FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2025107135> ?p ?o ?g. }

• W2025107135 endingPage "30525" @default.
• W2025107135 startingPage "30519" @default.
• W2025107135 abstract "We have evaluated the role of phosphatidylinositol 3-kinase (PI3-kinase) and Ca2+influx in ligand-stimulated internalization of the c-Kit receptor. The wild type (wt) c-Kit receptor and YF719, a mutant receptor in which the SH2-mediated binding site for the p85 subunit of PI3-kinase is disrupted, were expressed in DA-1 cells. YF719 internalized with similar kinetics as wt c-Kit although the receptor remained localized close to the plasma membrane. However, in the absence of extracellular Ca2+, or in the presence of the competitive Ca2+ influx blocker Ni2+, the YF719 mutant failed to internalize. Failure to internalize in the absence of Ca2+ was also observed for the wt c-Kit receptor in cells that were pretreated with the PI3-kinase inhibitor, wortmannin. Following stimulation with ligand, clathrin heavy chains were found to co-immunoprecipitate with c-Kit. However, under conditions in which PI3-kinase activity is inhibited and Ca2+ influx is blocked, clathrin failed to co-immunoprecipitate with c-Kit. Our results demonstrate that both Ca2+ influx and PI3-kinase activity influence c-Kit endocytosis, and inhibition of these two signals disrupts the earliest stages of ligand-mediated internalization. We have evaluated the role of phosphatidylinositol 3-kinase (PI3-kinase) and Ca2+influx in ligand-stimulated internalization of the c-Kit receptor. The wild type (wt) c-Kit receptor and YF719, a mutant receptor in which the SH2-mediated binding site for the p85 subunit of PI3-kinase is disrupted, were expressed in DA-1 cells. YF719 internalized with similar kinetics as wt c-Kit although the receptor remained localized close to the plasma membrane. However, in the absence of extracellular Ca2+, or in the presence of the competitive Ca2+ influx blocker Ni2+, the YF719 mutant failed to internalize. Failure to internalize in the absence of Ca2+ was also observed for the wt c-Kit receptor in cells that were pretreated with the PI3-kinase inhibitor, wortmannin. Following stimulation with ligand, clathrin heavy chains were found to co-immunoprecipitate with c-Kit. However, under conditions in which PI3-kinase activity is inhibited and Ca2+ influx is blocked, clathrin failed to co-immunoprecipitate with c-Kit. Our results demonstrate that both Ca2+ influx and PI3-kinase activity influence c-Kit endocytosis, and inhibition of these two signals disrupts the earliest stages of ligand-mediated internalization. c-Kit is a receptor tyrosine kinase and a member of the subfamily that includes the PDGF, 1The abbreviations used are: PDGF, platelet-derived growth factor; SLF, steel factor; PI3-kinase, phosphatidylinositol 3-kinase; BMMC, bone marrow-derived mast cells; wt, wild type; PBS, phosphate-buffered saline; FBS, fetal bovine serum; FITC, fluorescein isothiocyanate; TxR, Texas Red. CSF-1, and flt-3/flk-2 receptors. Together with its ligand steel factor (SLF), c-Kit is a key controlling receptor for a number of cell types including hematopoietic stem cells, mast cells, melanocytes, and germ cells. c-Kit is the gene product of the W locus in mice (1Huang E. Nocka K. Beier D.R. Chu T.Y. Buck J. Lahm H.W. Wellner D. Leder P. Besmer P. Cell. 1990; 63: 225-233Abstract Full Text PDF PubMed Scopus (941) Google Scholar), and its ligand SLF is the product of the sl locus (2Williams D.E. Eisenman J. Baird A. Rauch C. Van N.K. March C.J. Park L.S. Martin U. Mochizuki D.Y. Boswell H.S. Burgess G.S. Cosman D. Lyman S.D. Cell. 1990; 63: 167-174Abstract Full Text PDF PubMed Scopus (772) Google Scholar, 3Zsebo K.M. Williams D.A. Geissler E.N. Broudy V.C. Martin F.H. Atkins H.L. Hsu R.Y. Birkett N.C. Okino K.H. Murdock D.C. Jacobsen F.W. Langley K.E. Smith K.A. Takeishi T. Cattanach B.M. Galli S.J. Suggs S.V. Cell. 1990; 63: 213-224Abstract Full Text PDF PubMed Scopus (1216) Google Scholar). Mutations in either locus severely affect the growth and survival of stem cells of these tissues. As with other receptor tyrosine kinases, stimulation of the c-Kit receptor with SLF results in the recruitment and tyrosine phosphorylation of SH2-containing second messenger-generating enzymes (4Ullrich A. Schlessinger J. Cell. 1990; 61: 203-212Abstract Full Text PDF PubMed Scopus (4611) Google Scholar) such as phospholipase C-γ and PI3-kinase (5Rottapel R. Reedijk M. Williams D.E. Lyman S.D. Anderson D.M. Pawson T. Bernstein A. Mol. Cell. Biol. 1991; 11: 3043-3051Crossref PubMed Scopus (201) Google Scholar, 6Lev S. Givol D. Yarden Y. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 678-682Crossref PubMed Scopus (90) Google Scholar). The phosphorylated lipid products of these enzymes stimulate a variety of intracellular processes including Ca2+ mobilization and actin reorganization (7Wennstrom S. Hawkins P. Cooke F. Hara K. Yonezawa K. Kasuga M. Jackson T. Claesson W.L. Stephens L. Curr. Biol. 1994; 4: 385-393Abstract Full Text Full Text PDF PubMed Scopus (392) Google Scholar, 8Fukamachi H. Kawakami Y. Takei M. Ishizaka T. Ishizaka K. Kawakami T. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9524-9528Crossref PubMed Scopus (33) Google Scholar, 9Catt K.J. Hunyady L. Balla T. J. Bioenerg. Biomemb. 1991; 23: 7-27PubMed Google Scholar). Coincident with second messenger generation, the process of receptor internalization is also initiated. Within minutes following ligand binding, receptors cluster in dimers or oligomers and internalize by endocytosis, likely through clathrin-coated pits (10Sorkin A. Carpenter G. Science. 1993; 261: 612-615Crossref PubMed Scopus (213) Google Scholar, 11Schmid S.L. Damke H. FASEB J. 1995; 9: 1445-1453Crossref PubMed Scopus (63) Google Scholar, 12Robinson M.S. Curr. Opin. Cell Biol. 1994; 6: 538-544Crossref PubMed Scopus (306) Google Scholar). Eventually, clathrin coats are removed, and the remaining vesicles fuse with endosomes, late endosomes, and ultimately lysosomes, resulting in receptor degradation. Numerous deletion and mutagenesis studies have been carried out to map regions of receptor tyrosine kinases required for ligand-stimulated internalization (13Lin C.R. Chen W.S. Lazar C.S. Carpenter C.D. Gill G.N. Evans R.M. Rosenfeld M.G. Cell. 1986; 44: 839-848Abstract Full Text PDF PubMed Scopus (189) Google Scholar, 14Joly M. Kazlauskas A. Fay F.S. Corvera S. Science. 1994; 263: 684-687Crossref PubMed Scopus (252) Google Scholar, 15Mori S. Ronnstrand L. Claesson-Welsh L. Heldin C.H. J. Biol. Chem. 1994; 269: 4917-4921Abstract Full Text PDF PubMed Google Scholar, 16Severinsson L. Ek B. Mellstrom K. Claesson W.L. Heldin C.H. Mol. Cell. Biol. 1990; 10: 801-809Crossref PubMed Google Scholar, 17Glenney J.J. Chen W.S. Lazar C.S. Walton G.M. Zokas L.M. Rosenfeld M.G. Gill G.N. Cell. 1988; 52: 675-684Abstract Full Text PDF PubMed Scopus (157) Google Scholar, 18Chen W.S. Lazar C.S. Lund K.A. Welsh J.B. Chang C.P. Walton G.M. Der C.J. Wiley H.S. Gill G.N. Rosenfeld M.G. Cell. 1989; 59: 33-43Abstract Full Text PDF PubMed Scopus (258) Google Scholar, 19Chang C.P. Lazar C.S. Walsh B.J. Komuro M. Collawn J.F. Kuhn L.A. Tainer J.A. Trowbridge I.S. Farquhar M.G. Rosenfeld M.G. Wiley H.S. Gill G.N. J. Biol. Chem. 1993; 268: 19312-19320Abstract Full Text PDF PubMed Google Scholar, 20Dargemont C. Le B.A. Rothenberger S. Iacopetta B. Kuhn L.C. EMBO J. 1993; 12: 1713-1721Crossref PubMed Scopus (44) Google Scholar, 21Myles G.M. Brandt C.S. Carlberg K. Rohrschneider L.R. Mol. Cell. Biol. 1994; 14: 4843-4854Crossref PubMed Scopus (27) Google Scholar, 22Yamada K. Carpentier J.L. Cheatham B. Goncalves E. Shoelson S.E. Kahn C.R. J. Biol. Chem. 1995; 270: 3115-3122Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). Tyrosine kinase activity (23Lamaze C. Schmid S.L. Curr. Opin. Cell Biol. 1995; 7: 573-580Crossref PubMed Scopus (254) Google Scholar, 24Downing J.R. Roussel M.F. Sherr C.J. Mol. Cell. Biol. 1989; 9: 2890-2896Crossref PubMed Scopus (166) Google Scholar), autophosphorylation sites (17Glenney J.J. Chen W.S. Lazar C.S. Walton G.M. Zokas L.M. Rosenfeld M.G. Gill G.N. Cell. 1988; 52: 675-684Abstract Full Text PDF PubMed Scopus (157) Google Scholar, 18Chen W.S. Lazar C.S. Lund K.A. Welsh J.B. Chang C.P. Walton G.M. Der C.J. Wiley H.S. Gill G.N. Rosenfeld M.G. Cell. 1989; 59: 33-43Abstract Full Text PDF PubMed Scopus (258) Google Scholar), and interactions with second messenger-generating enzymes have been implicated. In particular, recruitment and activation of PI3-kinase have been associated with ligand-stimulated internalization of the PDGF receptor (14Joly M. Kazlauskas A. Fay F.S. Corvera S. Science. 1994; 263: 684-687Crossref PubMed Scopus (252) Google Scholar). Receptors carrying mutations within the cytoplasmic domain that disrupt the PI3-kinase-binding site are impaired in the later stages of endocytosis (25Joly M. Kazlauskas A. Corvera S. J. Biol. Chem. 1995; 270: 13225-13230Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). However, conflicting results with a deletion mutant encompassing this binding site demonstrate no impairment in any stage of endocytosis (16Severinsson L. Ek B. Mellstrom K. Claesson W.L. Heldin C.H. Mol. Cell. Biol. 1990; 10: 801-809Crossref PubMed Google Scholar). Ligand-stimulated endocytosis of c-Kit was investigated by Yee et al. (26Yee N.S. Hsiau C.W. Serve H. Vosseller K. Besmer P. J. Biol. Chem. 1994; 269: 31991-31998Abstract Full Text PDF PubMed Google Scholar). They reported that a c-Kit mutant with no kinase activity was impaired for ligand-stimulated internalization. Individual mutations converting tyrosines 719 and 821 to phenylalanines, however, failed to affect internalization, although Phe-821 was impaired for mitogenesis (27Serve H. Yee N.S. Stella G. Sepp L.L. Tan J.C. Besmer P. EMBO J. 1995; 14: 473-483Crossref PubMed Scopus (199) Google Scholar). Tyr-719 is located in the kinase insert region of the c-Kit catalytic domain. In its phosphorylated form, it forms part of a consensus binding site for the p85 subunit of PI3-kinase and has been shown to mediate this interaction in vitro and in vivo (6Lev S. Givol D. Yarden Y. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 678-682Crossref PubMed Scopus (90) Google Scholar). We have also examined the role of PI3-kinase activation on ligand-stimulated internalization of c-Kit. We found that in the absence of PI3-kinase activation, the c-Kit receptor internalizes but remains localized near the inner aspect of the plasma membrane. However, when both PI3-kinase and Ca2+ influx are inhibited, clathrin fails to co-immunoprecipitate with c-Kit, and receptor internalization is completely prevented. These results show that concurrent inhibition of PI3-kinase activity and Ca2+influx disrupts the earliest stages of c-Kit internalization. gp + e NIH 3T3 packaging cells were grown in Dulbecco's modified Eagle's medium (Life Technologies, Inc., Burlington ON) supplemented with 10% fetal bovine serum (FBS) and antibiotics. DA-1 cells are an interleukin-3-dependent, c-kit-negative murine lymphoma cell line (28Ihle J.N. Contemp. Top. Mol. Immunol. 1985; 10: 93-119PubMed Google Scholar). They were routinely grown in RPMI supplemented with 10% FBS, 10−5m β-mercaptoethanol, and 2% supernatant from WEHI-3 cells as a source of interleukin-3. Bone marrow-derived mast cells (BMMC) were isolated and cultured as described (29Berger S.A. Mak T.W. Paige C.J. J. Exp. Med. 1994; 180: 471-476Crossref PubMed Scopus (71) Google Scholar). The cDNAs for wild type (wt) murine c-kit or mutants in which tyrosine 719 was replaced with phenylalanine were cloned into the LXSN retroviral expression vector. These vectors were transferred into the gp + e NIH 3T3 packaging cells by electroporation, and transfectants were selected by growth in 400 μg/ml G418 (Life Technologies, Inc.). DA-1s were co-cultured with a confluent layer of pooled gp + e transfectants for 24 h. The non-adherent DA-1s were removed and cultured for an additional 5–10 days in interleukin-3. c-Kit-positive DA-1 cells were enriched by two rounds of cell sorting (FacsStar Plus, Becton Dickinson). Surface expression of c-Kit was detected using biotinylated SLF and streptavidin-conjugated phycoerythrin (Jackson, Westgrove PA). Stable populations of c-Kit expressing DA-1s were then cloned by limiting dilution. As reported by others (6Lev S. Givol D. Yarden Y. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 678-682Crossref PubMed Scopus (90) Google Scholar), we confirmed that the p85 subunit of PI3-kinase co-immunoprecipitates with c-Kit following stimulation of the wt receptor with SLF but fails to co-immunoprecipitate with YF719 (data not shown). We also confirmed that stimulation of DA-1 cells expressing wt or YF719 c-Kit receptors with SLF results in an equivalent mitogenic response (27Serve H. Yee N.S. Stella G. Sepp L.L. Tan J.C. Besmer P. EMBO J. 1995; 14: 473-483Crossref PubMed Scopus (199) Google Scholar) (data not shown), indicating that PI3-kinase activation is not essential for growth stimulation in this instance. Recombinant murine SLF was produced in soluble form in Escherichia coliusing the pFLAG.ATS isopropyl-1-thio-β-d-galactopyranoside-inducible secretion expression vector (Invitrogen). This vector includes an eight amino acid N-terminal FLAG epitope (InterScience, Markham, ON).E. coli containing the pFLAG.ATS plasmid was incubated overnight at 37 °C in LB with 100 μg/ml ampicillin. This culture was then diluted 20-fold and grown to an A 600 of 0.4–0.5 before being induced with 0.033 g/liter isopropyl-1-thio-β-d-galactopyranoside. The cultures were then incubated overnight at 37 °C before they were centrifuged at 10,000 rpm for 20 min. The bacterial supernatant was passed through a 0.22-micron filter and stored at −80 °C with 1 mmCaCl2 and 100 μm phenylmethylsulfonyl fluoride. FLAG-SLF was purified by passing supernatants over a column of Anti-FLAG M1 mouse monoclonal antibodies covalently attached to agarose gel. The column was first equilibrated with 30 ml PBS + 1 mm CaCl2. Bacterial supernatants were then passed over the M1 column three times. The FLAG-SLF fusion protein binding to the affinity column is Ca2+-dependent; therefore, elution of FLAG-SLF can be achieved by adding EDTA. Six elutions with 1 ml of PBS + 2 mm EDTA were performed. These were collected, concentrated, and checked for purity by silver stain. SLF was also conjugated with biotin (Sigma) as described (30Hu Q. Trevisan M. Xu Y. Dong W. Berger S.A. Lyman S.D. Minden M.D. J. Clin. Invest. 1995; 95: 2530-2538Crossref PubMed Scopus (18) Google Scholar). 1 × 106 cells of wt and YF719 DA-1 clones were resuspended in phosphate-buffered saline (Life Technologies, Inc.) + 0.5% FBS (PBS/FBS) and washed twice with the same solution. MgCl2and CaCl2, when added, were at 0.5 and 1 mm, respectively. Biotin-conjugated SLF (b-SLF) was then added to the cells in PBS/FBS at a concentration that allowed for maximal fluorescence- staining, typically 1:100 to 1:50 dilution. Cells were incubated on ice with b-SLF for 60 min. The kinetics of receptor internalization were the same whether cells were preincubated on ice or for a short time at 37 °C (not shown) indicating that the incubation on ice does not have any deleterious effects on the internalization process. Cells were incubated for various times at 37 °C and washed three times with ice-cold PBS/FBS. Cells were then fixed with 3% paraformaldehyde for 10 min on ice. This fixation procedure stabilized the staining pattern for several hours with little disruption of the cellular architecture. Following fixation, cells were washed three times with PBS/FBS and then incubated with streptavidin-conjugated phycoerythrin (Jackson) for 30 min on ice. Cells were then washed twice with PBS/FBS and analyzed by flow cytometry for expression and internalization of c-Kit. Actinomycin D (Sigma) stain was used to detect dead cells. These were gated out and not included in the analysis. The internalization of c-Kit was confirmed to be a ligand-dependent process using a FITC-conjugated anti-kit antibody (2B8) (Pharmingen, San Diego CA) directed at the extracellular domain of c-Kit. This antibody does not compete with SLF binding to the c-Kit receptor. Cells were either first incubated with (or without) SLF for 1 h at 4 °C, followed by a 10-min incubation at 37 °C, washed, and then labeled with FITC-conjugated anti-kit for 20 min at 4 °C. Cells were then analyzed by flow cytometry. In the absence of ligand, receptor levels did not decrease after incubation at 37 °C. However, addition of ligand caused a progressive loss of staining with the anti-kit antibody (data not shown) indicating that internalization of c-Kit is stimulated by ligand binding. 1 × 106cells of wt or YF719 DA-1 clones were resuspended in PBS + 0.5% FBS (PBS/FBS) and washed twice with the same solution. FITC-conjugated SLF was then added to the cells in PBS/FBS at a dilution of 1:100 to 1:50. Cells were incubated on ice with FITC-SLF for 40–60 min. Cells were then transferred to a 37 °C water bath for a short incubation time and then subsequently placed back on ice. Cells were washed three times with ice-cold PBS/FBS and then fixed with 3% paraformaldehyde for 10 min on ice. Control experiments showed that this fixation procedure is sufficient to prevent further movement of receptor into the cell (not shown). Following fixation, cells were washed three times with PBS/FBS. Cells were then labeled with an anti-FITC antibody conjugated with Texas Red (TxR) (Molecular Probes, Eugene, OR) for 45 min on ice. Cells were washed three times with PBS/FBS and then resuspended in 5 μl of 90% glycerol containing 1,4-diazobicyclo-[2.2.2]-octane (purchased from Aldrich) which significantly reduces photobleaching (31Johnson G.D. Davidson R.S. McNamee K.C. Russell G. Goodwin D. Holborow E.J. J. Immunol. Methods. 1982; 55: 231-242Crossref PubMed Scopus (653) Google Scholar). Cells were allowed to settle for 10 min on the microscope slide before applying a coverslip and nail polish. Incubation with glycerol was sufficient to immobilize the cells, while maintaining their three-dimensional structure relatively undistorted. Cells were observed by fluorescence microscope (Leitz DMR/BE) using a 100 × oil-immersion objective with filters for both FITC and TxR. Photographs were always exposed for 90 s. When cells were photographed using both filters, the Texas-Red image was exposed first since it experienced greater photobleaching. For experiments investigating the co-localization of the c-Kit receptor and SLF, cells were incubated with b-SLF and a FITC-conjugated anti-c-Kit antibody (2B8, Pharmingen) for 60 min at 4 °C either in the presence or absence of extracellular Ca2+. Cells were then incubated at 37 °C for 0, 7.5, or 15 min. Cells were washed with ice-cold PBS/FBS and fixed as described above. Cells were permeabilized with 0.1% saponin for 15 min at room temperature, washed, resuspended in glycerol/1,4-diazobicyclo-[2.2.2]-octane and plated on microscope slides as described above. Control experiments with a FITC-labeled isotype control antibody (Pharmingen) or streptavidin-conjugated TxR alone showed no staining (not shown). Wortmannin (purchased from Sigma) was dissolved at a concentration of 20 mm in Me2SO and stored in aliquots at −80 °C. Cells were prepared for immunofluorescence microscopy and flow cytometric analysis as described above with the exception that 100 nm wortmannin was added to the binding buffer minutes before the 37 °C incubation. For c-Kit immunoprecipitations, 2.5 × 106 BMMCs per sample were starved overnight in RPMI + 0.5% FBS and then washed twice in PBS + 0.5% FBS. Cells were then incubated with 500 ng/ml SLF at 37 °C for various time points using the indicated conditions and then immediately washed twice in ice-cold PBS/FBS. Cells were then lysed in lysis buffer containing 1% Triton X-100 (Caledon, Georgetown ON), 50 mmHEPES (pH 7.0), 150 mm NaCl, 10% glycerol, 1.5 mm MgCl2, and 1 mm EGTA with the following inhibitors: 500 μm sodium orthovanadate, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 1 mmphenylmethylsulfonyl fluoride, 10 μg/ml soybean trypsin inhibitor, 10 μm NaF, and 1 mm sodium molybdate (all from Sigma). Lysates were then spun at 10,000 rpm for 20 min, and supernatants were incubated with 50 μl of a 20% Protein A slurry (Pharmacia Biotech Inc.) and 5 μl of a polyclonal rabbit antibody raised against a glutathione S-transferase kit cytoplasmic tail fusion protein or 5 μl of preimmune serum and 50 μl of a 20% Protein A slurry. 2R. Rottapel, unpublished data. A whole cell lysate prepared from 1/10th the number of cells was used as a positive control on the Western blot. All the other lysates were incubated for 2 h at 4 °C, and the beads were washed three times in HNTG wash buffer containing 0.1% Triton-X, 20 mmHEPES (pH 7.0), 10% glycerol, 150 mm NaCl, and 1 mm sodium orthovanadate. Beads were resuspended in loading buffer with β2-mercaptoethanol and boiled for 5 min, and released proteins were resolved on a 7.5% acrylamide gel, transferred to nitrocellulose, and blocked in PBS plus 5% skim milk powder and 0.5% Tween 20. Blots were incubated with an anti-clathrin monoclonal antibodies (from Transduction Laboratories, Kentucky) at a dilution of 1:5,000 overnight followed by goat anti-mouse-conjugated horseradish peroxidase secondary antibodies (Jackson) at a dilution of 1:2,000 and visualized with chemiluminescence (NEN Life Science Products). Blots were stripped by acid treatment and re-probed with rabbit polyclonal anti-c-Kit antisera at a dilution of 1:500 followed by incubation with protein A-horseradish peroxidase (Amersham Corp.) at a dilution of 1:30,000. Visualization was again by chemiluminescence. We used labeled ligand in conjunction with flow cytometry to follow the average rate of internalization over a population of cells. Labeled ligand offers the advantage of following only those receptors that are activated, unlike the use of anti-receptor antibodies that cannot distinguish between activated and unactivated receptors. Cells were incubated with b-SLF at 4 °C and then further incubated for varying amounts of time at 37 °C. Internalization was arrested by adding ice-cold PBS/FBS and shifting the cells back to 4 °C. Following washing and fixing, the cells were then incubated with Streptavidin-conjugated phycoerythrin and analyzed for surface expression. Fig. 1demonstrates typical flow cytometry profiles with b-SLF, showing a decline in the intensity of the fluorescence signal as the bound ligand is internalized with the c-Kit receptor. Recruitment and activation of PI3-kinase has been implicated in both endocytic and exocytic processes in a number of different experimental systems. We therefore investigated the ligand-stimulated internalization kinetics of both wt and YF719, a c-Kit receptor mutant that fails to recruit and activate PI3-kinase. Fig. 2 depicts the average of three flow cytometry experiments. The mean fluorescence intensity of cells that were maintained at 4 °C is denoted as 100% surface receptor expression. As shown in Fig. 2 A, wt c-Kit and the YF719 mutant expressed in DA-1 cells internalize with similar kinetics. The half-life for loss of surface label is approximately 6 min. Another early signaling event following ligand-stimulated activation of c-Kit is the mobilization of Ca2+ from intracellular stores, followed by Ca2+ influx from the extracellular milieu (32Hoth M. Penner R. Nature. 1992; 355: 353-356Crossref PubMed Scopus (1491) Google Scholar). A number of Ca2+-binding proteins are associated with coated pits and endosomes, suggesting that high levels of intracellular Ca2+ may influence receptor endocytosis (33Mayorga L.S. Beron W. Sarrouf M.N. Colombo M.I. Creutz C. Stahl P.D. J. Biol. Chem. 1994; 269: 30927-30934Abstract Full Text PDF PubMed Google Scholar, 34Lin H.T. Sudhof T.C. Anderson R.G.W. Cell. 1992; 70: 283-291Abstract Full Text PDF PubMed Scopus (106) Google Scholar, 35Colombo M.I. Beron W. Stahl P.D. J. Biol. Chem. 1997; 272: 7707-7712Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). We therefore investigated the effect on receptor internalization of removing extracellular Ca2+. As shown in Fig.2 B, when Ca2+ is excluded from binding and wash buffers, wt c-Kit internalizes with kinetics similar to that observed in the presence of Ca2+; however, the YF719 receptor fails to internalize. To confirm that Ca2+ influx is required for YF719 internalization, Ni2+, a competitive blocker of Ca2+ influx channels (36Franzius D. Hoth M. Penner R. Pflugers Arch. Eur. J. Physiol. 1994; 428: 433-438Crossref PubMed Scopus (159) Google Scholar), was added to the incubation medium containing Ca2+, and the internalization kinetics were determined. As shown in Fig. 2 C, the inclusion of 2.5 mm Ni2+ in the binding and wash buffers blocks internalization of the YF719 mutant but has little effect on the kinetics or the extent of internalization of wt c-Kit. Taken together, these results demonstrate that internalization of YF719 is dependent on Ca2+ influx. Receptor endocytosis is a multi-step process involving transfer of receptor from clathrin-coated pits, to early and late endosomes (12Robinson M.S. Curr. Opin. Cell Biol. 1994; 6: 538-544Crossref PubMed Scopus (306) Google Scholar). PI3-kinase activity has been particularly associated with movement of receptor down later steps in the endocytic pathway (25Joly M. Kazlauskas A. Corvera S. J. Biol. Chem. 1995; 270: 13225-13230Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). We therefore examined the distribution pattern of internalized wt and mutant c-Kit receptors following activation with SLF using fluorescently labeled ligand and fluorescence microscopy. Cells were incubated with FITC-conjugated SLF at 4 °C to allow ligand binding. Cells were then incubated for a further period at 37 °C to allow for internalization. Following washing and fixing, the cells were further stained with anti-FITC antibody coupled with Texas Red. Because cells were not permeabilized, the anti-FITC antibody binds only those occupied receptors remaining on the surface of the cell. As shown in Fig.3 A, when wt cells are incubated with FITC-SLF at 4 °C, or for only a short time at 37 °C, the FITC and Texas Red stains are superimposable indicating that the ligand-receptor complex is on the cell surface. With longer incubations at 37 °C, the fluorescence becomes increasingly punctate, and there is an accompanying loss of Texas Red staining. Finally, after 10–15 min of incubation at 37 °C, little Texas Red fluorescence is observed, whereas the majority of the FITC staining is in large aggregates and appears to be internal to the cells. This pattern of staining is consistent with a process involving ligand-driven aggregation of the receptor, followed by internalization of the ligand-receptor complex and subsequent movement down the endocytic pathway. The receptor distribution pattern following ligand binding was also investigated for the YF719 mutant. As shown in Fig. 3 B, in the presence of Ca2+, the YF719 receptor was also observed to aggregate and internalize. However, unlike the wt receptor, the distribution pattern of the YF719 receptor remained punctate and was primarily located near the interior of the plasma membrane, even after a 15-min incubation. This suggests that although the YF719 receptor is internalizing, further movement down the endocytic pathway may be impaired. When Ca2+ was omitted from the binding and wash buffers, the wt receptor internalized and displayed a pattern of staining identical to that observed in the presence of Ca2+ and with similar kinetics (Fig. 4 A). In contrast, although the YF719 receptor was still observed to aggregate, the Texas Red and FITC stains remained co-localized on the surface of the cell (Fig. 4 B). This observation indicates that the receptor-ligand complexes are not internalizing, in agreement with our flow cytometric data which also demonstrated an inability of the YF719 receptor to internalize in the absence of Ca2+. We have extended our observations to time points as long as 30 min. At these times, the YF719 mutant still fails to internalize in the absence of Ca2+. The acidic environment of endosomes and lysosomes may, in some cases, lead to dissociation of ligand and receptor, resulting in ligand degradation and recycling of receptors back to the cell surface (37Mellman I. Fuchs R. Helenius A. Annu. Rev. Biochem. 1986; 55: 663-700Crossref PubMed Google Scholar). Since we used labeled ligand to follow the fate of the SLF·c-Kit complex, it was important to verify that SLF and c-Kit remained associated in intracellular compartments. Wild type and YF719 DA1 cells were incubated with both b-SLF and a FITC-conjugated anti-c-Kit antibody at 4 °C followed by 37 °C incubations. Cells were then fixed, permeabilized, and incubated with streptavidin-conjugated TxR. As shown in Fig.5, after a 7.5-min incubation at 37 °C, co-localization of the TxR and FITC labels is observed for both the wt and mutant YF719 receptor, either in the presence or absence of extracellular Ca2+. This co-localization is also observed after a 15-min incubation at 37 °C (data not shown). This observation therefore confirms that receptor and ligand remain associated during the internalization process. Our observation of impaired receptor internalization in the absence of Ca2+ for the YF719 mutant cannot distinguish between a dependence on PI3-kinase binding to the c-Kit receptor or a further requirement for PI3-kinase reaction products generated by enzymatic activation. To address this question, we performed our internalization experiments following pretreatment of the cells with wortmannin, a specific inhibitor of the PI3-kinase enzyme (38Arcaro A. Wymann M.P. Biochem. J. 1993; 296: 297-301Crossref PubMed Scopus (1053) Google Scholar). We prepared wt DA-1 cells for either flow cytometry or fluorescence microscopy. Minutes before incubation of the cells" @default.
• W2025107135 created "2016-06-24" @default.
• W2025107135 creator A5001335447 @default.
• W2025107135 creator A5021147315 @default.
• W2025107135 creator A5090218407 @default.
• W2025107135 date "1997-11-01" @default.
• W2025107135 modified "2023-10-16" @default.
• W2025107135 title "Phosphatidylinositol 3-Kinase and Ca2+ Influx Dependence for Ligand-stimulated Internalization of the c-Kit Receptor" @default.
• W2025107135 cites W1428006903 @default.
• W2025107135 cites W1487627529 @default.
• W2025107135 cites W1519692040 @default.
• W2025107135 cites W1534670700 @default.
• W2025107135 cites W1540264457 @default.
• W2025107135 cites W1562349797 @default.
• W2025107135 cites W1568586029 @default.
• W2025107135 cites W1601224295 @default.
• W2025107135 cites W1732879167 @default.
• W2025107135 cites W1808322538 @default.
• W2025107135 cites W1818313483 @default.
• W2025107135 cites W1971065575 @default.
• W2025107135 cites W1972960760 @default.
• W2025107135 cites W1981260497 @default.
• W2025107135 cites W1982437598 @default.
• W2025107135 cites W1999012962 @default.
• W2025107135 cites W2001922490 @default.
• W2025107135 cites W2001961027 @default.
• W2025107135 cites W2013131355 @default.
• W2025107135 cites W2015480034 @default.
• W2025107135 cites W2026082408 @default.
• W2025107135 cites W2032359283 @default.
• W2025107135 cites W2035584408 @default.
• W2025107135 cites W2037695813 @default.
• W2025107135 cites W2043053145 @default.
• W2025107135 cites W2044617353 @default.
• W2025107135 cites W2046974658 @default.
• W2025107135 cites W2053023240 @default.
• W2025107135 cites W2055963067 @default.
• W2025107135 cites W2061476447 @default.
• W2025107135 cites W2063385900 @default.
• W2025107135 cites W2070314562 @default.
• W2025107135 cites W2070360831 @default.
• W2025107135 cites W2073302858 @default.
• W2025107135 cites W2081113123 @default.
• W2025107135 cites W2113252814 @default.
• W2025107135 cites W2140922568 @default.
• W2025107135 cites W2151900782 @default.
• W2025107135 cites W2156475226 @default.
• W2025107135 cites W2161416030 @default.
• W2025107135 cites W2167597644 @default.
• W2025107135 cites W2174160083 @default.
• W2025107135 doi "https://doi.org/10.1074/jbc.272.48.30519" @default.
• W2025107135 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/9374546" @default.
• W2025107135 hasPublicationYear "1997" @default.
• W2025107135 type Work @default.
• W2025107135 sameAs 2025107135 @default.
• W2025107135 citedByCount "43" @default.
• W2025107135 countsByYear W20251071352012 @default.
• W2025107135 countsByYear W20251071352013 @default.
• W2025107135 countsByYear W20251071352014 @default.
• W2025107135 countsByYear W20251071352015 @default.
• W2025107135 countsByYear W20251071352016 @default.
• W2025107135 countsByYear W20251071352022 @default.
• W2025107135 countsByYear W20251071352023 @default.
• W2025107135 crossrefType "journal-article" @default.
• W2025107135 hasAuthorship W2025107135A5001335447 @default.
• W2025107135 hasAuthorship W2025107135A5021147315 @default.
• W2025107135 hasAuthorship W2025107135A5090218407 @default.
• W2025107135 hasBestOaLocation W20251071351 @default.
• W2025107135 hasConcept C116569031 @default.
• W2025107135 hasConcept C12554922 @default.
• W2025107135 hasConcept C139770010 @default.
• W2025107135 hasConcept C170493617 @default.
• W2025107135 hasConcept C184235292 @default.
• W2025107135 hasConcept C185592680 @default.
• W2025107135 hasConcept C2780610907 @default.
• W2025107135 hasConcept C55493867 @default.
• W2025107135 hasConcept C86803240 @default.
• W2025107135 hasConcept C95444343 @default.
• W2025107135 hasConceptScore W2025107135C116569031 @default.
• W2025107135 hasConceptScore W2025107135C12554922 @default.
• W2025107135 hasConceptScore W2025107135C139770010 @default.
• W2025107135 hasConceptScore W2025107135C170493617 @default.
• W2025107135 hasConceptScore W2025107135C184235292 @default.
• W2025107135 hasConceptScore W2025107135C185592680 @default.
• W2025107135 hasConceptScore W2025107135C2780610907 @default.
• W2025107135 hasConceptScore W2025107135C55493867 @default.
• W2025107135 hasConceptScore W2025107135C86803240 @default.
• W2025107135 hasConceptScore W2025107135C95444343 @default.
• W2025107135 hasIssue "48" @default.
• W2025107135 hasLocation W20251071351 @default.
• W2025107135 hasOpenAccess W2025107135 @default.
• W2025107135 hasPrimaryLocation W20251071351 @default.
• W2025107135 hasRelatedWork W1165872130 @default.
• W2025107135 hasRelatedWork W1487269337 @default.
• W2025107135 hasRelatedWork W1550990988 @default.
• W2025107135 hasRelatedWork W1964622351 @default.
• W2025107135 hasRelatedWork W1965030380 @default.
• W2025107135 hasRelatedWork W2000118052 @default.

• next