Modulation of TGF-beta type 1 receptor: flow cytometric detection with biotinylated TGF-beta.
Transforming growth factor beta type 1 (TGF-beta 1) was reacted with NHS-biotin to yield a derivative of TGF-beta 1 which was biotinylated on lysine residues. The biotinylated form of TGF-beta 1 was separated from the unreacted material by reverse phase chromatography. In three separate bioassays, the derivatized peptide was as active as the starting material. The use of FITC-avidin in conjunction with flow cytometry demonstrated that the binding of biotinylated TGF-beta 1 to its receptor is saturable, competable, and specific. A 100-fold molar excess of underivatized TGF-beta 1 gave 85% inhibition of binding of the biotinylated peptide to the mink lung cell line CCL-64, while TGF-beta 2 showed no inhibition of binding, nor did insulin, calcitonin, or TGF-alpha. Both CCL-64 cells and human umbilical vein endothelial cells showed a density-dependent down-regulation of receptor expression in culture. Several factors were examined that might mediate this effect. The down-regulation was shown not to be due to the secretion of an active form of TGF-beta 1. The extracellular matrix from high-density cells did not decrease expression of the receptor. Fibronectin, collagen, and gelatin were also unable to signal changes in receptor expression, even though in other systems such matrix components can regulate the responsiveness of cells to TGF-beta 1. Lastly, staining simultaneously for DNA content and TGF-beta 1 receptor expression showed that there was no correlation between cell cycle and receptor levels.
Newman
W
W
Department of Immunology, Otsuka Pharmaceutical Co., Rockville, Maryland 20850.
Beall
L D
LD
Bertolini
D R
DR
Cone
J L
JL
D000818:Animals; D001710:Biotin; D002453:Cell Cycle; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000378:metabolism; D004730:Endothelium, Vascular / Q000166:cytology; D005109:Extracellular Matrix / Q000502:physiology; D005434:Flow Cytometry / Q000379:methods; D007375:Interleukin-1 / Q000494:pharmacology; D008907:Mink; D011956:Receptors, Cell Surface / Q000502:physiology*; D018125:Receptors, Transforming Growth Factor beta; D015291:Transforming Growth Factors / Q000502:physiology*
D007375:Interleukin-1; D011956:Receptors, Cell Surface; D018125:Receptors, Transforming Growth Factor beta; D001710:Biotin; D015291:Transforming Growth Factors; D004247:DNA