-28919Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). The mechanism by which 5HT accomplishes this task remains to be explored. In platelets, plasma 5HT at high levels appears to be closely related to regulated exocytosis, in which Rho and Rab4 are implicated (30Symons M. Rusk N. Curr. Biol. 2003; 13: R409-R418Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 31Deneka M. Neeft M. van der Sluijs P. Crit. Rev. Biochem. Mol. Biol. 2003; 38: 121-142Crossref PubMed Scopus (118) Google Scholar, 32Walther D.J. Peter J.U. Winter S. Holtje M. Paulmann N. Grohmann M. Vowinckel J. Alamo-Bethencourt V. Wilhelm C.S. Ahnert-Hilger G. Bader M. Cell. 2003; 115: 851-862Abstract Full Text Full Text PDF PubMed Scopus (376) Google Scholar). The Rab protein family of small GTP-binding proteins regulates vesicular traffic (30Symons M. Rusk N. Curr. Biol. 2003; 13: R409-R418Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 32Walther D.J. Peter J.U. Winter S. Holtje M. Paulmann N. Grohmann M. Vowinckel J. Alamo-Bethencourt V. Wilhelm C.S. Ahnert-Hilger G. Bader M. Cell. 2003; 115: 851-862Abstract Full Text Full Text PDF PubMed Scopus (376) Google Scholar, 33Chavrier P. Parton R.G. Hauri H.P. Simons K. Zerial M. Cell. 1990; 62: 317-329Abstract Full Text PDF PubMed Scopus (888) Google Scholar). There are more than 60 Rab protein family members in mammalian cells, and individual Rab proteins are localized to the cytoplasmic leaflet of distinct compartments in both the endocytotic and exocytotic pathways (34Jones M.C. Caswell P.T. Norman J.C. Curr. Opin. Cell Biol. 2006; 18: 549-557Crossref PubMed Scopus (234) Google Scholar). Rab4 is associated with early endosomes and regulates membrane recycling (34Jones M.C. Caswell P.T. Norman J.C. Curr. Opin. Cell Biol. 2006; 18: 549-557Crossref PubMed Scopus (234) Google Scholar, 35Mohrmann K. van der Sluijs P. Mol. Membr. Biol. 1999; 16: 81-87Crossref PubMed Scopus (117) Google Scholar, 36Borner G.H. Harbour M. Hester S. Lilley K.S. Robinson M.S. J. Cell Biol. 2006; 175: 571-578Crossref PubMed Scopus (128) Google Scholar). Rab4 also regulates exocytic events from other post-Golgi compartments in cells with specialized functions (34Jones M.C. Caswell P.T. Norman J.C. Curr. Opin. Cell Biol. 2006; 18: 549-557Crossref PubMed Scopus (234) Google Scholar). In adipocytes, Rab4 controls recycling of the insulin-regulated glucose transporter GLUT4 (37Cormont M. Bortoluzzi M.N. Gautier N. Mari M. van Obberghen E. Le Marchand-Brustel Y. Mol. Cell. Biol. 1996; 16: 6879-6886Crossref PubMed Scopus (99) Google Scholar), and recombinant Rab4 stimulates α-granule secretion in platelets in vitro (38Shirakawa R. Yoshioka A. Horiuchi H. Nishioka H. Tabuchi A. Kita T. J. Biol. Chem. 2000; 275: 33844-33849Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). All Rab proteins contain highly conserved domains required for guanine nucleotide binding, GTP/GDP exchange, and GTP hydrolysis that are essential for their proper targeting and function (31Deneka M. Neeft M. van der Sluijs P. Crit. Rev. Biochem. Mol. Biol. 2003; 38: 121-142Crossref PubMed Scopus (118) Google Scholar). Stimulation of cells with 5HT activates the phosphatidylinositol pathway, which increases the intracellular Ca2+. As a consequence, transglutaminase is activated, which catalyzes transamidation of small GTPases with intracellular 5HT [5HT]in (32Walther D.J. Peter J.U. Winter S. Holtje M. Paulmann N. Grohmann M. Vowinckel J. Alamo-Bethencourt V. Wilhelm C.S. Ahnert-Hilger G. Bader M. Cell. 2003; 115: 851-862Abstract Full Text Full Text PDF PubMed Scopus (376) Google Scholar, 39Aktories K. Barbieri J.T. Nat. Rev. Microbiol. 2005; 3: 397-410Crossref PubMed Scopus (194) Google Scholar). Bacterial transglutaminase transamidates the Gln residue of the DTAGQE sequence within the GTP hydrolysis domain of Rho, which produces constitutively active Rho (39Aktories K. Barbieri J.T. Nat. Rev. Microbiol. 2005; 3: 397-410Crossref PubMed Scopus (194) Google Scholar). The DTAGQE signature is conserved in all Rab proteins. Rab4 and Rab27 are transamidated by transglutaminase (32Walther D.J. Peter J.U. Winter S. Holtje M. Paulmann N. Grohmann M. Vowinckel J. Alamo-Bethencourt V. Wilhelm C.S. Ahnert-Hilger G. Bader M. Cell. 2003; 115: 851-862Abstract Full Text Full Text PDF PubMed Scopus (376) Google Scholar). Thus, it has not been established whether the Rab4 effector network involved in actin-myosin dynamics and membrane trafficking is regulated through the transamidation to a constitutively active form of Rab4 (32Walther D.J. Peter J.U. Winter S. Holtje M. Paulmann N. Grohmann M. Vowinckel J. Alamo-Bethencourt V. Wilhelm C.S. Ahnert-Hilger G. Bader M. Cell. 2003; 115: 851-862Abstract Full Text Full Text PDF PubMed Scopus (376) Google Scholar). The present study investigates the mechanism by which high concentrations of [5HT]ex alter the redistribution of SERT. Using Rab4Q67L and Rab4N121I mutant forms of Rab4, here, we show that intracellular 5HTfirst binds to Rab4, allowing the GTPase to interact with SERT. Rab4N121I (41Lazzarino D.A. Blier P. Mellman I. J. Exp. Med. 1998; 188: 1769-1774Crossref PubMed Scopus (37) Google Scholar), which cannot bind to nucleotide, does not bind to SERT at all. This conclusion is enhanced by our finding that Rab4Q67L, which has impaired GTP-hydrolysis ability, therefore mimics the constitutively active GTP-bound form of Rab4 (47Gerez L. Mohrmann K. van Raak M. Jongeneelen M. Zhou X.Z. Lu K.P. van Der Sluijs P. Mol. Biol. Cell. 2000; 11: 2201-2211Crossref PubMed Scopus (41) Google Scholar). Our data clearly demonstrate that the SERT-Rab4Q67L association is not influenced by the concentration of [5HT]ex. The SERT-Rab4 interaction changes the distribution of transporters between intracellular locations versus those on the plasma membrane that modulate 5HT uptake. We propose that [5HT]ex controls the density of SERT molecules on the plasma membrane by enabling its association with Rab4. The Rab4-SERT interaction retains SERT molecules intracellularly. Materials—All restriction endonucleases and ligases were from New England Biolab (Beverly, MA). Expression vectors, cell culture materials, and Lipofectamine 2000 were from Invitrogen. Micro BCA Protein Assay, enhanced chemiluminescence (ECL) Western blotting (WB) system, NHS-SS-biotin, and immuno-pure horseradish peroxidase-conjugated streptavidin were from Pierce. 5HT was from Sigma-Aldrich. CHO cells were provided from American Type Culture Collection (Manassas, VA). [3H]5HT was purchased from PerkinElmer Life Sciences. Monoclonal anti-SERT-Ab was provided by MAbTechnology (Stone Mountain, GA). Plasmids, Constructs, and Cell Line Expression Systems—SERT cDNA was donated by Dr. H. H. Sitte (Medical University Vienna, Institute of Pharmacology) subcloned into Kpn1/Not1 sites of pcDNA3 and HindIII/XbaI sites of pEYFP-C1. CFP fusions of SERT do not alter the expression efficiency or 5HT uptake levels of transporter protein (40Schmid J.A. Scholze P. Kudlacek O. Freissmuth M. Singer E.A. Sitte H.H. J. Biol. Chem. 2001; 276: 3805-3810Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). Mutant transporters were constructed utilizing a Stratagene QuikChange XL site-directed mutagenesis kit. Rab4 and Rab4N121I constructs have been described previously (41Lazzarino D.A. Blier P. Mellman I. J. Exp. Med. 1998; 188: 1769-1774Crossref PubMed Scopus (37) Google Scholar). A list of primers used to construct each mutation is located in supplemental Table S1. All synthetic constructs were verified via DNA sequencing. CHO cells were maintained in α-minimal essential medium. Media were supplemented with 10% fetal bovine serum, 2 mm l-glutamine, penicillin, and streptomycin. For all studies, cells were transiently transfected with either SERT and/or Rab4 constructs using a 1:2.5 ratio of Lipofectamine 2000 reagent to DNA in Opti-MEM. SERT expression was determined with quantitative WB on a VersaDoc 1000 analysis system (42Ozaslan D. Wang S. Ahmed B. Bene A. Kocabas A.M. Kilic F. J. Biol. Chem. 2003; 278: 43991-44000Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 43Kocabas A.M. Rudnick G. Kilic F. J. Neurochem. 2003; 85: 1513-1520Crossref PubMed Scopus (49) Google Scholar, 44Kilic F. Rudnick G. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3106-3111Crossref PubMed Scopus (189) Google Scholar, 45Brenner B. Harney J.T. Ahmed B.A. Jeffus B.C. Unal R. Mehta J.L. Kilic F. J. Neurochem. 2007; 102: 206-216Crossref PubMed Scopus (144) Google Scholar). 5HT Uptake Assay in Heterologous Expression System—CHO cells in 24-well culture plates were transfected with SERT and assayed 24 h post-transfection (42Ozaslan D. Wang S. Ahmed B. Bene A. Kocabas A.M. Kilic F. J. Biol. Chem. 2003; 278: 43991-44000Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 43Kocabas A.M. Rudnick G. Kilic F. J. Neurochem. 2003; 85: 1513-1520Crossref PubMed Scopus (49) Google Scholar, 44Kilic F. Rudnick G. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3106-3111Crossref PubMed Scopus (189) Google Scholar, 45Brenner B. Harney J.T. Ahmed B.A. Jeffus B.C. Unal R. Mehta J.L. Kilic F. J. Neurochem. 2007; 102: 206-216Crossref PubMed Scopus (144) Google Scholar). Briefly, cells were washed with phosphate-buffered saline (PBS) containing 0.1 mm CaCl2 and 1 mm MgCl2 and incubated for 10 min with this buffer and then assayed for their 5HT uptake by incubating with 20.5 nm [1,2-3H]5HT (3400 cpm/pmol) as described previously (32Walther D.J. Peter J.U. Winter S. Holtje M. Paulmann N. Grohmann M. Vowinckel J. Alamo-Bethencourt V. Wilhelm C.S. Ahnert-Hilger G. Bader M. Cell. 2003; 115: 851-862Abstract Full Text Full Text PDF PubMed Scopus (376) Google Scholar, 33Chavrier P. Parton R.G. Hauri H.P. Simons K. Zerial M. Cell. 1990; 62: 317-329Abstract Full Text PDF PubMed Scopus (888) Google Scholar, 34Jones M.C. Caswell P.T. Norman J.C. Curr. Opin. Cell Biol. 2006; 18: 549-557Crossref PubMed Scopus (234) Google Scholar). Cells without plasmid DNA (mock transfected) served as negative controls. Protein concentration was determined using the Micro BCA Protein Assay Reagent Kit. The 5HT uptake rates were determined in triplicate in three independent experiments. WB Analysis and Quantitation of SERT Expression—Cells were solubilized in PBS containing 0.44% SDS, 1 mm phenylmethylsulfonyl fluoride, and protease inhibitor mixture (PIM). The PIM contained 5 μg/ml pepstatin, 50 μg/ml leupeptin, and 5 μg/ml aprotinin and was included with each lysis buffer (31Deneka M. Neeft M. van der Sluijs P. Crit. Rev. Biochem. Mol. Biol. 2003; 38: 121-142Crossref PubMed Scopus (118) Google Scholar). Samples were analyzed by SDS-PAGE and transferred to nitrocellulose. Blots were incubated first with SERT antibody (Ab) (diluted 1:400), and then with horseradish peroxidase-conjugated anti-rabbit IgG at a dilution of 1:5000. The signals were visualized using the ECL WB detection system. The SERT Ab gave one major band at 90 kDa in CHO or HeLa cells expressing SERT, which was absent in non-transfected cells. Signals of the 90-kDa band were quantitated with a VersaDoc 1000 analysis system (42Ozaslan D. Wang S. Ahmed B. Bene A. Kocabas A.M. Kilic F. J. Biol. Chem. 2003; 278: 43991-44000Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Using a standard curve of SERT prepared form the lysates of CHO cells expressing SERT, the integrated density value for each band was converted to an equivalent amount of SERT for Ab (44Kilic F. Rudnick G. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3106-3111Crossref PubMed Scopus (189) Google Scholar). Cell Surface Biotinylation—Cell surface expression of SERT was detected after biotinylation with membrane-impermeant NHS-SS-biotin as described previously (42Ozaslan D. Wang S. Ahmed B. Bene A. Kocabas A.M. Kilic F. J. Biol. Chem. 2003; 278: 43991-44000Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 43Kocabas A.M. Rudnick G. Kilic F. J. Neurochem. 2003; 85: 1513-1520Crossref PubMed Scopus (49) Google Scholar, 44Kilic F. Rudnick G. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3106-3111Crossref PubMed Scopus (189) Google Scholar, 45Brenner B. Harney J.T. Ahmed B.A. Jeffus B.C. Unal R. Mehta J.L. Kilic F. J. Neurochem. 2007; 102: 206-216Crossref PubMed Scopus (144) Google Scholar). Briefly, upon the biotinylation reaction, the cells were treated with 100 mm glycine to quench unreacted NHS-SS-biotin and lysed in Tris-buffered saline containing 1% SDS, 1% Triton X-100, and PIM/phenylmethylsulfonyl fluoride. The biotinylated proteins (500 μl) were recovered with an excess of streptavidin-agarose beads (400 μl) during overnight incubation. Biotinylated proteins were eluted in 100 μl of sample buffer, resolved by SDS-PAGE and transferred to nitrocellulose, and were detected with the SERT Ab as described (42Ozaslan D. Wang S. Ahmed B. Bene A. Kocabas A.M. Kilic F. J. Biol. Chem. 2003; 278: 43991-44000Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 43Kocabas A.M. Rudnick G. Kilic F. J. Neurochem. 2003; 85: 1513-1520Crossref PubMed Scopus (49) Google Scholar, 44Kilic F. Rudnick G. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3106-3111Crossref PubMed Scopus (189) Google Scholar, 45Brenner B. Harney J.T. Ahmed B.A. Jeffus B.C. Unal R. Mehta J.L. Kilic F. J. Neurochem. 2007; 102: 206-216Crossref PubMed Scopus (144) Google Scholar). Immunoprecipitation—Binding of Rab4 to SERT was assayed in CHO or HeLa cells co-transfected with their cDNA in a 1:1 ratio. Transfectants were pretreated with 5HT at indicated concentrations for 30 min. The cells were lysed in immunoprecipitation buffer (55 mm triethylamine (pH 7.5), 111 mm NaCl, 2.2 mm EDTA, and 0.44% SDS plus 1% Triton X-100, 1 mm phenylmethylsulfonyl fluoride, PIM), precleared by incubation with nonimmune rabbit serum and protein A for 1 h, and then centrifuged as described previously (42Ozaslan D. Wang S. Ahmed B. Bene A. Kocabas A.M. Kilic F. J. Biol. Chem. 2003; 278: 43991-44000Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 43Kocabas A.M. Rudnick G. Kilic F. J. Neurochem. 2003; 85: 1513-1520Crossref PubMed Scopus (49) Google Scholar, 44Kilic F. Rudnick G. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3106-3111Crossref PubMed Scopus (189) Google Scholar, 45Brenner B. Harney J.T. Ahmed B.A. Jeffus B.C. Unal R. Mehta J.L. Kilic F. J. Neurochem. 2007; 102: 206-216Crossref PubMed Scopus (144) Google Scholar). The precleared lysate was combined with an equal volume of a 1:1 slurry of protein A-Sepharose beads and mixed overnight at 4 °C with a polyclonal Rab4-Ab. Samples were separated on an SDS-PAGE and analyzed by WB with monoclonal anti-SERT Ab (1:1000 dilution). The signals were developed with the ECL detection system. For radioimmunoprecipitation, CHO cells (2.5 × 106/100 mm plate) were labeled with [3H]5HT (100 μCi/ml) in PBS containing 0.1 mm CaCl2 and 1 mm MgCl2 for 1 h at room temperature. After 1 h, the cells were washed with ice-cold PBS and harvested, and Rab4 was immunoprecipitated as described previously (46Kilic F. Dalton M.B. Burrell S.K. Mayer J.P. Patterson S.D. Sinensky M. J. Biol. Chem. 1997; 272: 5298-5304Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Immune precipitates were eluted from the beads by incubation in SDS sample buffer, analyzed by 12.5% SDS-PAGE, and visualized by fluorography. Fluorescence Microscopy—Cells were plated on gelatincoated coverslips at 50% confluence in 6-well culture plates. The next day, cells were transfected with CFP-SERT and/or YFP-Rab4. 24 h post-transfection, coverslips were removed for immunofluorescent processing. Cells were rinsed with PBS, fixed with 3% paraformaldehyde in PBS for 25 min, and washed with PBS. Coverslips were washed several times and mounted on glass slides using Vectashield. Fluorescence was observed with Texas Red (excitation, 560 nm; dichroic mirror, 590 nm; emission, 610 nm), YFP (excitation, 500 nm; diachronic mirror, 515 nm; emission, 535 nm), or CFP (excitation, 436 nm; dichroic mirror, 455 nm; emission, 480 nm) filter sets. Images were digitally photographed at 40× magnification using either Zeiss LSM510 laser confocal or Axioskop2 wide-field deconvolution fluorescence microscopes. During the processing stage, individual image channels were pseudocolored with RGB values corresponding to each of the fluorophore emission spectral profiles. Images were cropped using Adobe Photoshop 6.0 software. Data Analysis—Non-linear regression fits were performed with Origin (MicroCal Software, Northampton, MA), using the Marquardt-Levenberg non-linear least squares curve fitting algorithm. Each figure shows a representative experiment that was performed at least twice, and statistical analysis results are based on multiple experiments. Data with error bars represent the mean ± S.D. for triplicate samples. Reported p values are two-sided. Plasma 5HT level plays a role in altering the cellular uptake rate of 5HT by modulating the density of SERT molecules on the plasma membrane (45Brenner B. Harney J.T. Ahmed B.A. Jeffus B.C. Unal R. Mehta J.L. Kilic F. J. Neurochem. 2007; 102: 206-216Crossref PubMed Scopus (144) Google Scholar). Mechanisms that specify translocation of SERT to the plasma membrane are of therapeutic importance and are investigated by several groups. SERT trafficking is not the only cellular event that is under the control of extracellular 5HT. Depending on the concentration of extracellular 5HT, [5HT]ex, platelet Rab4 can be activated via a covalent modification with 5HT that is otherwise known as serotonylation or transamidation. Because Rab4 regulates recycling of many transporters and receptors from endosomes (36Borner G.H. Harbour M. Hester S. Lilley K.S. Robinson M.S. J. 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