Transforming growth factor-beta type-II receptor signalling: intrinsic/associated casein kinase activity, receptor interactions and functional effects of blocking antibodies.
The transforming growth factor beta (TGF-beta) family of growth factors control proliferation, extracellular matrix synthesis and/ or differentiation in a wide variety of cells. However, the molecular mechanisms governing ligand binding, receptor oligomerization and signal transduction remain incompletely understood. In this study, we utilized a set of antibodies selective for the extracellular and intracellular domains of the TGF-beta type-II receptor as probes to investigate the intrinsic kinase activity of this receptor and its physical association in multimeric complexes with type-I and type-III receptors. The type-II receptor immuno-precipitated from human osteosarcoma cells exhibited autophosphorylation and casein kinase activity that was markedly stimulated by polylysine yet was insensitive to heparin. Affinity cross-linking of 125I-TGF-beta 1 ligand to cellular receptors followed by specific immunoprecipitation demonstrated that type-II receptors form stable complexes with both type-I and type-III receptors expressed on the surfaces of both human osteosarcoma cells and rabbit chondrocytes. Pretreatment of the cultured cells with an antibody directed against a distinct extracellular segment of the type-II receptor (anti-TGF-beta-IIR-NT) effectively blocked the 125I-TGF-beta labelling of type-I receptors without preventing the affinity labelling of type-II or type-III receptors, indicating a selective disruption of the type-I/type-II hetero-oligomers. The anti-TGF-beta-IIR-NT antibodies also blocked the TGF-beta-dependent induction of the plasminogen activator inhibitor (PAI-1) promoter observed in mink lung epithelial cells. However, the same anti-TGF-beta-IIR-NT antibodies did not prevent the characteristic inhibition of cellular proliferation by TGF-beta 1, as determined by [3H]thymidine incorporation into DNA. The selective perturbation of PAI-1 promoter induction versus cell-cycle-negative regulation suggests that strategic disruption of TGF-beta type-I and -II receptor interactions can effectively alter specific cellular responses to TGF-beta signalling.
Hall
F L
FL
Department of Surgery, Childrens Hospital Los Angeles Research Institute, CA 90027, USA.
Benya
P D
PD
Padilla
S R
SR
Carbonaro-Hall
D
D
Williams
R
R
Buckley
S
S
Warburton
D
D
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D000918:Antibody Specificity; D002358:Cartilage, Articular / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D047388:Casein Kinases; D002454:Cell Differentiation; D002460:Cell Line; D002478:Cells, Cultured; D003432:Cross-Linking Reagents; D004247:DNA / Q000096:biosynthesis; D004261:DNA Replication / Q000187:drug effects; D006801:Humans; D008156:Luciferases / Q000096:biosynthesis; D008168:Lung; D008907:Mink; D008969:Molecular Sequence Data; D012516:Osteosarcoma; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D011494:Protein Kinases / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases; D011817:Rabbits; D000077294:Receptor, Transforming Growth Factor-beta Type II; D018125:Receptors, Transforming Growth Factor beta / Q000276:immunology / Q000502:physiology*; D011994:Recombinant Proteins / Q000096:biosynthesis; D015398:Signal Transduction; D013388:Succinimides; D013936:Thymidine / Q000378:metabolism; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000378:metabolism / Q000494:pharmacology*; D014212:Tretinoin / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D000906:Antibodies; D003432:Cross-Linking Reagents; D010446:Peptide Fragments; D018125:Receptors, Transforming Growth Factor beta; D011994:Recombinant Proteins; D013388:Succinimides; D016212:Transforming Growth Factor beta; D014212:Tretinoin; D004247:DNA; D008156:Luciferases; D011494:Protein Kinases; D047388:Casein Kinases; D017346:Protein Serine-Threonine Kinases; D000077294:Receptor, Transforming Growth Factor-beta Type II; C019358:disuccinimidyl suberate; D013936:Thymidine