Insulin/FGF-binding ciliary membrane glycoprotein from Tetrahymena.
Triton X-100 extracted ciliary membrane protein from isolated cilia, prepared from the protozoon Tetrahymena thermophila, were fractionated by affinity chromatography on columns with covalently bound fibroblast growth factor (FGF), insulin, or concanavalin A (ConA), respectively. The eluted proteins were further analyzed by electrophoresis on sodium dodecyl sulfate polyacrylamide gels, isoelectric focusing, and by immunoblotting techniques using antibodies against the FGF receptor, platetelet derived growth factor (PDGF) receptor alpha-subunit, and insulin receptor beta-subunit. The particular antibodies were chosen because the peptides PDGF, FGF, insulin, and ConA are chemoattractants in this organism and corresponding binding (receptor) proteins could be expected to be identified. A 66 kDa protein fraction was eluted from the FGF-MiniLeak agarose, insulin-MiniLeak agarose and ConA sepharose. This fraction responded in Western immunoblots to an antibody against the beta-subunit of the human insulin receptor, to an antibody against the PDGF receptor (PDGFR) and also to an antibody against the bovine FGF receptor (FGFR) that is known, in other systems, to inhibit FGF binding to its receptor. When analyzed by SDS-PAGE and stained with Coomassie blue the 66 kDa fraction appeared as a single component. However, in some experiments it appeared more heterogeneous when stained with silver indicating the presence of minor components that may be a procedural artifact or isoforms of the same glycoprotein. The 66 kDa protein(s) migrated in isoelectric focusing with a pI of 7.4. The results are discussed in terms of the possible role of the 66 kDa glycoprotein as a protein involved in peptide-mediated cell signalling.
Leick
V
V
Institute of Medical Biochemistry and Genetics Biochemistry Laboratory, Department B, University of Copenhagen, The Panum Institute, DK-2200 Copenhagen N, Denmark.
Bøg-Hansen
T C
TC
Juhl
H A
HA
D000818:Animals; D000918:Antibody Specificity; D015153:Blotting, Western; D002417:Cattle; D002458:Cell Fractionation; D002633:Chemotaxis / Q000502:physiology; D002846:Chromatography, Affinity; D002923:Cilia / Q000737:chemistry*; D003208:Concanavalin A / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000276:immunology; D005346:Fibroblast Growth Factors / Q000378:metabolism; D006801:Humans; D007328:Insulin / Q000378:metabolism; D007525:Isoelectric Focusing; D008565:Membrane Proteins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D051379:Mice; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D015800:Protozoan Proteins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D011972:Receptor, Insulin / Q000276:immunology; D020796:Receptor, Platelet-Derived Growth Factor alpha / Q000276:immunology; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D017468:Receptors, Fibroblast Growth Factor / Q000276:immunology; D016622:Silver Staining; D016808:Tetrahymena thermophila / Q000737:chemistry* / Q000276:immunology / Q000502:physiology
C434319:66 KDa protein, Tetrahymena thermophila; D000939:Epitopes; D007328:Insulin; D008565:Membrane Proteins; D015800:Protozoan Proteins; D011956:Receptors, Cell Surface; D017468:Receptors, Fibroblast Growth Factor; D003208:Concanavalin A; D005346:Fibroblast Growth Factors; D011972:Receptor, Insulin; D020796:Receptor, Platelet-Derived Growth Factor alpha