Home LiteratureArticle Details
PMID: 3063259 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

Identification of the glucose transporter in mammalian cell membranes with a 125I-forskolin photoaffinity label.

The Biochemical journal ·Vol. 255 ·No. 3 ·1988-11-01 ·Pages 983-90

Wadzinski BE, Shanahan MF, Clark RB, Ruoho AE

Abstract

The glucose transporter has been identified in a variety of mammalian cell membranes using a photoactivatable carrier-free radioiodinated derivative of forskolin, 3-[125I]iodo-4-azidophenethylamido-7-O-succinyldeacetylforskoli n ([125I]IAPS-forskolin) at 1-3 nM. The membranes that were photolabelled with [125I]IAPS-forskolin were human placental membranes, rat cortical and cerebellar synaptic membranes, rat cardiac sarcolemmal membranes, rat adipocyte plasma membranes, smooth-muscle membranes, and S49 wild-type (WT) lymphoma-cell membranes. The glucose transporter in plasma membranes prepared from the insulin-responsive rat cardiac sarcolemmal cells, rat adipocytes and smooth-muscle cells were determined to be approx. 45 kDa by SDS/polyacrylamide-gel electrophoresis (PAGE). Photolysis of human placental membranes, rat cortical and cerebellar synaptic membranes, and WT lymphoma membranes with [125I]-IAPS-forskolin, followed by SDS/PAGE, indicated specific derivatization of a broad band (43-55 kDa) in placental membranes and a narrower band (approx. 45 kDa) in synaptic membranes and WT lymphoma membranes. Digestion of the [125I]IAPS-forskolin-labelled placental and WT lymphoma membranes with endo-beta-galactosidase showed a reduction in the apparent molecular mass of the radiolabelled band to approx. 40 kDa. The membranes that were photolabelled with [125I]IAPS-forskolin and trypsin-treated produced a radiolabelled proteolytic fragment with an apparent molecular mass of 18 kDa. [125I]IAPS-forskolin is a highly effective probe for identifying low levels of glucose transporters in mammalian tissues.

MeSH Terms
Affinity Labels Animals Azides Carrier Proteins/metabolism Cell Line Cell Membrane/drug effects,metabolism Colforsin/analogs & derivatives Diterpenes Electrophoresis, Polyacrylamide Gel Female Glucose/metabolism Humans Insulin/pharmacology Iodine Radioisotopes Lymphoma/metabolism Membrane Proteins/metabolism Placenta/metabolism Rats Synaptic Membranes/metabolism Tumor Cells, Cultured/metabolism
Chemicals
Affinity Labels Azides Carrier Proteins Diterpenes Insulin Iodine Radioisotopes Membrane Proteins 3-iodo-4-azidophenethylamido-7-O-succinyldeacetylforskolin Colforsin Glucose
Authors & Affiliations
4 authors, click to expand affiliations / ORCID
Wadzinski B E
Department of Pharmacology, University of Wisconsin Medical School, Madison 53706.
Shanahan M F
Clark R B
Ruoho A E
References (58)
58 references, click to expand
  1. Inhibition of 3-O-methylglucose transport in human erythrocytes by forskolin.
    J Biol Chem. 1985 Nov 25;260(27):14677-82 PMID: 2997220
  2. Derivatization of the human erythrocyte glucose transporter using a novel forskolin photoaffinity label.
    J Biol Chem. 1987 Dec 25;262(36):17683-9 PMID: 3693367
  3. Glucose transport in human platelets and its inhibition by forskolin.
    J Pharmacol Exp Ther. 1986 Mar;236(3):585-9 PMID: 3005547
  4. Distribution of the glucose transporter in the mammalian brain.
    J Neurochem. 1986 May;46(5):1406-11 PMID: 3958712
  5. Interactions of insulin, catecholamines and adenosine in the regulation of glucose transport in isolated rat cardiac myocytes.
    Biochim Biophys Acta. 1986 Jun 16;887(1):121-9 PMID: 3518811
  6. Insulin-stimulated glucose transport in rat adipose cells. Modulation of transporter intrinsic activity by isoproterenol and adenosine.
    J Biol Chem. 1986 Aug 5;261(22):10033-6 PMID: 3525539
  7. Reversible association of cytochalasin B with the human erythrocyte membrane. Inhibition of glucose transport and the stoichiometry of cytochalasin binding.
    Biochim Biophys Acta. 1973 Oct 11;323(2):207-19 PMID: 4752283
  8. Isolation and partial characterization of rat brain synaptic plasma membranes.
    J Neurochem. 1974 Feb;22(2):281-90 PMID: 4364339
  9. Relationship between the beta-adrenergic receptor and adenylate cyclase.
    J Biol Chem. 1977 Aug 25;252(16):5761-75 PMID: 195960
  10. Purification and reconstitution of the adipocyte plasma membrane D-glucose transport system.
    J Biol Chem. 1977 Dec 10;252(23):8341-3 PMID: 924997
  11. Mechanism of insulin action on glucose transport in the isolated rat adipose cell. Enhancement of the number of functional transport systems.
    J Biol Chem. 1978 Nov 25;253(22):8002-5 PMID: 711732
  12. The monosaccharide transporter from human erythrocytes is heterogeneously glycosylated.
    Biochem Biophys Res Commun. 1979 Dec 14;91(3):955-61 PMID: 118753
  13. Evidence that insulin causes translocation of glucose transport activity to the plasma membrane from an intracellular storage site.
    Proc Natl Acad Sci U S A. 1980 May;77(5):2542-5 PMID: 6771756
  14. Genetic analysis of hormone-sensitive adenylate cyclase.
    Adv Cyclic Nucleotide Res. 1980;13:1-37 PMID: 6251703
  15. Unmasking effect of alamethicin on the (Na+,K+)-ATPase, beta-adrenergic receptor-coupled adenylate cyclase, and cAMP-dependent protein kinase activities of cardiac sarcolemmal vesicles.
    J Biol Chem. 1980 Oct 25;255(20):9971-80 PMID: 6253461
  16. Monosaccharide transport proteins of the human erythrocyte membrane.
    Biochim Biophys Acta. 1981 Jun 16;650(1):1-20 PMID: 7196262
  17. Photoaffinity labeling of the human erythrocyte D-glucose transporter.
    J Biol Chem. 1982 May 25;257(10):5419-25 PMID: 7200092
  18. Cytochalasin B. A natural photoaffinity ligand for labeling the human erythrocyte glucose transporter.
    J Biol Chem. 1982 Jul 10;257(13):7290-3 PMID: 7200980
  19. Detection of the rat adipose cell glucose transporter with antibody against the human red cell glucose transporter.
    Biochem Biophys Res Commun. 1982 Mar 15;105(1):89-95 PMID: 7046746
  20. Immunological identification of an insulin-responsive glucose transporter.
    Biochem Biophys Res Commun. 1982 Apr 14;105(3):1150-6 PMID: 7046752
  21. An improved method of reconstitution of adipocyte glucose transport activity.
    Anal Biochem. 1982 May 1;122(1):10-9 PMID: 7103076
  22. Photolabeling of glucose-sensitive cytochalasin B binding proteins in erythrocyte, fibroblast and adipocyte membranes.
    Biochem Biophys Res Commun. 1982 Jul 16;107(1):38-43 PMID: 6289825
  23. Identification of the glucose transport protein of the microvillous membrane of human placenta by photoaffinity labelling.
    Biochem Biophys Res Commun. 1982 Nov 30;109(2):408-13 PMID: 6891216
  24. Isolated cardiac myocytes. A new cellular model for studying insulin modulation of monosaccharide transport.
    Biochim Biophys Acta. 1982 Dec 30;721(4):385-93 PMID: 6760900
  25. D-Glucose-sensitive and -insensitive cytochalasin B binding proteins from microvillous plasma membranes of human placenta. Identification of the D-glucose transporter.
    Biochim Biophys Acta. 1983 Apr 21;730(1):57-63 PMID: 6681985
  26. Cloning and characterization of a cDNA encoding the rat brain glucose-transporter protein.
    Proc Natl Acad Sci U S A. 1986 Aug;83(16):5784-8 PMID: 3016720
  27. Improved methodology for analysis and quantitation of proteins on one-dimensional silver-stained slab gels.
    Anal Biochem. 1983 Mar;129(2):277-87 PMID: 6189421
  28. Photoaffinity labeling of the beta-adrenergic receptor from cultured lymphoma cells with [125I]iodoazidobenzylpindolol: loss of the label with desensitization.
    Proc Natl Acad Sci U S A. 1983 May;80(10):2849-53 PMID: 6134281
  29. Characterization of cytochalasin B photoincorporation into human erythrocyte D-glucose transporter and F-actin.
    Biochemistry. 1983 May 24;22(11):2750-6 PMID: 6683567
  30. Isolation and partial characterization of the basal cell membrane of human placental trophoblast.
    Biochim Biophys Acta. 1983 Sep 21;734(1):91-8 PMID: 6615829
  31. Identification of the glucose transporter in rat skeletal muscle.
    Arch Biochem Biophys. 1983 Oct 1;226(1):198-205 PMID: 6685458
  32. The regulation of glucose transport by cAMP stimulators via three different mechanisms in rat and human adipocytes.
    J Biol Chem. 1983 Nov 25;258(22):13685-92 PMID: 6196354
  33. Insulin-stimulated translocation of glucose transporters in the isolated rat adipose cells: characterization of subcellular fractions.
    Biochim Biophys Acta. 1983 Dec 19;763(4):393-407 PMID: 6360220
  34. Proteolytic and chemical dissection of the human erythrocyte glucose transporter.
    Biochem J. 1984 Jul 1;221(1):179-88 PMID: 6431970
  35. Proteolytic cleavages of cytochalasin B binding components of band 4.5 proteins of the human red blood cell membrane.
    Biochim Biophys Acta. 1984 Sep 19;776(1):10-20 PMID: 6541055
  36. Insulin action on glucose transport in cardiac muscle.
    J Biol Chem. 1984 Nov 10;259(21):13117-22 PMID: 6386808
  37. Orientation of the glucose transporter in the human erythrocyte membrane. Investigation by in situ proteolytic dissection.
    J Biol Chem. 1984 Nov 25;259(22):13878-84 PMID: 6150039
  38. Identification and characterization of the glucose transporter of the blood-brain barrier by cytochalasin B binding and immunological reactivity.
    Proc Natl Acad Sci U S A. 1984 Nov;81(22):7233-7 PMID: 6150484
  39. Characterization of a photosensitive glucose derivative. A photoaffinity reagent for the erythrocyte hexose transporter.
    Biochim Biophys Acta. 1985 Jan 25;812(2):503-11 PMID: 3838144
  40. The glucose transporter of mammalian cells.
    Annu Rev Physiol. 1985;47:503-17 PMID: 3888079
  41. Phorbol ester provokes insulin-like effects on glucose transport, amino acid uptake, and pyruvate dehydrogenase activity in BC3H-1 cultured myocytes.
    Endocrinology. 1985 Jun;116(6):2650-5 PMID: 3158511
  42. A D-glucose-sensitive cytochalasin B binding component of cerebral microvessels.
    J Neurochem. 1985 Aug;45(2):650-2 PMID: 4009179
  43. Sequence and structure of a human glucose transporter.
    Science. 1985 Sep 6;229(4717):941-5 PMID: 3839598
  44. Reconstitution of the glucose transporter from bovine heart.
    Biochim Biophys Acta. 1985 Aug 27;818(2):171-82 PMID: 4040772
  45. Photoaffinity labeling of the human erythrocyte monosaccharide transporter with an aryl azide derivative of D-glucose.
    J Biol Chem. 1985 Sep 15;260(20):10897-900 PMID: 4040910
  46. The topology of the major band 4.5 protein component of the human erythrocyte membrane: characterization of reactive cysteine residues.
    Biochim Biophys Acta. 1985 Sep 25;819(1):83-92 PMID: 4041454
  47. Facilitated glucose transporter of human erythrocyte: proteolytic mapping of the [3H]cytochalasin B photoaffinity-labeled transporter polypeptide.
    Biochim Biophys Acta. 1985 Nov 8;832(1):14-21 PMID: 3902090
  48. Hormonal regulation of mammalian glucose transport.
    Annu Rev Biochem. 1986;55:1059-89 PMID: 3527041
  49. Regulation of insulin-stimulated glucose transport in the isolated rat adipocyte. cAMP-independent effects of lipolytic and antilipolytic agents.
    J Biol Chem. 1987 Jan 5;262(1):245-53 PMID: 3025204
  50. Desensitization of hormonal stimuli coupled to regulation of cyclic AMP levels.
    Adv Cyclic Nucleotide Protein Phosphorylation Res. 1986;20:151-209 PMID: 3028084
  51. Chemical and genetic comparison of the glucose and nucleoside transporters.
    Biochem Cell Biol. 1986 Nov;64(11):1170-80 PMID: 3828109
  52. Photolabeling of the adipocyte hexose carrier with an aryl azide derivative of maltose.
    Mol Cell Endocrinol. 1987 Feb;49(2-3):181-8 PMID: 3556753
  53. Forskolin inhibits insulin-stimulated glucose transport in rat adipose cells by a direct interaction with the glucose transporter.
    Mol Pharmacol. 1987 Mar;31(3):279-83 PMID: 3470598
  54. Molecular characteristics of rat brain glucose transporter: a novel species with Mr 45,000.
    J Biochem. 1987 Jan;101(1):43-52 PMID: 3571209
  55. [3H]forskolin. Direct photoaffinity labeling of the erythrocyte D-glucose transporter.
    J Biol Chem. 1987 May 5;262(13):5978-84 PMID: 3106349
  56. Beta-adrenergic receptor desensitization of wild-type but not cyc lymphoma cells unmasked by submillimolar Mg2+.
    FASEB J. 1987 Oct;1(4):289-97 PMID: 2820824
  57. Evidence that forskolin binds to the glucose transporter of human erythrocytes.
    J Biol Chem. 1987 Oct 25;262(30):14571-5 PMID: 3667590
  58. Exofacial photoaffinity labelling of the human erythrocyte sugar transporter.
    Biochim Biophys Acta. 1986 Feb 13;855(1):115-26 PMID: 3753652
Article Info
Journal
The Biochemical journal
Abbr.
Biochem J
ISSN
0264-6021
DOI
10.1042/bj2550983
Published
1988-11-01
Pages
983-90
Language
English
Region
England
NLM ID
2984726R
PMCID
PMC1135338
Subset
IM
Grants
NIDDK NIH HHS · DK 36855 · United States
NIGMS NIH HHS · GM 33138 · United States
NHLBI NIH HHS · HL 36070 · United States
External Links
PubMed source DOI: 10.1042/bj2550983 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