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• W1968053555 abstract "Understanding the molecular mechanisms of agonist-induced trafficking of G-protein-coupled receptors is important because of the essential role of trafficking in signal transduction. We examined the role of the GTPases dynamin 1 and Rab5a in substance P (SP)-induced trafficking and signaling of the neurokinin 1 receptor (NK1R), an important mediator of pain, depression, and inflammation, by studying transfected cells and enteric neurons that naturally express the NK1R. In unstimulated cells, the NK1R colocalized with dynamin at the plasma membrane, and Rab5a was detected in endosomes. SP induced translocation of the receptor into endosomes containing Rab5a immediately beneath the plasma membrane and then in a perinuclear location. Expression of the dominant negative mutants dynamin 1 K44E and Rab5aS34N inhibited endocytosis of SP by 45 and 32%, respectively. Dynamin K44E caused membrane retention of the NK1R, whereas Rab5aS34N also impeded the translocation of the receptor from superficially located to perinuclear endosomes. Both dynamin K44E and Rab5aS34N strongly inhibited resensitization of SP-induced Ca2+ mobilization by 60 and 85%, respectively, but had no effect on NK1R desensitization. Dynamin K44E but not Rab5aS34N markedly reduced SP-induced phosphorylation of extracellular signal regulated kinases 1 and 2. Thus, dynamin mediates the formation of endosomes containing the NK1R, and Rab5a mediates both endosomal formation and their translocation from a superficial to a perinuclear location. Dynamin and Rab5a-dependent trafficking is essential for NK1R resensitization but is not necessary for desensitization of signaling. Dynamin-dependent but not Rab5a-dependent trafficking is required for coupling of the NK1R to the mitogen-activated protein kinase cascade. These processes may regulate the nociceptive, depressive, and proinflammatory effects of SP. Understanding the molecular mechanisms of agonist-induced trafficking of G-protein-coupled receptors is important because of the essential role of trafficking in signal transduction. We examined the role of the GTPases dynamin 1 and Rab5a in substance P (SP)-induced trafficking and signaling of the neurokinin 1 receptor (NK1R), an important mediator of pain, depression, and inflammation, by studying transfected cells and enteric neurons that naturally express the NK1R. In unstimulated cells, the NK1R colocalized with dynamin at the plasma membrane, and Rab5a was detected in endosomes. SP induced translocation of the receptor into endosomes containing Rab5a immediately beneath the plasma membrane and then in a perinuclear location. Expression of the dominant negative mutants dynamin 1 K44E and Rab5aS34N inhibited endocytosis of SP by 45 and 32%, respectively. Dynamin K44E caused membrane retention of the NK1R, whereas Rab5aS34N also impeded the translocation of the receptor from superficially located to perinuclear endosomes. Both dynamin K44E and Rab5aS34N strongly inhibited resensitization of SP-induced Ca2+ mobilization by 60 and 85%, respectively, but had no effect on NK1R desensitization. Dynamin K44E but not Rab5aS34N markedly reduced SP-induced phosphorylation of extracellular signal regulated kinases 1 and 2. Thus, dynamin mediates the formation of endosomes containing the NK1R, and Rab5a mediates both endosomal formation and their translocation from a superficial to a perinuclear location. Dynamin and Rab5a-dependent trafficking is essential for NK1R resensitization but is not necessary for desensitization of signaling. Dynamin-dependent but not Rab5a-dependent trafficking is required for coupling of the NK1R to the mitogen-activated protein kinase cascade. These processes may regulate the nociceptive, depressive, and proinflammatory effects of SP. G-protein-coupled receptor substance P neurokinin 1 receptor β2-adrenergic receptor extracellular signal-regulated kinase phosphorylated ERK mitogen-activated protein green fluorescent protein enhanced GFP polymerase chain reaction Agonist-induced trafficking of G-protein-coupled receptors (GPCRs)1 and associated proteins plays an essential role in signal transduction (reviewed in Refs. 1Böhm S. Grady E.F. Bunnett N.W. Biochem. J. 1997; 322: 1-18Crossref PubMed Scopus (462) Google Scholar and 2Luttrell L.M. Daaka Y. Lefkowitz R.J. Curr. Opin. Cell Biol. 1999; 11: 177-183Crossref PubMed Scopus (603) Google Scholar). Within seconds of stimulation, G-protein receptor kinases and second messenger kinases translocate from the cytoplasm to the plasma membrane, where they phosphorylate receptors (3Pitcher J.A. Inglese J. Higgins J.B. Arriza J.L. Casey P.J. Kim C. Benovic J.L. Kwatra M.M. Caron M.G. Lefkowitz R.J. Science. 1992; 257: 1264-1267Crossref PubMed Scopus (567) Google Scholar, 4Daaka Y. Pitcher J.A. Richardson M. Stoffel R.H. Robishaw J.D. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2180-2185Crossref PubMed Scopus (154) Google Scholar, 5Barak L.S. Warabi K. Feng X. Caron M.G. Kwatra M.M. J. Biol. Chem. 1999; 274: 7565-7569Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). β-Arrestins are multifunctional proteins that interact with G-protein receptor kinase-phosphorylated receptors at the plasma membrane. This interaction disrupts association of GPCRs with heterotrimeric G-proteins and thereby mediates uncoupling and desensitization (6Lohse M.J. Benovic J.L. Codina J. Caron M.G. Lefkowitz R.J. Science. 1990; 248: 1547-1550Crossref PubMed Scopus (900) Google Scholar, 7Attramadal H. Arriza J.L. Aoki C. Dawson T.M. Codina J. Kwatra M.M. Snyder S.H. Caron M.G. Lefkowitz R.J. J. Biol. 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Chem. 1996; 271: 18302-18305Abstract Full Text Full Text PDF PubMed Scopus (398) Google Scholar, 13Lee K.B. Pals-Rylaarsdam R. Benovic J.L. Hosey M.M. J. Biol. Chem. 1998; 273: 12967-12972Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). In addition, β-arrestins are scaffolds that recruit components of the MAP kinase cascade into endosomes, which is necessary for mitogenic signaling (14Luttrell L.M. Ferguson S.S. Daaka Y. Miller W.E. Maudsley S. Della Rocca G.J. Lin F. Kawakatsu H. Owada K. Luttrell D.K. Caron M.G. Lefkowitz R.J. Science. 1999; 283: 655-661Crossref PubMed Scopus (1255) Google Scholar, 15DeFea K.A. Zalevsky J. Thoma M.S. Dery O. Mullins R.D. Bunnett N.W. J. Cell Biol. 2000; 148: 1267-1281Crossref PubMed Scopus (683) Google Scholar, 16DeFea K.A. Vaughn Z.D. O'Bryan E.M. Nishijima D. Dery O. Bunnett N.W. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 11086-11091Crossref PubMed Scopus (351) Google Scholar). Once internalized, GPCRs recycle to the cell surface, which mediates resensitization (17von Zastrow M. Kobilka B.K. J. Biol. Chem. 1992; 267: 3530-3538Abstract Full Text PDF PubMed Google Scholar, 18Grady E.F. Garland A.G. Gamp P.D. Lovett M. Payan D.G. Bunnett N.W. Mol. Biol. Cell. 1995; 6: 509-524Crossref PubMed Scopus (201) Google Scholar, 19Yu S.S. Lefkowitz R.J. Hausdorff W.P. J. Biol. Chem. 1993; 268: 337-341Abstract Full Text PDF PubMed Google Scholar, 20Garland A.M. Grady E.F. Lovett M. Vigna S.R. Frucht M.M. Krause J.E. Bunnett N.W. Mol. Pharmacol. 1996; 49: 438-446PubMed Google Scholar), or are degraded in lysosomes, which down-regulates receptors (21Brass L.F. Pizarro S. Ahuja M. Belmonte E. Blanchard N. Stadel J.M. Hoxie J.A. J. Biol. Chem. 1994; 269: 2943-2952Abstract Full Text PDF PubMed Google Scholar, 22Böhm S.K. Khitin L.M. Grady E.F. Aponte G. Payan D.G. Bunnett N.W. J. Biol. Chem. 1996; 271: 22003-22016Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar, 23Dery O. Thoma M.S. Wong H. Grady E.F. Bunnett N.W. J. Biol. Chem. 1999; 274: 18524-18535Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar, 24Li J.G. Benovic J.L. Liu-Chen L.Y. Mol. Pharmacol. 2000; 58: 795-801Crossref PubMed Scopus (91) Google Scholar). However, despite the importance of receptor trafficking for signal transduction, the molecular mechanisms are not fully understood. Most information derives from studies of very few receptors and is not necessarily applicable to all GPCRs. Furthermore, most observations are made using transfected cells expressing very high levels of GPCRs, which need not represent cells that naturally express these receptors at physiological levels. We investigated the mechanisms and function of substance P (SP)-induced endocytosis of the neurokinin-1 receptor (NK1R) in transfected cell lines and enteric neurons that naturally express this receptor. An understanding of the mechanisms that regulate this system is important, because SP and the NK1R mediate pain, depression, inflammation, smooth muscle contraction, and exocrine secretion (25Otsuka M. Yoshioka K. Physiol. Rev. 1993; 73: 229-308Crossref PubMed Scopus (1042) Google Scholar, 26Kramer M.S. Cutler N. Feighner J. Shrivastava R. Carman J. Sramek J.J. Reines S.A. Liu G. Snavely D. Wyatt-Knowles E. Hale J.J. Mills S.G. MacCoss M. Swain C.J. Harrison T. Hill R.G. Hefti F. Scolnick E.M. Cascieri M.A. Chicchi G.G. Sadowski S. Williams A.R. Hewson L. Smith D. Carlson E.J. et al.Science. 1998; 281: 1640-1645Crossref PubMed Scopus (975) Google Scholar). SP induces endocytosis and recycling of the NK1R in neurons and endothelial cells that participate in pain and inflammation (11McConalogue K. Dery O. Lovett M. Wong H. Walsh J.H. Grady E.F. Bunnett N.W. J. Biol. Chem. 1999; 274: 16257-16268Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 18Grady E.F. Garland A.G. Gamp P.D. Lovett M. Payan D.G. Bunnett N.W. Mol. Biol. Cell. 1995; 6: 509-524Crossref PubMed Scopus (201) Google Scholar, 27Garland A.M. Grady E.F. Payan D.G. Vigna S.R. Bunnett N.W. Biochem. J. 1994; 303: 177-186Crossref PubMed Scopus (128) Google Scholar, 28Bowden J.J. Garland A.M. Baluk P. Lefevre P. Grady E.F. Vigna S.R. Bunnett N.W. McDonald D.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8964-8968Crossref PubMed Scopus (247) Google Scholar, 29Mantyh P.W. DeMaster E. Malhotra A. Ghilardi J.R. Rogers S.D. Mantyh C.R. Liu H. Basbaum A.I. Vigna S.R. Maggio J.E. Science. 1995; 268: 1629-1632Crossref PubMed Scopus (431) Google Scholar, 30Grady E.F. Gamp P.D. Baluk P. McDonald D.M. Payan D.G. Bunnett N.W. Neuroscience. 1996; 16: 1239-1254Crossref PubMed Scopus (71) Google Scholar, 31McConalogue K. Corvera C.U. Gamp P.D. Grady E.F. Bunnett N.W. Mol. Biol. Cell. 1998; 9: 2305-2324Crossref PubMed Scopus (76) Google Scholar). However, the molecular mechanisms of this trafficking and its importance in signal transduction are unknown. We examined the role of the GTPases dynamin and ras-related GTPase 5a (Rab5a) in SP-induced endocytosis of the NK1R and determined the importance of this trafficking for desensitization, resensitization, and mitogenic signaling. The cytosolic GTPase dynamin mediates the first steps of endosome formation at sites of clathrin-coated pits and caveoli (32McClure S.J. Robinson P.J. Mol. Membr. Biol. 1996; 13: 189-215Crossref PubMed Scopus (74) Google Scholar, 33McNiven M.A. Cao H. Pitts K.R. Yoon Y. Trends Biochem. Sci. 2000; 25: 115-120Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar). Dynamin is required for constitutive endocytosis of the transferrin receptor as well as agonist-induced endocytosis of some GPCRs (12Zhang J. Ferguson S.S.G. Barak L.S. Menard L. Caron M.G. J. Biol. Chem. 1996; 271: 18302-18305Abstract Full Text Full Text PDF PubMed Scopus (398) Google Scholar, 13Lee K.B. Pals-Rylaarsdam R. Benovic J.L. Hosey M.M. J. Biol. Chem. 1998; 273: 12967-12972Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 34Herskovits J.S. Burgess C.C. Obar R.A. Vallee R.B. J. Cell Biol. 1993; 122: 565-578Crossref PubMed Scopus (396) Google Scholar, 35Chu P. Murray S. Lissin D. von Zastrow M. J. Biol. Chem. 1997; 272: 27124-27130Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 36Vogler O. Nolte B. Voss M. Schmidt M. Jakobs K.H. van Koppen C.J. J. Biol. Chem. 1999; 274: 12333-12338Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 37Vickery R.G. von Zastrow M. J. Cell Biol. 1999; 144: 31-43Crossref PubMed Scopus (197) Google Scholar, 38Iwata K. Ito K. Fukuzaki A. Inaki K. Haga T. Eur. J. Biochem. 1999; 263: 596-602Crossref PubMed Scopus (92) Google Scholar). However, some GPCRs internalize by dynamin-independent processes (12Zhang J. Ferguson S.S.G. Barak L.S. Menard L. Caron M.G. J. Biol. Chem. 1996; 271: 18302-18305Abstract Full Text Full Text PDF PubMed Scopus (398) Google Scholar, 37Vickery R.G. von Zastrow M. J. Cell Biol. 1999; 144: 31-43Crossref PubMed Scopus (197) Google Scholar, 39Vogler O. Bogatkewitsch G.S. Wriske C. Krummenerl P. Jakobs K.H. van Koppen C.J. J. Biol. Chem. 1998; 273: 12155-12160Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 40Schramm N.L. Limbird L.E. J. Biol. Chem. 1999; 274: 24935-24940Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). Rab5a mediates later steps of formation of endosomes containing the transferrin receptor and is required for endosomal translocation to a perinuclear region (41Bucci 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, 42Roberts R.L. Barbieri M.A. Pryse K.M. Chua M. Morisaki J.H. Stahl P.D. J. Cell Sci. 1999; 112: 3667-3675Crossref PubMed Google Scholar, 43Trischler M. Stoorvogel W. Ullrich O. J. Cell Sci. 1999; 112: 4773-4783Crossref PubMed Google Scholar). However, very little is known about the role of Rab5a in trafficking of GPCRs other than recent observations that Rab5a participates in the formation of endosomes containing the dopamine D2 and β2-adrenergic (β2-AR) receptors (38Iwata K. Ito K. Fukuzaki A. Inaki K. Haga T. Eur. J. Biochem. 1999; 263: 596-602Crossref PubMed Scopus (92) Google Scholar,44Seachrist J.L. Anborgh P.H. Ferguson S.S. J. Biol. Chem. 2000; 275: 27221-27228Abstract Full Text Full Text PDF PubMed Google Scholar) and contributes to down-regulation of the κ-opioid receptor (24Li J.G. Benovic J.L. Liu-Chen L.Y. Mol. Pharmacol. 2000; 58: 795-801Crossref PubMed Scopus (91) Google Scholar). Nothing is known about the role of dynamin and Rab5a in trafficking of the NK1R, and the relative roles of dynamin and Rab5a in desensitization, resensitization and mitogenic signaling of GPCRs are unknown. Our aims were to (a) determine the role of dynamin and Rab5a in SP-induced endocytosis and intracellular trafficking of the NK1R by expressing dominant negative mutants of these GTPases; (b) compare the importance of dynamin and Rab5a-dependent trafficking for desensitization and resensitization of signal transduction; (c) define the role of this trafficking for mitogenic signaling; and (d) establish whether dynamin and Rab5a could contribute to trafficking of the NK1R in neurons that naturally express this receptor. Enhanced green fluorescent protein (EGFP) expression vector pEGFP-C1, expression vector pIRES2-EGFP, and JM109 bacteria were from CLONTECH (Palo Alto, CA). Restriction enzymes, T4 ligase, and Lipofectin were from Life Technologies, Inc. or New England Biolabs, Inc. (Beverly, MA). Exp Tag DNA polymerase was from Stratagene (La Jolla, CA). A QiaEx extraction kit was from Qiagen (Hilden, Germany). G418 was from Gemini Bio-Products, Inc. (Calabashes, CA). Kanamycin and protease inhibitor mixture were from Calbiochem. Glutathione-agarose was from Amersham Pharmacia Biotech. The ECL detection kit was from Amersham Pharmacia Biotech. The Alexa594 protein labeling kit was from Molecular Probes, Inc. (Eugene, OR). SP or the NK1R-selective agonist [Sar9,MetO211]SP was labeled with Alexa594 as described (45Bunnett N.W. Dazin P.F. Payan D.G. Grady E.F. Peptides. 1995; 16: 733-740Crossref PubMed Scopus (27) Google Scholar). 125I-SP (2,000 μCi/mol) was from Amersham Pharmacia Biotech. Antibodies to Rab5a, dynamin, extracellular signal-regulated kinases 1 and 2 (ERK1/2), and phosphorylated ERK1/2 (pERK1/2) were from Transduction Laboratories (Santa Cruz, CA). The sources of other reagents and of antibodies to FLAG and 12CA5 epitopes, NK1R, EGFP, and fluorescence- or enzyme-tagged secondary antibodies have been described (11McConalogue K. Dery O. Lovett M. Wong H. Walsh J.H. Grady E.F. Bunnett N.W. J. Biol. Chem. 1999; 274: 16257-16268Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 22Böhm S.K. Khitin L.M. Grady E.F. Aponte G. Payan D.G. Bunnett N.W. J. Biol. Chem. 1996; 271: 22003-22016Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar, 23Dery O. Thoma M.S. Wong H. Grady E.F. Bunnett N.W. J. Biol. Chem. 1999; 274: 18524-18535Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar,46Grady E.F. Baluk P. Böhm S. Gamp P. Wong H. Payan D.G. Ansel J. Portbury A.L. Furness J.B. McDonald D.M. Bunnett N.W. J. Neurosci. 1996; 16: 6975-6986Crossref PubMed Google Scholar, 47McConalogue K. Grady E.F. Minnis J. Balestra B. Tonini M. Brecha N.C. Bunnett N.W. Sternini C. Neuroscience. 1999; 90: 1051-1059Crossref PubMed Scopus (75) Google Scholar, 48Sternini C. Brecha N.C. Minnis J. D'Agostino G. Balestra B. Fiori E. Tonini M. Neuroscience. 2000; 98: 233-241Crossref PubMed Scopus (46) Google Scholar). Canine Rab5a and the GTPase-defective binding mutant Rab5aS34N were fused to EGFP at the N terminus by PCR using the forward primer 5′-GGCCGGAATTCCATGGCTAATCGAGGAGCAACA-3′ (EcoRI site underlined followed by translation initiation site) and the reverse primer 5′-GGCCGGGGATCCTTAGTTACTACAACACTGACTCCT-3′ (BamHI restriction site underlined followed by stop codon). Rab5a cDNA was used as template to amplify an 800-bp fragment that was separated on an agarose gel and purified using a QiaEx extraction kit. The PCR fragment and the vector pEGFP-C1 were digested withEcoRI and BamHI and ligated with the T4 ligase overnight at 16 °C. Chemically competent JM109 bacteria were transformed using the heat shock method and selected on LB medium containing 30 μg/ml of Kanamycin. Correct sequences were verified. Rab5a tagged with EGFP is fully functional (42Roberts R.L. Barbieri M.A. Pryse K.M. Chua M. Morisaki J.H. Stahl P.D. J. Cell Sci. 1999; 112: 3667-3675Crossref PubMed Google Scholar). Generation and use of pcDNA3.1-HA-dynamin I and HA-dynamin I K44E (N-terminal HA.11 epitopes), a dominant negative mutant that lacks GTPase activity, have been described (34Herskovits J.S. Burgess C.C. Obar R.A. Vallee R.B. J. Cell Biol. 1993; 122: 565-578Crossref PubMed Scopus (396) Google Scholar). HA-dynamin-pIRES-EGFP constructs were generated by enzymatic digestion of pcDNA3.1-HA-dynamin with XhoI andXbaI and insertion of DNA into pIRES2-EGFP. The pIRES-EGFP construct enabled convenient identification of transfected cells using EGFP while avoiding attachment of EGFP to dynamin, which impairs its function (49Labrousse A.M. Shurland D.L. van der Bliek A.M. Mol. Biol. Cell. 1998; 9: 3227-3239Crossref PubMed Scopus (23) Google Scholar). Kirsten murine sarcoma virus-transformed rat kidney epithelial cells (KNRK) were from the American Type Tissue Culture Collection (Manassas, VA). Generation of KNRK cells stably expressing rat NK1R with N-terminal FLAG epitope (KNRK-FLAGNK1R cells), which does not affect receptor function, has been described (50Vigna S. Bowden G. McDonald D.M. Fisher J. Okamoto A. McVey D.C. Payan D. Bunnett N.W. J. Neurosci. 1994; 14: 834-845Crossref PubMed Google Scholar). To generate a cell line stably expressing FLAGNK1R plus Rab5a-GFP or Rab5aS34N-GFP, KNRK-FLAGNK1R cells were transfected with cDNA encoding Rab5a-GFP or Rab5aS34N-GFP (11McConalogue K. Dery O. Lovett M. Wong H. Walsh J.H. Grady E.F. Bunnett N.W. J. Biol. Chem. 1999; 274: 16257-16268Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Clones were screened by fluorescence microscopy and flow cytometry to detect NK1R and Rab5a. Cells were also transiently transfected with Rab5a and dynamin, since stable expression of dominant negative dynamin caused cell death, and to enable simultaneous comparison of transfected and untransfected cells by microscopy. KNRK-FLAGNK1R cells were transiently transfected by overnight incubation with 5 μg/ml cDNA encoding Rab5a-GFP, Rab5aS34N-GFP, HA-dynamin, or HA-dynamin K44E by lipofection. The medium was replaced, and cells were studied 24 h later. To transiently express HA-dynamin-pIRES-EGFP or HA-dynamin K44E-pIRES-EGFP, KNRK-FLAGNK1R cells were transfected by electroporation (Bio-Rad) and plated for 18 h, and positive transfected cells were sorted and plated 24 h before experiments. Cell lines are designated KNRK-NK1R+Rab5a, KNRK-NK1R+Rab5aS34N, KNRK-NK1R+DYN, or KNRK-NK1R+DYNK44E (where DYN represents dynamin). Cells were prepared for experiments as described (11McConalogue K. Dery O. Lovett M. Wong H. Walsh J.H. Grady E.F. Bunnett N.W. J. Biol. Chem. 1999; 274: 16257-16268Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 27Garland A.M. Grady E.F. Payan D.G. Vigna S.R. Bunnett N.W. Biochem. J. 1994; 303: 177-186Crossref PubMed Scopus (128) Google Scholar) and were usually incubated with 5 mm sodium butyrate for 18 h before use to boost expression of transfected genes. As a control, KNRK-FLAGNK1R cells were transfected with vectors without the Rab5a or dynamin inserts. Expression of empty vectors or EGFP alone did not affect signaling or trafficking of the NK1R (see Ref. 11McConalogue K. Dery O. Lovett M. Wong H. Walsh J.H. Grady E.F. Bunnett N.W. J. Biol. Chem. 1999; 274: 16257-16268Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar; data not shown) or related receptors (23Dery O. Thoma M.S. Wong H. Grady E.F. Bunnett N.W. J. Biol. Chem. 1999; 274: 18524-18535Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). Flow cytometry was used to monitor expression of NK1R, Rab5a, and dynamin and to enrich populations of transfected cells (11McConalogue K. Dery O. Lovett M. Wong H. Walsh J.H. Grady E.F. Bunnett N.W. J. Biol. Chem. 1999; 274: 16257-16268Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 23Dery O. Thoma M.S. Wong H. Grady E.F. Bunnett N.W. J. Biol. Chem. 1999; 274: 18524-18535Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). To detect the NK1R, cells were resuspended in 1 ml of Iscove's medium containing 1 mg/ml bovine serum albumin, with 10 μg/ml M2 antibody to the extracellular FLAG for 1 h at 4 °C, washed, and incubated with 2 μg/ml phycoerythrin-conjugated goat anti-mouse IgG for 1 h at 4 °C. Expression of other constructs was determined using EGFP. Cells were analyzed using a Facscan flow cytometer (Becton Dickinson Co., Franklin Lakes, NJ). Fluorophores were excited at 488 nm, and emission was collected at 530/30 nm for EGFP and 575/25 nm for phycoerythrin. Western blotting was used to confirm expression of Rab5a and dynamin (11McConalogue K. Dery O. Lovett M. Wong H. Walsh J.H. Grady E.F. Bunnett N.W. J. Biol. Chem. 1999; 274: 16257-16268Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 23Dery O. Thoma M.S. Wong H. Grady E.F. Bunnett N.W. J. Biol. Chem. 1999; 274: 18524-18535Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). Cells (107) were pelleted and lysed in 1 ml of Laemmli buffer. Lysates were fractionated by SDS-PAGE (4–15%), and proteins were transferred to nitrocellulose. Membranes were incubated with 5% nonfat milk in 100 mmPBS, pH 7.4, overnight and with antibodies to EGFP (9708; 1:1,000–1:20,000, 8 h, 4 °C), Rab5a, or dynamin (1:1,000, 2 h, room temperature). Membranes were washed and incubated with goat anti-rabbit IgG conjugated to horseradish peroxidase (1:8,000, 1 h, room temperature). Proteins were detected on film using an ECL detection kit. Controls included preabsorption of the diluted primary antibody with the EGFP fusion protein (1–2 μg/ml) for 1 h at 37 °C and use of nontransfected KNRK cells. The rate of NK1R endocytosis was quantified with 125I-SP (27Garland A.M. Grady E.F. Payan D.G. Vigna S.R. Bunnett N.W. Biochem. J. 1994; 303: 177-186Crossref PubMed Scopus (128) Google Scholar). Cells were incubated in Hanks' balanced salt solution containing 50 pm125I-SP, 0.1% bovine serum albumin for 60 min at 4 °C. They were washed and incubated at 37 °C for 0–10 min. Cells were washed with ice-cold PBS and incubated in 250 μl of ice-cold 0.2 m acetic acid containing 500 mmNaCl (pH 2.5) on ice for 5 min to separate acid-labile (cell surface) from acid-resistant (internalized) label. Nonspecific binding was measured in the presence of 1 μm SP and was subtracted to give specific binding. Observations were in triplicate inn > 3 experiments. KNRK cells expressing empty vector without NK1R insert do not bind or take up 125I-SP (50Vigna S. Bowden G. McDonald D.M. Fisher J. Okamoto A. McVey D.C. Payan D. Bunnett N.W. J. Neurosci. 1994; 14: 834-845Crossref PubMed Google Scholar). [Ca2+]i was measured in populations of transfected cells using Fura-2/AM (11McConalogue K. Dery O. Lovett M. Wong H. Walsh J.H. Grady E.F. Bunnett N.W. J. Biol. Chem. 1999; 274: 16257-16268Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar,27Garland A.M. Grady E.F. Payan D.G. Vigna S.R. Bunnett N.W. Biochem. J. 1994; 303: 177-186Crossref PubMed Scopus (128) Google Scholar). Fluorescence was measured at 340- and 380-nm excitation and 510-nm emission, and the results were expressed as the ratio of the fluorescence at the two excitation wavelengths, which is proportional to the [Ca2+]i . Cells were exposed once to SP to generate concentration-response curves. For desensitization experiments, cells were exposed to SP or vehicle (control) for 2 min, washed, and exposed again to SP 5 min after the first exposure. To examine resensitization, cells were incubated with SP or vehicle (control) for 10 min, washed, and challenged with SP 0–180 min after washing. All observations were in n > 3 experiments. KNRK cells expressing empty vector without the NK1R insert do not mobilize [Ca2+]i in response to SP (50Vigna S. Bowden G. McDonald D.M. Fisher J. Okamoto A. McVey D.C. Payan D. Bunnett N.W. J. Neurosci. 1994; 14: 834-845Crossref PubMed Google Scholar). SP-induced phosphorylation of ERK1/2 was determined by Western blotting (15DeFea K.A. Zalevsky J. Thoma M.S. Dery O. Mullins R.D. Bunnett N.W. J. Cell Biol. 2000; 148: 1267-1281Crossref PubMed Scopus (683) Google Scholar, 16DeFea K.A. Vaughn Z.D. O'Bryan E.M. Nishijima D. Dery O. Bunnett N.W. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 11086-11091Crossref PubMed Scopus (351) Google Scholar). Similar results were obtained in kinase assays using myelin basic protein as a substrate (data not shown). Cells were maintained serum-free medium overnight and incubated with 10 nm SP for 0–30 min at 37 °C. Cells were lysed in boiling 20 mm Tris-HCl, pH 8, 10 mm EDTA, 0.3% SDS, 67 mm DTT. Lysates (20 μg of protein) were analyzed by 12% SDS-PAGE and transferred to polyvinylidene difluoride membranes. Membranes were incubated with pERK1/2 (pERK) antibody (1:1000, overnight, 4 °C), followed by goat anti-mouse IgG conjugated to horseradish peroxidase (1:30,000, 1 h, room temperature). Proteins were visualized by ECL. Blots were stripped and reprobed with antibody to total ERK1/2 to ensure that equal levels of ERK1/2 were present at each time point. The intensities of the combined ERK1 and ERK2 bands for phosphorylated and total proteins were determined by histogram analysis (mean density × number pixels) (15DeFea K.A. Zalevsky J. Thoma M.S. Dery O. Mullins R.D. Bunnett N.W. J. Cell Biol. 2000; 148: 1267-1281Crossref PubMed Scopus (683) Google Scholar, 16DeFea K.A. Vaughn Z.D. O'Bryan E.M. Nishijima D. Dery O. Bunnett N.W. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 11086-11091Crossref PubMed Scopus (351) Google Scholar). Intensities of the pERK bands were normalized to total ERKs to correct for any differences in loading. Results are expressed as the -fold increase in pERK1/2 relative to unstimulated cells. All observations were in n = 4 experiments. Cells were incubated with 10–100 nm Alexa594-SP for 60 min at 4 °C (for equilibrium binding), washed at 4 °C, and either fixed immediately or incubated in SP-free medium at 37 °C for 2.5–10 min (for trafficking to proceed) (11McConalogue K. Dery O. Lovett M. Wong H. Walsh J.H. Grady E.F. Bunnett N.W. J. Biol. Chem. 1999; 274: 16257-16268Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 18Grady E.F. Garland A.G. Gamp P.D. Lovett M. Payan D.G. Bunnett N.W. Mol. Biol. Cell. 1995; 6: 509-524Crossref PubMed Scopus (201) Google Scholar). Cells were fixed with 4% paraformaldehyde in 100 mm PBS, pH 7.4, for 20 min at 4 °C. NK1R was localized using Alexa-SP or by immunofluorescence using the M2 FLAG antibody (10 μg/ml, 18 h, 4 °C) or an antibody to the C terminus of the rat NK1R (1:1,000, 18 h, 4 °C). Rab5a was localized using EGFP, and dynamin was localized by immunofluorescence using the HA antibody (4 μg/ml, 18 h, 4 °C)" @default.
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• W1968053555 title "Dynamin and Rab5a-dependent Trafficking and Signaling of the Neurokinin 1 Receptor" @default.
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