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. 1996 Sep 1;134(5):1229–1240. doi: 10.1083/jcb.134.5.1229

Targeting of P-selectin to two regulated secretory organelles in PC12 cells

PMCID: PMC2120975  PMID: 8794864

Abstract

Targeting of P-selectin to the regulated secretory organelles (RSOs) of phaeochromocytoma PC12 cells has been investigated. By expressing from cDNA a chimera composed of HRP and P-selectin, and then following HRP activity through subcellular fractionation, we have discovered that P- selectin contains signals that target HRP to the synaptic-like microvesicles (SLMV) as well as the dense-core granules (DCGs) of these cells. Mutagenesis of the chimera followed by transient expression in PC12 cells shows that at least two different sequences within the carboxy-terminal cytoplasmic tail of P-selectin are necessary, but that neither is sufficient for trafficking to the SLMV. One of these sequences is centred on the 10 amino acids of the membrane-proximal C1 exon that is also implicated in lysosomal targeting. The other sequence needed for trafficking to the SLMV includes the last four amino acids of the protein. The same series of mutations have a different effect on DCG targeting, showing that traffic to the two different RSOs depends on different features within the cytoplasmic domain of P-selectin.

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Selected References

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  1. Bauerfeind R., Régnier-Vigouroux A., Flatmark T., Huttner W. B. Selective storage of acetylcholine, but not catecholamines, in neuroendocrine synaptic-like microvesicles of early endosomal origin. Neuron. 1993 Jul;11(1):105–121. doi: 10.1016/0896-6273(93)90275-v. [DOI] [PubMed] [Google Scholar]
  2. Blackstone C. D., Moss S. J., Martin L. J., Levey A. I., Price D. L., Huganir R. L. Biochemical characterization and localization of a non-N-methyl-D-aspartate glutamate receptor in rat brain. J Neurochem. 1992 Mar;58(3):1118–1126. doi: 10.1111/j.1471-4159.1992.tb09370.x. [DOI] [PubMed] [Google Scholar]
  3. Buckley K. M., Floor E., Kelly R. B. Cloning and sequence analysis of cDNA encoding p38, a major synaptic vesicle protein. J Cell Biol. 1987 Dec;105(6 Pt 1):2447–2456. doi: 10.1083/jcb.105.6.2447. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cameron P. L., Südhof T. C., Jahn R., De Camilli P. Colocalization of synaptophysin with transferrin receptors: implications for synaptic vesicle biogenesis. J Cell Biol. 1991 Oct;115(1):151–164. doi: 10.1083/jcb.115.1.151. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chilcote T. J., Galli T., Mundigl O., Edelmann L., McPherson P. S., Takei K., De Camilli P. Cellubrevin and synaptobrevins: similar subcellular localization and biochemical properties in PC12 cells. J Cell Biol. 1995 Apr;129(1):219–231. doi: 10.1083/jcb.129.1.219. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Clift-O'Grady L., Linstedt A. D., Lowe A. W., Grote E., Kelly R. B. Biogenesis of synaptic vesicle-like structures in a pheochromocytoma cell line PC-12. J Cell Biol. 1990 May;110(5):1693–1703. doi: 10.1083/jcb.110.5.1693. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Connolly C. N., Futter C. E., Gibson A., Hopkins C. R., Cutler D. F. Transport into and out of the Golgi complex studied by transfecting cells with cDNAs encoding horseradish peroxidase. J Cell Biol. 1994 Nov;127(3):641–652. doi: 10.1083/jcb.127.3.641. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cutler D. F., Cramer L. P. Sorting during transport to the surface of PC12 cells: divergence of synaptic vesicle and secretory granule proteins. J Cell Biol. 1990 Mar;110(3):721–730. doi: 10.1083/jcb.110.3.721. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Desnos C., Clift-O'Grady L., Kelly R. B. Biogenesis of synaptic vesicles in vitro. J Cell Biol. 1995 Sep;130(5):1041–1049. doi: 10.1083/jcb.130.5.1041. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Disdier M., Morrissey J. H., Fugate R. D., Bainton D. F., McEver R. P. Cytoplasmic domain of P-selectin (CD62) contains the signal for sorting into the regulated secretory pathway. Mol Biol Cell. 1992 Mar;3(3):309–321. doi: 10.1091/mbc.3.3.309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Elferink L. A., Trimble W. S., Scheller R. H. Two vesicle-associated membrane protein genes are differentially expressed in the rat central nervous system. J Biol Chem. 1989 Jul 5;264(19):11061–11064. [PubMed] [Google Scholar]
  12. Feany M. B., Yee A. G., Delvy M. L., Buckley K. M. The synaptic vesicle proteins SV2, synaptotagmin and synaptophysin are sorted to separate cellular compartments in CHO fibroblasts. J Cell Biol. 1993 Nov;123(3):575–584. doi: 10.1083/jcb.123.3.575. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Fischer von Mollard G., Stahl B., Walch-Solimena C., Takei K., Daniels L., Khoklatchev A., De Camilli P., Südhof T. C., Jahn R. Localization of Rab5 to synaptic vesicles identifies endosomal intermediate in synaptic vesicle recycling pathway. Eur J Cell Biol. 1994 Dec;65(2):319–326. [PubMed] [Google Scholar]
  14. Green S. A., Setiadi H., McEver R. P., Kelly R. B. The cytoplasmic domain of P-selectin contains a sorting determinant that mediates rapid degradation in lysosomes. J Cell Biol. 1994 Feb;124(4):435–448. doi: 10.1083/jcb.124.4.435. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Greene L. A., Tischler A. S. Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor. Proc Natl Acad Sci U S A. 1976 Jul;73(7):2424–2428. doi: 10.1073/pnas.73.7.2424. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Grote E., Hao J. C., Bennett M. K., Kelly R. B. A targeting signal in VAMP regulating transport to synaptic vesicles. Cell. 1995 May 19;81(4):581–589. doi: 10.1016/0092-8674(95)90079-9. [DOI] [PubMed] [Google Scholar]
  17. Grote E., Kelly R. B. Endocytosis of VAMP is facilitated by a synaptic vesicle targeting signal. J Cell Biol. 1996 Feb;132(4):537–547. doi: 10.1083/jcb.132.4.537. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hurtley S. M. Recycling of a secretory granule membrane protein after stimulated secretion. J Cell Sci. 1993 Oct;106(Pt 2):649–655. doi: 10.1242/jcs.106.2.649. [DOI] [PubMed] [Google Scholar]
  19. Jahn R., Schiebler W., Ouimet C., Greengard P. A 38,000-dalton membrane protein (p38) present in synaptic vesicles. Proc Natl Acad Sci U S A. 1985 Jun;82(12):4137–4141. doi: 10.1073/pnas.82.12.4137. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Johnston G. I., Bliss G. A., Newman P. J., McEver R. P. Structure of the human gene encoding granule membrane protein-140, a member of the selectin family of adhesion receptors for leukocytes. J Biol Chem. 1990 Dec 5;265(34):21381–21385. [PubMed] [Google Scholar]
  21. Johnston G. I., Cook R. G., McEver R. P. Cloning of GMP-140, a granule membrane protein of platelets and endothelium: sequence similarity to proteins involved in cell adhesion and inflammation. Cell. 1989 Mar 24;56(6):1033–1044. doi: 10.1016/0092-8674(89)90636-3. [DOI] [PubMed] [Google Scholar]
  22. Johnston P. A., Cameron P. L., Stukenbrok H., Jahn R., De Camilli P., Südhof T. C. Synaptophysin is targeted to similar microvesicles in CHO and PC12 cells. EMBO J. 1989 Oct;8(10):2863–2872. doi: 10.1002/j.1460-2075.1989.tb08434.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Koedam J. A., Cramer E. M., Briend E., Furie B., Furie B. C., Wagner D. D. P-selectin, a granule membrane protein of platelets and endothelial cells, follows the regulated secretory pathway in AtT-20 cells. J Cell Biol. 1992 Feb;116(3):617–625. doi: 10.1083/jcb.116.3.617. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Lah J. J., Burry R. W. Synaptophysin has a selective distribution in early endosomes of PC12 cells. J Neurocytol. 1993 Feb;22(2):92–101. doi: 10.1007/BF01181573. [DOI] [PubMed] [Google Scholar]
  25. Leube R. E., Leimer U., Grund C., Franke W. W., Harth N., Wiedenmann B. Sorting of synaptophysin into special vesicles in nonneuroendocrine epithelial cells. J Cell Biol. 1994 Dec;127(6 Pt 1):1589–1601. doi: 10.1083/jcb.127.6.1589. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Linstedt A. D., Kelly R. B. Endocytosis of the synaptic vesicle protein, synaptophysin, requires the COOH-terminal tail. J Physiol (Paris) 1991;85(2):90–96. [PubMed] [Google Scholar]
  27. McEver R. P. Regulation of function and expression of P-selectin. Agents Actions Suppl. 1995;47:117–119. doi: 10.1007/978-3-0348-7343-7_10. [DOI] [PubMed] [Google Scholar]
  28. Milgram S. L., Eipper B. A., Mains R. E. Differential trafficking of soluble and integral membrane secretory granule-associated proteins. J Cell Biol. 1994 Jan;124(1-2):33–41. doi: 10.1083/jcb.124.1.33. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Milgram S. L., Johnson R. C., Mains R. E. Expression of individual forms of peptidylglycine alpha-amidating monooxygenase in AtT-20 cells: endoproteolytic processing and routing to secretory granules. J Cell Biol. 1992 May;117(4):717–728. doi: 10.1083/jcb.117.4.717. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Milgram S. L., Mains R. E., Eipper B. A. COOH-terminal signals mediate the trafficking of a peptide processing enzyme in endocrine cells. J Cell Biol. 1993 Apr;121(1):23–36. doi: 10.1083/jcb.121.1.23. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Mundigl O., Matteoli M., Daniell L., Thomas-Reetz A., Metcalf A., Jahn R., De Camilli P. Synaptic vesicle proteins and early endosomes in cultured hippocampal neurons: differential effects of Brefeldin A in axon and dendrites. J Cell Biol. 1993 Sep;122(6):1207–1221. doi: 10.1083/jcb.122.6.1207. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Navone F., Di Gioia G., Jahn R., Browning M., Greengard P., De Camilli P. Microvesicles of the neurohypophysis are biochemically related to small synaptic vesicles of presynaptic nerve terminals. J Cell Biol. 1989 Dec;109(6 Pt 2):3425–3433. doi: 10.1083/jcb.109.6.3425. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Papini E., Rossetto O., Cutler D. F. Vesicle-associated membrane protein (VAMP)/synaptobrevin-2 is associated with dense core secretory granules in PC12 neuroendocrine cells. J Biol Chem. 1995 Jan 20;270(3):1332–1336. doi: 10.1074/jbc.270.3.1332. [DOI] [PubMed] [Google Scholar]
  34. Régnier-Vigouroux A., Tooze S. A., Huttner W. B. Newly synthesized synaptophysin is transported to synaptic-like microvesicles via constitutive secretory vesicles and the plasma membrane. EMBO J. 1991 Dec;10(12):3589–3601. doi: 10.1002/j.1460-2075.1991.tb04925.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Sandoval I. V., Bakke O. Targeting of membrane proteins to endosomes and lysosomes. Trends Cell Biol. 1994 Aug;4(8):292–297. doi: 10.1016/0962-8924(94)90220-8. [DOI] [PubMed] [Google Scholar]
  36. Setiadi H., Disdier M., Green S. A., Canfield W. M., McEver R. P. Residues throughout the cytoplasmic domain affect the internalization efficiency of P-selectin. J Biol Chem. 1995 Nov 10;270(45):26818–26826. doi: 10.1074/jbc.270.45.26818. [DOI] [PubMed] [Google Scholar]
  37. Stinchcombe J. C., Nomoto H., Cutler D. F., Hopkins C. R. Anterograde and retrograde traffic between the rough endoplasmic reticulum and the Golgi complex. J Cell Biol. 1995 Dec;131(6 Pt 1):1387–1401. doi: 10.1083/jcb.131.6.1387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Subramaniam M., Koedam J. A., Wagner D. D. Divergent fates of P- and E-selectins after their expression on the plasma membrane. Mol Biol Cell. 1993 Aug;4(8):791–801. doi: 10.1091/mbc.4.8.791. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Südhof T. C., Lottspeich F., Greengard P., Mehl E., Jahn R. A synaptic vesicle protein with a novel cytoplasmic domain and four transmembrane regions. Science. 1987 Nov 20;238(4830):1142–1144. doi: 10.1126/science.3120313. [DOI] [PubMed] [Google Scholar]
  40. Trowbridge I. S., Collawn J. F., Hopkins C. R. Signal-dependent membrane protein trafficking in the endocytic pathway. Annu Rev Cell Biol. 1993;9:129–161. doi: 10.1146/annurev.cb.09.110193.001021. [DOI] [PubMed] [Google Scholar]
  41. Wiedenmann B., Franke W. W. Identification and localization of synaptophysin, an integral membrane glycoprotein of Mr 38,000 characteristic of presynaptic vesicles. Cell. 1985 Jul;41(3):1017–1028. doi: 10.1016/s0092-8674(85)80082-9. [DOI] [PubMed] [Google Scholar]
  42. de Hoop M. J., Huber L. A., Stenmark H., Williamson E., Zerial M., Parton R. G., Dotti C. G. The involvement of the small GTP-binding protein Rab5a in neuronal endocytosis. Neuron. 1994 Jul;13(1):11–22. doi: 10.1016/0896-6273(94)90456-1. [DOI] [PubMed] [Google Scholar]

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