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. 1996 Sep 1;134(5):1169–1177. doi: 10.1083/jcb.134.5.1169

Clustered folate receptors deliver 5-methyltetrahydrofolate to cytoplasm of MA104 cells

PMCID: PMC2120971  PMID: 8794859

Abstract

Previously, a high affinity, glycosylphosphatidylinositol-anchored receptor for folate and a caveolae internalization cycle have been found necessary for potocytosis of 5-methyltetrahydrofolate in MA104. We now show by cell fractionation that folate receptors also must be clustered in caveolae for potocytosis. An enriched fraction of caveolae from control cells retained 65-70% of the [3H]folic acid bound to cells in culture. Exposure of cells to the cholesterol-binding drug, filipin, which is known to uncluster receptors, shifted approximately 50% of the bound [3H]folic acid from the caveolae fraction to the noncaveolae membrane fraction and markedly inhibited internalization of [3H]folic acid. An mAb directed against the folate receptor also shifted approximately 50% of the caveolae-associated [3H]folic acid to noncaveolae membrane, indicating the antibody perturbs the normal receptor distribution. Concordantly, the mAb inhibited the delivery of 5-methyl[3H]tetrahydrofolate to the cytoplasm. Receptor bound 5- methyl[3H]tetrahydrofolate moved directly from caveolae to the cytoplasm and was not blocked by phenylarsine oxide, an inhibitor of receptor-mediated endocytosis. These results suggest cell fractionation can be used to study the uptake of molecules by caveolae.

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Selected References

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  1. Anderson R. G., Brown M. S., Goldstein J. L. Role of the coated endocytic vesicle in the uptake of receptor-bound low density lipoprotein in human fibroblasts. Cell. 1977 Mar;10(3):351–364. doi: 10.1016/0092-8674(77)90022-8. [DOI] [PubMed] [Google Scholar]
  2. Anderson R. G., Goldstein J. L., Brown M. S. A mutation that impairs the ability of lipoprotein receptors to localise in coated pits on the cell surface of human fibroblasts. Nature. 1977 Dec 22;270(5639):695–699. doi: 10.1038/270695a0. [DOI] [PubMed] [Google Scholar]
  3. Anderson R. G., Kamen B. A., Rothberg K. G., Lacey S. W. Potocytosis: sequestration and transport of small molecules by caveolae. Science. 1992 Jan 24;255(5043):410–411. doi: 10.1126/science.1310359. [DOI] [PubMed] [Google Scholar]
  4. Andreasen P. A., Sottrup-Jensen L., Kjøller L., Nykjaer A., Moestrup S. K., Petersen C. M., Gliemann J. Receptor-mediated endocytosis of plasminogen activators and activator/inhibitor complexes. FEBS Lett. 1994 Feb 7;338(3):239–245. doi: 10.1016/0014-5793(94)80276-9. [DOI] [PubMed] [Google Scholar]
  5. Chang W. J., Rothberg K. G., Kamen B. A., Anderson R. G. Lowering the cholesterol content of MA104 cells inhibits receptor-mediated transport of folate. J Cell Biol. 1992 Jul;118(1):63–69. doi: 10.1083/jcb.118.1.63. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chang W. J., Ying Y. S., Rothberg K. G., Hooper N. M., Turner A. J., Gambliel H. A., De Gunzburg J., Mumby S. M., Gilman A. G., Anderson R. G. Purification and characterization of smooth muscle cell caveolae. J Cell Biol. 1994 Jul;126(1):127–138. doi: 10.1083/jcb.126.1.127. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Davis C. G., Lehrman M. A., Russell D. W., Anderson R. G., Brown M. S., Goldstein J. L. The J.D. mutation in familial hypercholesterolemia: amino acid substitution in cytoplasmic domain impedes internalization of LDL receptors. Cell. 1986 Apr 11;45(1):15–24. doi: 10.1016/0092-8674(86)90533-7. [DOI] [PubMed] [Google Scholar]
  8. Jemmerson R., Agre M. Monoclonal antibodies to different epitopes on a cell-surface enzyme, human placental alkaline phosphatase, effect different patterns of labeling with protein A-colloidal gold. J Histochem Cytochem. 1987 Nov;35(11):1277–1284. doi: 10.1177/35.11.2443558. [DOI] [PubMed] [Google Scholar]
  9. Kamen B. A., Capdevila A. Receptor-mediated folate accumulation is regulated by the cellular folate content. Proc Natl Acad Sci U S A. 1986 Aug;83(16):5983–5987. doi: 10.1073/pnas.83.16.5983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Kamen B. A., Johnson C. A., Wang M. T., Anderson R. G. Regulation of the cytoplasmic accumulation of 5-methyltetrahydrofolate in MA104 cells is independent of folate receptor regulation. J Clin Invest. 1989 Nov;84(5):1379–1386. doi: 10.1172/JCI114310. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Kamen B. A., Smith A. K., Anderson R. G. The folate receptor works in tandem with a probenecid-sensitive carrier in MA104 cells in vitro. J Clin Invest. 1991 Apr;87(4):1442–1449. doi: 10.1172/JCI115150. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kamen B. A., Wang M. T., Streckfuss A. J., Peryea X., Anderson R. G. Delivery of folates to the cytoplasm of MA104 cells is mediated by a surface membrane receptor that recycles. J Biol Chem. 1988 Sep 25;263(27):13602–13609. [PubMed] [Google Scholar]
  13. Kaplan J., Ward D. M., Wiley H. S. Phenylarsine oxide-induced increase in alveolar macrophage surface receptors: evidence for fusion of internal receptor pools with the cell surface. J Cell Biol. 1985 Jul;101(1):121–129. doi: 10.1083/jcb.101.1.121. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  15. Liu P., Anderson R. G. Compartmentalized production of ceramide at the cell surface. J Biol Chem. 1995 Nov 10;270(45):27179–27185. doi: 10.1074/jbc.270.45.27179. [DOI] [PubMed] [Google Scholar]
  16. Liu P., Ying Y., Ko Y. G., Anderson R. G. Localization of platelet-derived growth factor-stimulated phosphorylation cascade to caveolae. J Biol Chem. 1996 Apr 26;271(17):10299–10303. doi: 10.1074/jbc.271.17.10299. [DOI] [PubMed] [Google Scholar]
  17. Mayor S., Rothberg K. G., Maxfield F. R. Sequestration of GPI-anchored proteins in caveolae triggered by cross-linking. Science. 1994 Jun 24;264(5167):1948–1951. doi: 10.1126/science.7516582. [DOI] [PubMed] [Google Scholar]
  18. Mello R. J., Brown M. S., Goldstein J. L., Anderson R. G. LDL receptors in coated vesicles isolated from bovine adrenal cortex: binding sites unmasked by detergent treatment. Cell. 1980 Jul;20(3):829–837. doi: 10.1016/0092-8674(80)90329-3. [DOI] [PubMed] [Google Scholar]
  19. Mineo C., James G. L., Smart E. J., Anderson R. G. Localization of epidermal growth factor-stimulated Ras/Raf-1 interaction to caveolae membrane. J Biol Chem. 1996 May 17;271(20):11930–11935. doi: 10.1074/jbc.271.20.11930. [DOI] [PubMed] [Google Scholar]
  20. Parton R. G., Joggerst B., Simons K. Regulated internalization of caveolae. J Cell Biol. 1994 Dec;127(5):1199–1215. doi: 10.1083/jcb.127.5.1199. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Ritter T. E., Fajardo O., Matsue H., Anderson R. G., Lacey S. W. Folate receptors targeted to clathrin-coated pits cannot regulate vitamin uptake. Proc Natl Acad Sci U S A. 1995 Apr 25;92(9):3824–3828. doi: 10.1073/pnas.92.9.3824. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Rothberg K. G., Heuser J. E., Donzell W. C., Ying Y. S., Glenney J. R., Anderson R. G. Caveolin, a protein component of caveolae membrane coats. Cell. 1992 Feb 21;68(4):673–682. doi: 10.1016/0092-8674(92)90143-z. [DOI] [PubMed] [Google Scholar]
  23. Rothberg K. G., Ying Y. S., Kamen B. A., Anderson R. G. Cholesterol controls the clustering of the glycophospholipid-anchored membrane receptor for 5-methyltetrahydrofolate. J Cell Biol. 1990 Dec;111(6 Pt 2):2931–2938. doi: 10.1083/jcb.111.6.2931. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Rothberg K. G., Ying Y. S., Kolhouse J. F., Kamen B. A., Anderson R. G. The glycophospholipid-linked folate receptor internalizes folate without entering the clathrin-coated pit endocytic pathway. J Cell Biol. 1990 Mar;110(3):637–649. doi: 10.1083/jcb.110.3.637. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Sargiacomo M., Sudol M., Tang Z., Lisanti M. P. Signal transducing molecules and glycosyl-phosphatidylinositol-linked proteins form a caveolin-rich insoluble complex in MDCK cells. J Cell Biol. 1993 Aug;122(4):789–807. doi: 10.1083/jcb.122.4.789. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Schroeder R., London E., Brown D. Interactions between saturated acyl chains confer detergent resistance on lipids and glycosylphosphatidylinositol (GPI)-anchored proteins: GPI-anchored proteins in liposomes and cells show similar behavior. Proc Natl Acad Sci U S A. 1994 Dec 6;91(25):12130–12134. doi: 10.1073/pnas.91.25.12130. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Shaul P. W., Smart E. J., Robinson L. J., German Z., Yuhanna I. S., Ying Y., Anderson R. G., Michel T. Acylation targets emdothelial nitric-oxide synthase to plasmalemmal caveolae. J Biol Chem. 1996 Mar 15;271(11):6518–6522. doi: 10.1074/jbc.271.11.6518. [DOI] [PubMed] [Google Scholar]
  28. Smart E. J., Foster D. C., Ying Y. S., Kamen B. A., Anderson R. G. Protein kinase C activators inhibit receptor-mediated potocytosis by preventing internalization of caveolae. J Cell Biol. 1994 Feb;124(3):307–313. doi: 10.1083/jcb.124.3.307. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Smart E. J., Ying Y. S., Anderson R. G. Hormonal regulation of caveolae internalization. J Cell Biol. 1995 Nov;131(4):929–938. doi: 10.1083/jcb.131.4.929. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Smart E. J., Ying Y. S., Conrad P. A., Anderson R. G. Caveolin moves from caveolae to the Golgi apparatus in response to cholesterol oxidation. J Cell Biol. 1994 Dec;127(5):1185–1197. doi: 10.1083/jcb.127.5.1185. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Smart E. J., Ying Y. S., Mineo C., Anderson R. G. A detergent-free method for purifying caveolae membrane from tissue culture cells. Proc Natl Acad Sci U S A. 1995 Oct 24;92(22):10104–10108. doi: 10.1073/pnas.92.22.10104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. YAMADA E. The fine structure of the gall bladder epithelium of the mouse. J Biophys Biochem Cytol. 1955 Sep 25;1(5):445–458. doi: 10.1083/jcb.1.5.445. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. van den Berg C. W., Cinek T., Hallett M. B., Horejsi V., Morgan B. P. Exogenous glycosyl phosphatidylinositol-anchored CD59 associates with kinases in membrane clusters on U937 cells and becomes Ca(2+)-signaling competent. J Cell Biol. 1995 Nov;131(3):669–677. doi: 10.1083/jcb.131.3.669. [DOI] [PMC free article] [PubMed] [Google Scholar]

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