Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1994 Aug 2;126(4):991–1004. doi: 10.1083/jcb.126.4.991

Structural requirements and sequence motifs for polarized sorting and endocytosis of LDL and Fc receptors in MDCK cells

PMCID: PMC2120116  PMID: 8051216

Abstract

In MDCK cells, basolateral sorting of most membrane proteins has been shown to depend on distinct cytoplasmic domain determinants. These signals can be divided into those which are related to signals for localization at clathrin-coated pits and those which are unrelated. The LDL receptor bears two tyrosine-containing signals, one of each class, that can independently target receptors from the Golgi complex and from endosomes to the basolateral plasma membrane. We have now investigated the other structural features required for the activity of both determinants. We find that both depend, at least in part, on clusters of 1-3 acidic amino acids located on the COOH-terminal side of each tyrosine. While single residues adjacent to each tyrosine were also found to be critical, the two signals differed in that only the coated pit-unrelated signal could tolerate a phenylalanine in place of its tyrosine residue. We also found that the structural requirements for basolateral targeting of the "coated pit-related" signal were distinct from those required for rapid endocytosis. Apart from sharing a common tyrosine residue, no feature of the NPXY motif for coated pit localization was required for basolateral targeting. We also investigated basolateral targeting of the mouse macrophage Fc receptor (FcRII-B2) which contains a tyrosine-independent coated pit localization signal. Basolateral transport and endocytosis were found to depend on a common dileucine-type motif. Thus, basolateral targeting determinants, like coated pit domains, can contain either tyrosine- or di-leucine-containing signals. The amino acids in the vicinity of these motifs determine whether they function as determinants for endocytosis, basolateral targeting, or both.

Full Text

The Full Text of this article is available as a PDF (2.1 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Aroeti B., Kosen P. A., Kuntz I. D., Cohen F. E., Mostov K. E. Mutational and secondary structural analysis of the basolateral sorting signal of the polymeric immunoglobulin receptor. J Cell Biol. 1993 Dec;123(5):1149–1160. doi: 10.1083/jcb.123.5.1149. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Beisiegel U., Schneider W. J., Goldstein J. L., Anderson R. G., Brown M. S. Monoclonal antibodies to the low density lipoprotein receptor as probes for study of receptor-mediated endocytosis and the genetics of familial hypercholesterolemia. J Biol Chem. 1981 Nov 25;256(22):11923–11931. [PubMed] [Google Scholar]
  3. Brewer C. B., Roth M. G. A single amino acid change in the cytoplasmic domain alters the polarized delivery of influenza virus hemagglutinin. J Cell Biol. 1991 Aug;114(3):413–421. doi: 10.1083/jcb.114.3.413. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Casanova J. E., Apodaca G., Mostov K. E. An autonomous signal for basolateral sorting in the cytoplasmic domain of the polymeric immunoglobulin receptor. Cell. 1991 Jul 12;66(1):65–75. doi: 10.1016/0092-8674(91)90139-p. [DOI] [PubMed] [Google Scholar]
  5. Casanova J. E., Breitfeld P. P., Ross S. A., Mostov K. E. Phosphorylation of the polymeric immunoglobulin receptor required for its efficient transcytosis. Science. 1990 May 11;248(4956):742–745. doi: 10.1126/science.2110383. [DOI] [PubMed] [Google Scholar]
  6. Chen W. J., Goldstein J. L., Brown M. S. NPXY, a sequence often found in cytoplasmic tails, is required for coated pit-mediated internalization of the low density lipoprotein receptor. J Biol Chem. 1990 Feb 25;265(6):3116–3123. [PubMed] [Google Scholar]
  7. Dargemont C., Le Bivic A., Rothenberger S., Iacopetta B., Kühn L. C. The internalization signal and the phosphorylation site of transferrin receptor are distinct from the main basolateral sorting information. EMBO J. 1993 Apr;12(4):1713–1721. doi: 10.1002/j.1460-2075.1993.tb05816.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Davis C. G., van Driel I. R., Russell D. W., Brown M. S., Goldstein J. L. The low density lipoprotein receptor. Identification of amino acids in cytoplasmic domain required for rapid endocytosis. J Biol Chem. 1987 Mar 25;262(9):4075–4082. [PubMed] [Google Scholar]
  9. Dotti C. G., Simons K. Polarized sorting of viral glycoproteins to the axon and dendrites of hippocampal neurons in culture. Cell. 1990 Jul 13;62(1):63–72. doi: 10.1016/0092-8674(90)90240-f. [DOI] [PubMed] [Google Scholar]
  10. Geffen I., Fuhrer C., Leitinger B., Weiss M., Huggel K., Griffiths G., Spiess M. Related signals for endocytosis and basolateral sorting of the asialoglycoprotein receptor. J Biol Chem. 1993 Oct 5;268(28):20772–20777. [PubMed] [Google Scholar]
  11. Harter C., Mellman I. Transport of the lysosomal membrane glycoprotein lgp120 (lgp-A) to lysosomes does not require appearance on the plasma membrane. J Cell Biol. 1992 Apr;117(2):311–325. doi: 10.1083/jcb.117.2.311. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hirt R. P., Hughes G. J., Frutiger S., Michetti P., Perregaux C., Poulain-Godefroy O., Jeanguenat N., Neutra M. R., Kraehenbuhl J. P. Transcytosis of the polymeric Ig receptor requires phosphorylation of serine 664 in the absence but not the presence of dimeric IgA. Cell. 1993 Jul 30;74(2):245–255. doi: 10.1016/0092-8674(93)90416-n. [DOI] [PubMed] [Google Scholar]
  13. Hunziker W., Harter C., Matter K., Mellman I. Basolateral sorting in MDCK cells requires a distinct cytoplasmic domain determinant. Cell. 1991 Sep 6;66(5):907–920. doi: 10.1016/0092-8674(91)90437-4. [DOI] [PubMed] [Google Scholar]
  14. Hunziker W., Mellman I. Expression of macrophage-lymphocyte Fc receptors in Madin-Darby canine kidney cells: polarity and transcytosis differ for isoforms with or without coated pit localization domains. J Cell Biol. 1989 Dec;109(6 Pt 2):3291–3302. doi: 10.1083/jcb.109.6.3291. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hunziker W., Mellman I. Relationships between sorting in the exocytic and endocytic pathways of MDCK cells. Semin Cell Biol. 1991 Dec;2(6):397–410. [PubMed] [Google Scholar]
  16. Johnson K. F., Kornfeld S. A His-Leu-Leu sequence near the carboxyl terminus of the cytoplasmic domain of the cation-dependent mannose 6-phosphate receptor is necessary for the lysosomal enzyme sorting function. J Biol Chem. 1992 Aug 25;267(24):17110–17115. [PubMed] [Google Scholar]
  17. Johnson K. F., Kornfeld S. The cytoplasmic tail of the mannose 6-phosphate/insulin-like growth factor-II receptor has two signals for lysosomal enzyme sorting in the Golgi. J Cell Biol. 1992 Oct;119(2):249–257. doi: 10.1083/jcb.119.2.249. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kornfeld S., Mellman I. The biogenesis of lysosomes. Annu Rev Cell Biol. 1989;5:483–525. doi: 10.1146/annurev.cb.05.110189.002411. [DOI] [PubMed] [Google Scholar]
  19. Le Bivic A., Sambuy Y., Patzak A., Patil N., Chao M., Rodriguez-Boulan E. An internal deletion in the cytoplasmic tail reverses the apical localization of human NGF receptor in transfected MDCK cells. J Cell Biol. 1991 Nov;115(3):607–618. doi: 10.1083/jcb.115.3.607. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Letourneur F., Klausner R. D. A novel di-leucine motif and a tyrosine-based motif independently mediate lysosomal targeting and endocytosis of CD3 chains. Cell. 1992 Jun 26;69(7):1143–1157. doi: 10.1016/0092-8674(92)90636-q. [DOI] [PubMed] [Google Scholar]
  21. Lewis V. A., Koch T., Plutner H., Mellman I. A complementary DNA clone for a macrophage-lymphocyte Fc receptor. 1986 Nov 27-Dec 3Nature. 324(6095):372–375. doi: 10.1038/324372a0. [DOI] [PubMed] [Google Scholar]
  22. Lobel P., Dahms N. M., Kornfeld S. Cloning and sequence analysis of the cation-independent mannose 6-phosphate receptor. J Biol Chem. 1988 Feb 15;263(5):2563–2570. [PubMed] [Google Scholar]
  23. Matter K., Hunziker W., Mellman I. Basolateral sorting of LDL receptor in MDCK cells: the cytoplasmic domain contains two tyrosine-dependent targeting determinants. Cell. 1992 Nov 27;71(5):741–753. doi: 10.1016/0092-8674(92)90551-m. [DOI] [PubMed] [Google Scholar]
  24. Matter K., Whitney J. A., Yamamoto E. M., Mellman I. Common signals control low density lipoprotein receptor sorting in endosomes and the Golgi complex of MDCK cells. Cell. 1993 Sep 24;74(6):1053–1064. doi: 10.1016/0092-8674(93)90727-8. [DOI] [PubMed] [Google Scholar]
  25. McClelland A., Kühn L. C., Ruddle F. H. The human transferrin receptor gene: genomic organization, and the complete primary structure of the receptor deduced from a cDNA sequence. Cell. 1984 Dec;39(2 Pt 1):267–274. doi: 10.1016/0092-8674(84)90004-7. [DOI] [PubMed] [Google Scholar]
  26. Miettinen H. M., Matter K., Hunziker W., Rose J. K., Mellman I. Fc receptor endocytosis is controlled by a cytoplasmic domain determinant that actively prevents coated pit localization. J Cell Biol. 1992 Feb;116(4):875–888. doi: 10.1083/jcb.116.4.875. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Mostov K. E., Friedlander M., Blobel G. The receptor for transepithelial transport of IgA and IgM contains multiple immunoglobulin-like domains. Nature. 1984 Mar 1;308(5954):37–43. doi: 10.1038/308037a0. [DOI] [PubMed] [Google Scholar]
  28. Nagafuchi A., Shirayoshi Y., Okazaki K., Yasuda K., Takeichi M. Transformation of cell adhesion properties by exogenously introduced E-cadherin cDNA. Nature. 1987 Sep 24;329(6137):341–343. doi: 10.1038/329341a0. [DOI] [PubMed] [Google Scholar]
  29. Ogata S., Fukuda M. Lysosomal targeting of Limp II membrane glycoprotein requires a novel Leu-Ile motif at a particular position in its cytoplasmic tail. J Biol Chem. 1994 Feb 18;269(7):5210–5217. [PubMed] [Google Scholar]
  30. Oshima A., Nolan C. M., Kyle J. W., Grubb J. H., Sly W. S. The human cation-independent mannose 6-phosphate receptor. Cloning and sequence of the full-length cDNA and expression of functional receptor in COS cells. J Biol Chem. 1988 Feb 15;263(5):2553–2562. [PubMed] [Google Scholar]
  31. Pearse B. M., Robinson M. S. Clathrin, adaptors, and sorting. Annu Rev Cell Biol. 1990;6:151–171. doi: 10.1146/annurev.cb.06.110190.001055. [DOI] [PubMed] [Google Scholar]
  32. Pelham H. R., Munro S. Sorting of membrane proteins in the secretory pathway. Cell. 1993 Nov 19;75(4):603–605. doi: 10.1016/0092-8674(93)90479-A. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Pohlmann R., Krentler C., Schmidt B., Schröder W., Lorkowski G., Culley J., Mersmann G., Geier C., Waheed A., Gottschalk S. Human lysosomal acid phosphatase: cloning, expression and chromosomal assignment. EMBO J. 1988 Aug;7(8):2343–2350. doi: 10.1002/j.1460-2075.1988.tb03078.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Pohlmann R., Nagel G., Schmidt B., Stein M., Lorkowski G., Krentler C., Cully J., Meyer H. E., Grzeschik K. H., Mersmann G. Cloning of a cDNA encoding the human cation-dependent mannose 6-phosphate-specific receptor. Proc Natl Acad Sci U S A. 1987 Aug;84(16):5575–5579. doi: 10.1073/pnas.84.16.5575. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Prill V., Lehmann L., von Figura K., Peters C. The cytoplasmic tail of lysosomal acid phosphatase contains overlapping but distinct signals for basolateral sorting and rapid internalization in polarized MDCK cells. EMBO J. 1993 May;12(5):2181–2193. doi: 10.1002/j.1460-2075.1993.tb05866.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Ringwald M., Schuh R., Vestweber D., Eistetter H., Lottspeich F., Engel J., Dölz R., Jähnig F., Epplen J., Mayer S. The structure of cell adhesion molecule uvomorulin. Insights into the molecular mechanism of Ca2+-dependent cell adhesion. EMBO J. 1987 Dec 1;6(12):3647–3653. doi: 10.1002/j.1460-2075.1987.tb02697.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Rodriguez-Boulan E., Powell S. K. Polarity of epithelial and neuronal cells. Annu Rev Cell Biol. 1992;8:395–427. doi: 10.1146/annurev.cb.08.110192.002143. [DOI] [PubMed] [Google Scholar]
  38. Thomas D. C., Brewer C. B., Roth M. G. Vesicular stomatitis virus glycoprotein contains a dominant cytoplasmic basolateral sorting signal critically dependent upon a tyrosine. J Biol Chem. 1993 Feb 15;268(5):3313–3320. [PubMed] [Google Scholar]
  39. Trowbridge I. S. Endocytosis and signals for internalization. Curr Opin Cell Biol. 1991 Aug;3(4):634–641. doi: 10.1016/0955-0674(91)90034-v. [DOI] [PubMed] [Google Scholar]
  40. Unkeless J. C. Characterization of a monoclonal antibody directed against mouse macrophage and lymphocyte Fc receptors. J Exp Med. 1979 Sep 19;150(3):580–596. doi: 10.1084/jem.150.3.580. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Vaux D. The structure of an endocytosis signal. Trends Cell Biol. 1992 Jul;2(7):189–192. doi: 10.1016/0962-8924(92)90232-c. [DOI] [PubMed] [Google Scholar]
  42. Vega M. A., Rodriguez F., Seguí B., Calés C., Alcalde J., Sandoval I. V. Targeting of lysosomal integral membrane protein LIMP II. The tyrosine-lacking carboxyl cytoplasmic tail of LIMP II is sufficient for direct targeting to lysosomes. J Biol Chem. 1991 Sep 5;266(25):16269–16272. [PubMed] [Google Scholar]
  43. Wandinger-Ness A., Bennett M. K., Antony C., Simons K. Distinct transport vesicles mediate the delivery of plasma membrane proteins to the apical and basolateral domains of MDCK cells. J Cell Biol. 1990 Sep;111(3):987–1000. doi: 10.1083/jcb.111.3.987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Williams M. A., Fukuda M. Accumulation of membrane glycoproteins in lysosomes requires a tyrosine residue at a particular position in the cytoplasmic tail. J Cell Biol. 1990 Sep;111(3):955–966. doi: 10.1083/jcb.111.3.955. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Yokode M., Pathak R. K., Hammer R. E., Brown M. S., Goldstein J. L., Anderson R. G. Cytoplasmic sequence required for basolateral targeting of LDL receptor in livers of transgenic mice. J Cell Biol. 1992 Apr;117(1):39–46. doi: 10.1083/jcb.117.1.39. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES