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. 1993 May;12(5):2169–2180. doi: 10.1002/j.1460-2075.1993.tb05865.x

Adaptor self-aggregation, adaptor-receptor recognition and binding of alpha-adaptin subunits to the plasma membrane contribute to recruitment of adaptor (AP2) components of clathrin-coated pits.

M P Chang 1, W G Mallet 1, K E Mostov 1, F M Brodsky 1
PMCID: PMC413438  PMID: 8491205

Abstract

Initiation of receptor-mediated endocytosis by nucleation of clathrin-coated pits involves binding of AP2 adaptor molecules to the plasma membrane. This process was reconstituted in vitro, using plasma membrane fragments, prepared by freeze-thaw lysis of cells, and stripped of their endogenous coat proteins, as targets for binding of purified adaptor molecules and their dissociated subunits. The dissociated alpha-adaptin subunit of AP2 bound to plasma membrane fragments, while the dissociated beta-adaptin subunit did not, suggesting that plasma membrane localization of AP2 adaptors is mediated by alpha-adaptin. Membrane binding of intact AP2 adaptor molecules was enhanced by adaptor self-aggregation, which can be modulated by physiological concentrations of inositol phosphates, and may therefore be sensitive to receptor signaling. Adaptor binding was partially inhibited by soluble peptides representing the cytoplasmic domains of the asialoglycoprotein receptor and the polymeric immunoglobulin receptor. These results indicate that direct binding of adaptors to the cytoplasmic domains of receptors contributes to coated pit nucleation but this appears to be a weak interaction, suggesting that an additional recognition signal could be required for high affinity adaptor binding.

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Selected References

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

  1. Ahle S., Mann A., Eichelsbacher U., Ungewickell E. Structural relationships between clathrin assembly proteins from the Golgi and the plasma membrane. EMBO J. 1988 Apr;7(4):919–929. doi: 10.1002/j.1460-2075.1988.tb02897.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Ahle S., Ungewickell E. Identification of a clathrin binding subunit in the HA2 adaptor protein complex. J Biol Chem. 1989 Nov 25;264(33):20089–20093. [PubMed] [Google Scholar]
  3. Beck K. A., Chang M., Brodsky F. M., Keen J. H. Clathrin assembly protein AP-2 induces aggregation of membrane vesicles: a possible role for AP-2 in endosome formation. J Cell Biol. 1992 Nov;119(4):787–796. doi: 10.1083/jcb.119.4.787. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Beck K. A., Keen J. H. Interaction of phosphoinositide cycle intermediates with the plasma membrane-associated clathrin assembly protein AP-2. J Biol Chem. 1991 Mar 5;266(7):4442–4447. [PubMed] [Google Scholar]
  5. Beck K. A., Keen J. H. Self-association of the plasma membrane-associated clathrin assembly protein AP-2. J Biol Chem. 1991 Mar 5;266(7):4437–4441. [PubMed] [Google Scholar]
  6. Beltzer J. P., Spiess M. In vitro binding of the asialoglycoprotein receptor to the beta adaptin of plasma membrane coated vesicles. EMBO J. 1991 Dec;10(12):3735–3742. doi: 10.1002/j.1460-2075.1991.tb04942.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Berridge M. J., Irvine R. F. Inositol trisphosphate, a novel second messenger in cellular signal transduction. Nature. 1984 Nov 22;312(5992):315–321. doi: 10.1038/312315a0. [DOI] [PubMed] [Google Scholar]
  8. Brodsky F. M. Living with clathrin: its role in intracellular membrane traffic. Science. 1988 Dec 9;242(4884):1396–1402. doi: 10.1126/science.2904698. [DOI] [PubMed] [Google Scholar]
  9. Collawn J. F., Stangel M., Kuhn L. A., Esekogwu V., Jing S. Q., Trowbridge I. S., Tainer J. A. Transferrin receptor internalization sequence YXRF implicates a tight turn as the structural recognition motif for endocytosis. Cell. 1990 Nov 30;63(5):1061–1072. doi: 10.1016/0092-8674(90)90509-d. [DOI] [PubMed] [Google Scholar]
  10. Fuhrer C., Geffen I., Spiess M. Endocytosis of the ASGP receptor H1 is reduced by mutation of tyrosine-5 but still occurs via coated pits. J Cell Biol. 1991 Aug;114(3):423–431. doi: 10.1083/jcb.114.3.423. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Geuze H. J., Slot J. W., Strous G. J., Peppard J., von Figura K., Hasilik A., Schwartz A. L. Intracellular receptor sorting during endocytosis: comparative immunoelectron microscopy of multiple receptors in rat liver. Cell. 1984 May;37(1):195–204. doi: 10.1016/0092-8674(84)90315-5. [DOI] [PubMed] [Google Scholar]
  12. Goldstein J. L., Brown M. S., Anderson R. G., Russell D. W., Schneider W. J. Receptor-mediated endocytosis: concepts emerging from the LDL receptor system. Annu Rev Cell Biol. 1985;1:1–39. doi: 10.1146/annurev.cb.01.110185.000245. [DOI] [PubMed] [Google Scholar]
  13. Guagliardi L. E., Koppelman B., Blum J. S., Marks M. S., Cresswell P., Brodsky F. M. Co-localization of molecules involved in antigen processing and presentation in an early endocytic compartment. Nature. 1990 Jan 11;343(6254):133–139. doi: 10.1038/343133a0. [DOI] [PubMed] [Google Scholar]
  14. Keen J. H. Clathrin and associated assembly and disassembly proteins. Annu Rev Biochem. 1990;59:415–438. doi: 10.1146/annurev.bi.59.070190.002215. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. Mahaffey D. T., Moore M. S., Brodsky F. M., Anderson R. G. Coat proteins isolated from clathrin coated vesicles can assemble into coated pits. J Cell Biol. 1989 May;108(5):1615–1624. doi: 10.1083/jcb.108.5.1615. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Mahaffey D. T., Peeler J. S., Brodsky F. M., Anderson R. G. Clathrin-coated pits contain an integral membrane protein that binds the AP-2 subunit with high affinity. J Biol Chem. 1990 Sep 25;265(27):16514–16520. [PubMed] [Google Scholar]
  18. Manfredi J. J., Bazari W. L. Purification and characterization of two distinct complexes of assembly polypeptides from calf brain coated vesicles that differ in their polypeptide composition and kinase activities. J Biol Chem. 1987 Sep 5;262(25):12182–12188. [PubMed] [Google Scholar]
  19. Moore M. S., Mahaffey D. T., Brodsky F. M., Anderson R. G. Assembly of clathrin-coated pits onto purified plasma membranes. Science. 1987 May 1;236(4801):558–563. doi: 10.1126/science.2883727. [DOI] [PubMed] [Google Scholar]
  20. Morris S. A., Mann A., Ungewickell E. Analysis of 100-180-kDa phosphoproteins in clathrin-coated vesicles from bovine brain. J Biol Chem. 1990 Feb 25;265(6):3354–3357. [PubMed] [Google Scholar]
  21. Okamoto C. T., Shia S. P., Bird C., Mostov K. E., Roth M. G. The cytoplasmic domain of the polymeric immunoglobulin receptor contains two internalization signals that are distinct from its basolateral sorting signal. J Biol Chem. 1992 May 15;267(14):9925–9932. [PubMed] [Google Scholar]
  22. 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]
  23. Peeler J. S., Donzell W. C., Anderson R. G. The appendage domain of the AP-2 subunit is not required for assembly or invagination of clathrin-coated pits. J Cell Biol. 1993 Jan;120(1):47–54. doi: 10.1083/jcb.120.1.47. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Prasad K., Keen J. H. Interaction of assembly protein AP-2 and its isolated subunits with clathrin. Biochemistry. 1991 Jun 4;30(22):5590–5597. doi: 10.1021/bi00236a036. [DOI] [PubMed] [Google Scholar]
  25. Robinson M. S. 100-kD coated vesicle proteins: molecular heterogeneity and intracellular distribution studied with monoclonal antibodies. J Cell Biol. 1987 Apr;104(4):887–895. doi: 10.1083/jcb.104.4.887. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Robinson M. S. Cloning and expression of gamma-adaptin, a component of clathrin-coated vesicles associated with the Golgi apparatus. J Cell Biol. 1990 Dec;111(6 Pt 1):2319–2326. doi: 10.1083/jcb.111.6.2319. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Robinson M. S., Kreis T. E. Recruitment of coat proteins onto Golgi membranes in intact and permeabilized cells: effects of brefeldin A and G protein activators. Cell. 1992 Apr 3;69(1):129–138. doi: 10.1016/0092-8674(92)90124-u. [DOI] [PubMed] [Google Scholar]
  28. Schröder S., Ungewickell E. Subunit interaction and function of clathrin-coated vesicle adaptors from the Golgi and the plasma membrane. J Biol Chem. 1991 Apr 25;266(12):7910–7918. [PubMed] [Google Scholar]
  29. Solari R., Kraehenbuhl J. P. Biosynthesis of the IgA antibody receptor: a model for the transepithelial sorting of a membrane glycoprotein. Cell. 1984 Jan;36(1):61–71. doi: 10.1016/0092-8674(84)90074-6. [DOI] [PubMed] [Google Scholar]
  30. 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]
  31. 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]
  32. Wong D. H., Brodsky F. M. 100-kD proteins of Golgi- and trans-Golgi network-associated coated vesicles have related but distinct membrane binding properties. J Cell Biol. 1992 Jun;117(6):1171–1179. doi: 10.1083/jcb.117.6.1171. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Zeitlin P. L., Hubbard A. L. Cell surface distribution and intracellular fate of asialoglycoproteins: a morphological and biochemical study of isolated rat hepatocytes and monolayer cultures. J Cell Biol. 1982 Mar;92(3):634–647. doi: 10.1083/jcb.92.3.634. [DOI] [PMC free article] [PubMed] [Google Scholar]

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