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J Cell Biol. 1993 August 2; 122(4): 789–807.
PMCID: PMC2119592

Signal transducing molecules and glycosyl-phosphatidylinositol-linked proteins form a caveolin-rich insoluble complex in MDCK cells


GPI-linked protein molecules become Triton-insoluble during polarized sorting to the apical cell surface of epithelial cells. These insoluble complexes, enriched in cholesterol, glycolipids, and GPI-linked proteins, have been isolated by flotation on sucrose density gradients and are thought to contain the putative GPI-sorting machinery. As the cellular origin and molecular protein components of this complex remain unknown, we have begun to characterize these low-density insoluble complexes isolated from MDCK cells. We find that these complexes, which represent 0.4-0.8% of the plasma membrane, ultrastructurally resemble caveolae and are over 150-fold enriched in a model GPI-anchored protein and caveolin, a caveolar marker protein. However, they exclude many other plasma membrane associated molecules and organelle-specific marker enzymes, suggesting that they represent microdomains of the plasma membrane. In addition to caveolin, these insoluble complexes contain a subset of hydrophobic plasma membrane proteins and cytoplasmically-oriented signaling molecules, including: (a) GTP- binding proteins--both small and heterotrimeric; (b) annex II--an apical calcium-regulated phospholipid binding protein with a demonstrated role in exocytic fusion events; (c) c-Yes--an apically localized member of the Src family of non-receptor type protein- tyrosine kinases; and (d) an unidentified serine-kinase activity. As we demonstrate that caveolin is both a transmembrane molecule and a major phospho-acceptor component of these complexes, we propose that caveolin could function as a transmembrane adaptor molecule that couples luminal GPI-linked proteins with cytoplasmically oriented signaling molecules during GPI-membrane trafficking or GPI-mediated signal transduction events. In addition, our results have implications for understanding v- Src transformation and the actions of cholera and pertussis toxins on hetero-trimeric G proteins.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Akiyama T, Ishida J, Nakagawa S, Ogawara H, Watanabe S, Itoh N, Shibuya M, Fukami Y. Genistein, a specific inhibitor of tyrosine-specific protein kinases. J Biol Chem. 1987 Apr 25;262(12):5592–5595. [PubMed]
  • Ali SM, Geisow MJ, Burgoyne RD. A role for calpactin in calcium-dependent exocytosis in adrenal chromaffin cells. Nature. 1989 Jul 27;340(6231):313–315. [PubMed]
  • Anderson RG, Pathak RK. Vesicles and cisternae in the trans Golgi apparatus of human fibroblasts are acidic compartments. Cell. 1985 Mar;40(3):635–643. [PubMed]
  • Anderson RG, Kamen BA, Rothberg KG, Lacey SW. Potocytosis: sequestration and transport of small molecules by caveolae. Science. 1992 Jan 24;255(5043):410–411. [PubMed]
  • van den Berghe N, Nieuwkoop NJ, Vaandrager AB, de Jonge HR. Asymmetrical distribution of G-proteins among the apical and basolateral membranes of rat enterocytes. Biochem J. 1991 Sep 1;278(Pt 2):565–571. [PubMed]
  • Bomsel M, Mostov K. Role of heterotrimeric G proteins in membrane traffic. Mol Biol Cell. 1992 Dec;3(12):1317–1328. [PMC free article] [PubMed]
  • Boyle WJ, van der Geer P, Hunter T. Phosphopeptide mapping and phosphoamino acid analysis by two-dimensional separation on thin-layer cellulose plates. Methods Enzymol. 1991;201:110–149. [PubMed]
  • Brada D, Dubach UC. Isolation of a homogeneous glucosidase II from pig kidney microsomes. Eur J Biochem. 1984 May 15;141(1):149–156. [PubMed]
  • Brown DA, Rose JK. Sorting of GPI-anchored proteins to glycolipid-enriched membrane subdomains during transport to the apical cell surface. Cell. 1992 Feb 7;68(3):533–544. [PubMed]
  • Brown DA, Crise B, Rose JK. Mechanism of membrane anchoring affects polarized expression of two proteins in MDCK cells. Science. 1989 Sep 29;245(4925):1499–1501. [PubMed]
  • Brugge JS, Erikson RL. Identification of a transformation-specific antigen induced by an avian sarcoma virus. Nature. 1977 Sep 22;269(5626):346–348. [PubMed]
  • Campbell KS, Hager EJ, Friedrich RJ, Cambier JC. IgM antigen receptor complex contains phosphoprotein products of B29 and mb-1 genes. Proc Natl Acad Sci U S A. 1991 May 1;88(9):3982–3986. [PubMed]
  • Chamberlain JP. Fluorographic detection of radioactivity in polyacrylamide gels with the water-soluble fluor, sodium salicylate. Anal Biochem. 1979 Sep 15;98(1):132–135. [PubMed]
  • Cross GA. Glycolipid anchoring of plasma membrane proteins. Annu Rev Cell Biol. 1990;6:1–39. [PubMed]
  • Domínguez P, Velasco G, Barros F, Lazo PS. Intestinal brush border membranes contain regulatory subunits of adenylyl cyclase. Proc Natl Acad Sci U S A. 1987 Oct;84(20):6965–6969. [PubMed]
  • Drust DS, Creutz CE. Aggregation of chromaffin granules by calpactin at micromolar levels of calcium. Nature. 1988 Jan 7;331(6151):88–91. [PubMed]
  • Dupree P, Parton RG, Raposo G, Kurzchalia TV, Simons K. Caveolae and sorting in the trans-Golgi network of epithelial cells. EMBO J. 1993 Apr;12(4):1597–1605. [PubMed]
  • Emans N, Gorvel JP, Walter C, Gerke V, Kellner R, Griffiths G, Gruenberg J. Annexin II is a major component of fusogenic endosomal vesicles. J Cell Biol. 1993 Mar;120(6):1357–1369. [PMC free article] [PubMed]
  • Ercolani L, Stow JL, Boyle JF, Holtzman EJ, Lin H, Grove JR, Ausiello DA. Membrane localization of the pertussis toxin-sensitive G-protein subunits alpha i-2 and alpha i-3 and expression of a metallothionein-alpha i-2 fusion gene in LLC-PK1 cells. Proc Natl Acad Sci U S A. 1990 Jun;87(12):4635–4639. [PubMed]
  • Fujimoto T. Calcium pump of the plasma membrane is localized in caveolae. J Cell Biol. 1993 Mar;120(5):1147–1157. [PMC free article] [PubMed]
  • Fujimoto T, Nakade S, Miyawaki A, Mikoshiba K, Ogawa K. Localization of inositol 1,4,5-trisphosphate receptor-like protein in plasmalemmal caveolae. J Cell Biol. 1992 Dec;119(6):1507–1513. [PMC free article] [PubMed]
  • Garcia M, Mirre C, Quaroni A, Reggio H, Le Bivic A. GPI-anchored proteins associate to form microdomains during their intracellular transport in Caco-2 cells. J Cell Sci. 1993 Apr;104(Pt 4):1281–1290. [PubMed]
  • Gerke V, Weber K. Identity of p36K phosphorylated upon Rous sarcoma virus transformation with a protein purified from brush borders; calcium-dependent binding to non-erythroid spectrin and F-actin. EMBO J. 1984 Jan;3(1):227–233. [PubMed]
  • Gilbert T, Le Bivic A, Quaroni A, Rodriguez-Boulan E. Microtubular organization and its involvement in the biogenetic pathways of plasma membrane proteins in Caco-2 intestinal epithelial cells. J Cell Biol. 1991 Apr;113(2):275–288. [PMC free article] [PubMed]
  • Glenney JR., Jr Tyrosine phosphorylation of a 22-kDa protein is correlated with transformation by Rous sarcoma virus. J Biol Chem. 1989 Dec 5;264(34):20163–20166. [PubMed]
  • Glenney JR., Jr Microinjection of antibodies to the calpactin I light chain in MDBK cells causes precipition of the cytoskeletal calpactin I complex without affecting the distribution of related proteins. Prog Clin Biol Res. 1990;349:135–146. [PubMed]
  • Glenney JR., Jr The sequence of human caveolin reveals identity with VIP21, a component of transport vesicles. FEBS Lett. 1992 Dec 7;314(1):45–48. [PubMed]
  • Glenney JR, Jr, Zokas L. Novel tyrosine kinase substrates from Rous sarcoma virus-transformed cells are present in the membrane skeleton. J Cell Biol. 1989 Jun;108(6):2401–2408. [PMC free article] [PubMed]
  • Glenney JR, Jr, Soppet D. Sequence and expression of caveolin, a protein component of caveolae plasma membrane domains phosphorylated on tyrosine in Rous sarcoma virus-transformed fibroblasts. Proc Natl Acad Sci U S A. 1992 Nov 1;89(21):10517–10521. [PubMed]
  • Gottesman MM, Roth C, Vlahakis G, Pastan I. Cholera toxin treatment stimulates tumorigenicity of Rous sarcoma virus-transformed cells. Mol Cell Biol. 1984 Dec;4(12):2639–2642. [PMC free article] [PubMed]
  • Grandori C, Sudol M, Hanafusa H. c-yes protein kinase is associated with a 38 kD protein in cerebellum. Oncogene. 1991 Jun;6(6):1063–1066. [PubMed]
  • Hagmann J, Fishman PH. Detergent extraction of cholera toxin and gangliosides from cultured cells and isolated membranes. Biochim Biophys Acta. 1982 Apr 29;720(2):181–187. [PubMed]
  • Hannan LA, Lisanti MP, Rodriguez-Boulan E, Edidin M. Correctly sorted molecules of a GPI-anchored protein are clustered and immobile when they arrive at the apical surface of MDCK cells. J Cell Biol. 1993 Jan;120(2):353–358. [PMC free article] [PubMed]
  • Hoessli D, Rungger-Brändle E. Association of specific cell-surface glycoproteins with a triton X-100-resistant complex of plasma membrane proteins isolated from T-lymphoma cells (P1798). Exp Cell Res. 1985 Jan;156(1):239–250. [PubMed]
  • Hooper NM, Turner AJ. Ectoenzymes of the kidney microvillar membrane. Differential solubilization by detergents can predict a glycosyl-phosphatidylinositol membrane anchor. Biochem J. 1988 Mar 15;250(3):865–869. [PubMed]
  • Huang MM, Bolen JB, Barnwell JW, Shattil SJ, Brugge JS. Membrane glycoprotein IV (CD36) is physically associated with the Fyn, Lyn, and Yes protein-tyrosine kinases in human platelets. Proc Natl Acad Sci U S A. 1991 Sep 1;88(17):7844–7848. [PubMed]
  • Kurzchalia TV, Dupree P, Parton RG, Kellner R, Virta H, Lehnert M, Simons K. VIP21, a 21-kD membrane protein is an integral component of trans-Golgi-network-derived transport vesicles. J Cell Biol. 1992 Sep;118(5):1003–1014. [PMC free article] [PubMed]
  • Le Bivic A, Quaroni A, Nichols B, Rodriguez-Boulan E. Biogenetic pathways of plasma membrane proteins in Caco-2, a human intestinal epithelial cell line. J Cell Biol. 1990 Oct;111(4):1351–1361. [PMC free article] [PubMed]
  • Lisanti MP, Sargiacomo M, Graeve L, Saltiel AR, Rodriguez-Boulan E. Polarized apical distribution of glycosyl-phosphatidylinositol-anchored proteins in a renal epithelial cell line. Proc Natl Acad Sci U S A. 1988 Dec;85(24):9557–9561. [PubMed]
  • Lisanti MP, Caras IW, Davitz MA, Rodriguez-Boulan E. A glycophospholipid membrane anchor acts as an apical targeting signal in polarized epithelial cells. J Cell Biol. 1989 Nov;109(5):2145–2156. [PMC free article] [PubMed]
  • Lisanti MP, Rodriguez-Boulan E. Glycophospholipid membrane anchoring provides clues to the mechanism of protein sorting in polarized epithelial cells. Trends Biochem Sci. 1990 Mar;15(3):113–118. [PubMed]
  • Lisanti MP, Caras IW, Gilbert T, Hanzel D, Rodriguez-Boulan E. Vectorial apical delivery and slow endocytosis of a glycolipid-anchored fusion protein in transfected MDCK cells. Proc Natl Acad Sci U S A. 1990 Oct;87(19):7419–7423. [PubMed]
  • Lisanti MP, Le Bivic A, Saltiel AR, Rodriguez-Boulan E. Preferred apical distribution of glycosyl-phosphatidylinositol (GPI) anchored proteins: a highly conserved feature of the polarized epithelial cell phenotype. J Membr Biol. 1990 Feb;113(2):155–167. [PubMed]
  • Lisanti MP, Caras IW, Rodriguez-Boulan E. Fusion proteins containing a minimal GPI-attachment signal are apically expressed in transfected MDCK cells. J Cell Sci. 1991 Jul;99(Pt 3):637–640. [PubMed]
  • Lisanti MP, Field MC, Caras IW, Menon AK, Rodriguez-Boulan E. Mannosamine, a novel inhibitor of glycosylphosphatidylinositol incorporation into proteins. EMBO J. 1991 Aug;10(8):1969–1977. [PubMed]
  • Low MG. Glycosyl-phosphatidylinositol: a versatile anchor for cell surface proteins. FASEB J. 1989 Mar;3(5):1600–1608. [PubMed]
  • Löw A, Faulhammer HG, Sprinzl M. Affinity labeling of GTP-binding proteins in cellular extracts. FEBS Lett. 1992 May 25;303(1):64–68. [PubMed]
  • Luo K, Hurley TR, Sefton BM. Transfer of proteins to membranes facilitates both cyanogen bromide cleavage and two-dimensional proteolytic mapping. Oncogene. 1990 Jun;5(6):921–923. [PubMed]
  • Melançon P. Vesicle traffic: G whizz. Curr Biol. 1993 Apr 1;3(4):230–233. [PubMed]
  • Montesano R, Roth J, Robert A, Orci L. Non-coated membrane invaginations are involved in binding and internalization of cholera and tetanus toxins. Nature. 1982 Apr 15;296(5858):651–653. [PubMed]
  • Nakata T, Sobue K, Hirokawa N. Conformational change and localization of calpactin I complex involved in exocytosis as revealed by quick-freeze, deep-etch electron microscopy and immunocytochemistry. J Cell Biol. 1990 Jan;110(1):13–25. [PMC free article] [PubMed]
  • Nigam SK, Rodriguez-Boulan E, Silver RB. Changes in intracellular calcium during the development of epithelial polarity and junctions. Proc Natl Acad Sci U S A. 1992 Jul 1;89(13):6162–6166. [PubMed]
  • Peter ME, Hall C, Rühlmann A, Sancho J, Terhorst C. The T-cell receptor zeta chain contains a GTP/GDP binding site. EMBO J. 1992 Mar;11(3):933–941. [PubMed]
  • Peters KR, Carley WW, Palade GE. Endothelial plasmalemmal vesicles have a characteristic striped bipolar surface structure. J Cell Biol. 1985 Dec;101(6):2233–2238. [PMC free article] [PubMed]
  • Pimplikar SW, Simons K. Regulation of apical transport in epithelial cells by a Gs class of heterotrimeric G protein. Nature. 1993 Apr 1;362(6419):456–458. [PubMed]
  • Radke K, Carter VC, Moss P, Dehazya P, Schliwa M, Martin GS. Membrane association of a 36,000-dalton substrate for tyrosine phosphorylation in chicken embryo fibroblasts transformed by avian sarcoma viruses. J Cell Biol. 1983 Nov;97(5 Pt 1):1601–1611. [PMC free article] [PubMed]
  • Robinson PJ, Millrain M, Antoniou J, Simpson E, Mellor AL. A glycophospholipid anchor is required for Qa-2-mediated T cell activation. Nature. 1989 Nov 2;342(6245):85–87. [PubMed]
  • Rodriguez-Boulan E. Polarized assembly of enveloped viruses from cultured epithelial cells. Methods Enzymol. 1983;98:486–501. [PubMed]
  • Rodriguez-Boulan E, Nelson WJ. Morphogenesis of the polarized epithelial cell phenotype. Science. 1989 Aug 18;245(4919):718–725. [PubMed]
  • Rodriguez-Boulan E, Powell SK. Polarity of epithelial and neuronal cells. Annu Rev Cell Biol. 1992;8:395–427. [PubMed]
  • Rothberg KG, Heuser JE, Donzell WC, Ying YS, Glenney JR, Anderson RG. Caveolin, a protein component of caveolae membrane coats. Cell. 1992 Feb 21;68(4):673–682. [PubMed]
  • Rothberg KG, Ying YS, Kamen BA, Anderson RG. Cholesterol controls the clustering of the glycophospholipid-anchored membrane receptor for 5-methyltetrahydrofolate. J Cell Biol. 1990 Dec;111(6 Pt 2):2931–2938. [PMC free article] [PubMed]
  • Sargiacomo M, Lisanti M, Graeve L, Le Bivic A, Rodriguez-Boulan E. Integral and peripheral protein composition of the apical and basolateral membrane domains in MDCK cells. J Membr Biol. 1989 Mar;107(3):277–286. [PubMed]
  • Saukkonen K, Burnette WN, Mar VL, Masure HR, Tuomanen EI. Pertussis toxin has eukaryotic-like carbohydrate recognition domains. Proc Natl Acad Sci U S A. 1992 Jan 1;89(1):118–122. [PubMed]
  • Schell MJ, Maurice M, Stieger B, Hubbard AL. 5'nucleotidase is sorted to the apical domain of hepatocytes via an indirect route. J Cell Biol. 1992 Dec;119(5):1173–1182. [PMC free article] [PubMed]
  • Severs NJ. Caveolae: static inpocketings of the plasma membrane, dynamic vesicles or plain artifact? J Cell Sci. 1988 Jul;90(Pt 3):341–348. [PubMed]
  • Shenker A, Goldsmith P, Unson CG, Spiegel AM. The G protein coupled to the thromboxane A2 receptor in human platelets is a member of the novel Gq family. J Biol Chem. 1991 May 15;266(14):9309–9313. [PubMed]
  • Shpetner HS, Vallee RB. Dynamin is a GTPase stimulated to high levels of activity by microtubules. Nature. 1992 Feb 20;355(6362):733–735. [PubMed]
  • Simionescu N, Siminoescu M, Palade GE. Permeability of muscle capillaries to small heme-peptides. Evidence for the existence of patent transendothelial channels. J Cell Biol. 1975 Mar;64(3):586–607. [PMC free article] [PubMed]
  • Simons K, Wandinger-Ness A. Polarized sorting in epithelia. Cell. 1990 Jul 27;62(2):207–210. [PubMed]
  • Stefanová I, Horejsí V, Ansotegui IJ, Knapp W, Stockinger H. GPI-anchored cell-surface molecules complexed to protein tyrosine kinases. Science. 1991 Nov 15;254(5034):1016–1019. [PubMed]
  • Stow JL, de Almeida JB, Narula N, Holtzman EJ, Ercolani L, Ausiello DA. A heterotrimeric G protein, G alpha i-3, on Golgi membranes regulates the secretion of a heparan sulfate proteoglycan in LLC-PK1 epithelial cells. J Cell Biol. 1991 Sep;114(6):1113–1124. [PMC free article] [PubMed]
  • Streuli CH, Patel B, Critchley DR. The cholera toxin receptor ganglioside GM remains associated with triton X-100 cytoskeletons of BALB/c-3T3 cells. Exp Cell Res. 1981 Dec;136(2):247–254. [PubMed]
  • Su B, Waneck GL, Flavell RA, Bothwell AL. The glycosyl phosphatidylinositol anchor is critical for Ly-6A/E-mediated T cell activation. J Cell Biol. 1991 Feb;112(3):377–384. [PMC free article] [PubMed]
  • Sudol M, Hanafusa H. Cellular proteins homologous to the viral yes gene product. Mol Cell Biol. 1986 Aug;6(8):2839–2846. [PMC free article] [PubMed]
  • Thomas PM, Samelson LE. The glycophosphatidylinositol-anchored Thy-1 molecule interacts with the p60fyn protein tyrosine kinase in T cells. J Biol Chem. 1992 Jun 15;267(17):12317–12322. [PubMed]
  • Thompson JA, Lau AL, Cunningham DD. Selective radiolabeling of cell surface proteins to a high specific activity. Biochemistry. 1987 Feb 10;26(3):743–750. [PubMed]
  • Vidugiriene J, Menon AK. Early lipid intermediates in glycosyl-phosphatidylinositol anchor assembly are synthesized in the ER and located in the cytoplasmic leaflet of the ER membrane bilayer. J Cell Biol. 1993 Jun;121(5):987–996. [PMC free article] [PubMed]
  • Wandinger-Ness A, Bennett MK, 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. [PMC free article] [PubMed]
  • Yu J, Fischman DA, Steck TL. Selective solubilization of proteins and phospholipids from red blood cell membranes by nonionic detergents. J Supramol Struct. 1973;1(3):233–248. [PubMed]
  • Zachary I, Sinnett-Smith J, Rozengurt E. Stimulation of tyrosine kinase activity in anti-phosphotyrosine immune complexes of Swiss 3T3 cell lysates occurs rapidly after addition of bombesin, vasopressin, and endothelin to intact cells. J Biol Chem. 1991 Dec 15;266(35):24126–24133. [PubMed]
  • Zhao YH, Krueger JG, Sudol M. Expression of cellular-yes protein in mammalian tissues. Oncogene. 1990 Nov;5(11):1629–1635. [PubMed]
  • Zurzolo C, Lisanti MP, Caras IW, Nitsch L, Rodriguez-Boulan E. Glycosylphosphatidylinositol-anchored proteins are preferentially targeted to the basolateral surface in Fischer rat thyroid epithelial cells. J Cell Biol. 1993 Jun;121(5):1031–1039. [PMC free article] [PubMed]

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