Home LiteratureArticle Details
PMID: 8027172 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

Purification and characterization of smooth muscle cell caveolae.

The Journal of cell biology ·Vol. 126 ·No. 1 ·1994-07-00 ·Pages 127-38

Chang WJ, Ying YS, Rothberg KG, Hooper NM, Turner AJ, Gambliel HA, De Gunzburg J, Mumby SM, Gilman AG, Anderson RG

Abstract

Plasmalemmal caveolae are a membrane specialization that mediates transcytosis across endothelial cells and the uptake of small molecules and ions by both epithelial and connective tissue cells. Recent findings suggest that caveolae may, in addition, be involved in signal transduction. To better understand the molecular composition of this membrane specialization, we have developed a biochemical method for purifying caveolae from chicken smooth muscle cells. Biochemical and morphological markers indicate that we can obtain approximately 1.5 mg of protein in the caveolae fraction from approximately 100 g of chicken gizzard. Gel electrophoresis shows that there are more than 30 proteins enriched in caveolae relative to the plasma membrane. Among these proteins are: caveolin, a structural molecule of the caveolae coat; multiple, glycosylphosphatidylinositol-anchored membrane proteins; both G alpha and G beta subunits of heterotrimeric GTP-binding protein; and the Ras-related GTP-binding protein, Rap1A/B. The method we have developed will facilitate future studies on the structure and function of caveolae.

MeSH Terms
Animals Caveolin 1 Caveolins Cell Compartmentation Cell Fractionation/methods Cell Membrane/chemistry,ultrastructure Chickens GTP-Binding Proteins/isolation & purification Gizzard, Avian/cytology Glycosylphosphatidylinositols Immunohistochemistry Membrane Proteins/chemistry Microscopy, Immunoelectron Muscle, Smooth/chemistry,ultrastructure Subcellular Fractions/chemistry,ultrastructure
Chemicals
Caveolin 1 Caveolins Glycosylphosphatidylinositols Membrane Proteins GTP-Binding Proteins
Authors & Affiliations
10 authors, click to expand affiliations / ORCID
Chang W J
Department of Cell Biology and Neuroscience, University of Texas Southwestern Medical Center at Dallas 75235.
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
References (49)
49 references, click to expand
  1. VIP21, a 21-kD membrane protein is an integral component of trans-Golgi-network-derived transport vesicles.
    J Cell Biol. 1992 Sep;118(5):1003-14 PMID: 1512286
  2. Localization of inositol 1,4,5-trisphosphate receptor-like protein in plasmalemmal caveolae.
    J Cell Biol. 1992 Dec;119(6):1507-13 PMID: 1334960
  3. The nature of large noncovalent complexes containing glycosyl-phosphatidylinositol-anchored membrane glycoproteins and protein tyrosine kinases.
    J Immunol. 1992 Oct 1;149(7):2262-70 PMID: 1382093
  4. Isolation of two proteins with high affinity for guanine nucleotides from membranes of bovine brain.
    J Biol Chem. 1984 Nov 25;259(22):13806-13 PMID: 6438083
  5. Novel tyrosine kinase substrates from Rous sarcoma virus-transformed cells are present in the membrane skeleton.
    J Cell Biol. 1989 Jun;108(6):2401-8 PMID: 2472406
  6. Palmitylation of an amino-terminal cysteine motif of protein tyrosine kinases p56lck and p59fyn mediates interaction with glycosyl-phosphatidylinositol-anchored proteins.
    Mol Cell Biol. 1993 Oct;13(10):6385-92 PMID: 8413237
  7. Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
    Nature. 1970 Aug 15;227(5259):680-5 PMID: 5432063
  8. Permeability of intestinal capillaries. Pathway followed by dextrans and glycogens.
    J Cell Biol. 1972 May;53(2):365-92 PMID: 4112540
  9. Specific binding of [alpha-32P]GTP to cytosolic and membrane-bound proteins of human platelets correlates with the activation of phospholipase C.
    Proc Natl Acad Sci U S A. 1987 Apr;84(8):2261-5 PMID: 3470789
  10. Inositol glycan mimics the action of insulin on glucose utilization in rat adipocytes.
    Biochem Biophys Res Commun. 1987 Dec 31;149(3):1084-92 PMID: 3322285
  11. Use of dinitrophenol-IgG conjugates to detect sparse antigens by immunogold labeling.
    J Histochem Cytochem. 1989 Jan;37(1):69-74 PMID: 2491753
  12. A ras-related gene with transformation suppressor activity.
    Cell. 1989 Jan 13;56(1):77-84 PMID: 2642744
  13. Characterization of antibodies to the glycosyl-phosphatidylinositol membrane anchors of mammalian proteins.
    Biochem J. 1991 Jan 15;273(Pt 2):301-6 PMID: 1703760
  14. Detergent insolubility of alkaline phosphatase during biosynthetic transport and endocytosis. Role of cholesterol.
    J Biol Chem. 1993 Feb 15;268(5):3150-5 PMID: 8428991
  15. Regulation of the cytoplasmic accumulation of 5-methyltetrahydrofolate in MA104 cells is independent of folate receptor regulation.
    J Clin Invest. 1989 Nov;84(5):1379-86 PMID: 2478584
  16. The glycophospholipid-linked folate receptor internalizes folate without entering the clathrin-coated pit endocytic pathway.
    J Cell Biol. 1990 Mar;110(3):637-49 PMID: 1968465
  17. Evidence for a role of rap1 protein in the regulation of human platelet Ca2+ fluxes.
    Biochem J. 1992 Jan 15;281 ( Pt 2):325-31 PMID: 1310590
  18. Cholesterol controls the clustering of the glycophospholipid-anchored membrane receptor for 5-methyltetrahydrofolate.
    J Cell Biol. 1990 Dec;111(6 Pt 2):2931-8 PMID: 2148564
  19. Potocytosis: sequestration and transport of small molecules by caveolae.
    Science. 1992 Jan 24;255(5043):410-1 PMID: 1310359
  20. Isolation of rat hepatocyte plasma membranes. I. Presence of the three major domains.
    J Cell Biol. 1983 Jan;96(1):217-29 PMID: 6298249
  21. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.
    Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350-4 PMID: 388439
  22. Endothelial plasmalemmal vesicles have a characteristic striped bipolar surface structure.
    J Cell Biol. 1985 Dec;101(6):2233-8 PMID: 4066756
  23. 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 PMID: 239003
  24. Human cDNAs rap1 and rap2 homologous to the Drosophila gene Dras3 encode proteins closely related to ras in the 'effector' region.
    Oncogene. 1988 Aug;3(2):201-4 PMID: 3045729
  25. The glycophosphatidylinositol-anchored Thy-1 molecule interacts with the p60fyn protein tyrosine kinase in T cells.
    J Biol Chem. 1992 Jun 15;267(17):12317-22 PMID: 1351058
  26. Assessment of transport capacity of plasmalemmal Ca2+ pump in smooth muscle.
    Am J Physiol. 1988 Aug;255(2 Pt 1):C226-36 PMID: 3136657
  27. The folate receptor works in tandem with a probenecid-sensitive carrier in MA104 cells in vitro.
    J Clin Invest. 1991 Apr;87(4):1442-9 PMID: 1849150
  28. A novel N-terminal motif for palmitoylation of G-protein alpha subunits.
    Biochem J. 1993 Apr 15;291 ( Pt 2):349-53 PMID: 8484716
  29. The fine structure of the gall bladder epithelium of the mouse.
    J Biophys Biochem Cytol. 1955 Sep 25;1(5):445-58 PMID: 13263332
  30. Complementary DNA for the folate binding protein correctly predicts anchoring to the membrane by glycosyl-phosphatidylinositol.
    J Clin Invest. 1989 Aug;84(2):715-20 PMID: 2527252
  31. Palmitoylation is required for signaling functions and membrane attachment of Gq alpha and Gs alpha.
    J Biol Chem. 1993 Nov 25;268(33):25001-8 PMID: 8227063
  32. Signal transduction through decay-accelerating factor. Interaction of glycosyl-phosphatidylinositol anchor and protein tyrosine kinases p56lck and p59fyn 1.
    J Immunol. 1992 Dec 1;149(11):3535-41 PMID: 1385527
  33. Lowering the cholesterol content of MA104 cells inhibits receptor-mediated transport of folate.
    J Cell Biol. 1992 Jul;118(1):63-9 PMID: 1618907
  34. Caveolae and sorting in the trans-Golgi network of epithelial cells.
    EMBO J. 1993 Apr;12(4):1597-605 PMID: 8385608
  35. GPI-anchored cell-surface molecules complexed to protein tyrosine kinases.
    Science. 1991 Nov 15;254(5034):1016-9 PMID: 1719635
  36. Potocytosis of small molecules and ions by caveolae.
    Trends Cell Biol. 1993 Mar;3(3):69-72 PMID: 14731772
  37. Lipid modifications of G proteins: alpha subunits are palmitoylated.
    Proc Natl Acad Sci U S A. 1993 Apr 15;90(8):3675-9 PMID: 8475115
  38. 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):13602-9 PMID: 3417674
  39. Calcium pump of the plasma membrane is localized in caveolae.
    J Cell Biol. 1993 Mar;120(5):1147-57 PMID: 8382206
  40. Each caveola contains multiple glycosyl-phosphatidylinositol-anchored membrane proteins.
    Cold Spring Harb Symp Quant Biol. 1992;57:593-604 PMID: 1364076
  41. Tyrosine phosphorylation of a 22-kDa protein is correlated with transformation by Rous sarcoma virus.
    J Biol Chem. 1989 Dec 5;264(34):20163-6 PMID: 2479645
  42. Phosphatidylinositol-glycan anchors of membrane proteins: potential precursors of insulin mediators.
    Science. 1988 Apr 22;240(4851):509-11 PMID: 3282305
  43. Protein measurement with the Folin phenol reagent.
    J Biol Chem. 1951 Nov;193(1):265-75 PMID: 14907713
  44. 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):9557-61 PMID: 2974157
  45. Caveolin, a protein component of caveolae membrane coats.
    Cell. 1992 Feb 21;68(4):673-82 PMID: 1739974
  46. Synthetic peptide antisera with determined specificity for G protein alpha or beta subunits.
    Methods Enzymol. 1991;195:215-33 PMID: 1903488
  47. The G protein alpha s subunit incorporates [3H]palmitic acid and mutation of cysteine-3 prevents this modification.
    Biochemistry. 1993 Aug 17;32(32):8057-61 PMID: 8347607
  48. Association of the Ras-antagonistic Rap1/Krev-1 proteins with the Golgi complex.
    Proc Natl Acad Sci U S A. 1991 Mar 1;88(5):1606-10 PMID: 1900364
  49. 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 PMID: 8349730
Article Info
Journal
The Journal of cell biology
Abbr.
J Cell Biol
ISSN
0021-9525
DOI
10.1083/jcb.126.1.127
Published
1994-07-00
Pages
127-38
Language
English
Region
United States
NLM ID
0375356
PMCID
PMC2120085
Subset
IM
Grants
NIGMS NIH HHS · GM 34497 · United States
NIGMS NIH HHS · GM 43169 · United States
NHLBI NIH HHS · HL 20948 · United States
External Links
PubMed source DOI: 10.1083/jcb.126.1.127 PMC Full Text View journal details
Analysis Services
Analysis Services

Contact

No. 2 Wenbo Road, Zhangqiu District, Jinan, Shandong

Qilu Normal University · Genelibs Bioinformatics Lab

750 Shunhua Rd, Jinan

2F, Bldg F, University Science Park

Tel: 0531-88819269

WeChat Official Account

Follow our WeChat subscription account for real-time updates and the latest in medical and biological research.


Business Email

E-mail: [email protected]

Copyright © 2026.Genelibs Jinan Honghui Biotechnology Co., Ltd. 鲁ICP备20007159号-2