title: Small-molecule control of insulin and PDGF receptor signaling and the role of membrane attachment.
abstract: BACKGROUND
  Receptor tyrosine kinases (RTKs) regulate the proliferation, differentiation and metabolism of cells, and play key roles in tissue repair, tumorigenesis and development. To facilitate the study of RTKs, we have made conditional alleles that encode monomeric forms of the normally heterotetrameric insulin receptor and monomeric platelet-derived growth factor (PDGF) beta receptors fused to the FK506-binding protein 12 (FKBP12). The chimeric receptors can be induced to undergo dimerization or oligomerization by a small synthetic molecule called FK1012, and the consequences were studied in cells and embryonic tissues.
  
  
  RESULTS
  When equipped with an amino-terminal plasma membrane localization sequence and expressed in HEK293 cells, these chimeric receptors could signal to downstream targets as indicated by the FK1012-dependent activation of p70 S6 kinase (p70(S6k)) and mitogen-activated protein (MAP) kinase. In Xenopus embryos, the engineered PDGF receptor protein induced the formation of mesoderm from animal-pole explants in an FK1012-dependent manner. A cytosolic variant of the protein underwent efficient transphosphorylation, yet failed to activate appreciably either p70(S6k) or MAP kinase following treatment with FK1012. These results provide evidence of a requirement for membrane localization of RTKs, consistent with current models of RTK signaling.
  
  
  CONCLUSIONS
  We have developed an approach using the small molecule FK1012 to conditionally activate chimeric proteins containing FKBP fused to the insulin receptor or to the PDGF beta receptor. Using this system, we were able to induce mesoderm formation in Xenopus animal-cap tissue and to demonstrate that membrane localization is required for RTK signaling in transfected cells. This system should allow the further dissection of RTK-mediated pathways.
authors.affiliation: Howard Hughes Medical Institute, Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford Street, Cambridge, Massachusetts, 02138, USA.
authors.forename: J
authors.initials: J
authors.lastname: Yang
authors.forename: K
authors.initials: K
authors.lastname: Symes
authors.forename: M
authors.initials: M
authors.lastname: Mercola
authors.forename: S L
authors.initials: SL
authors.lastname: Schreiber
mesh_terms: D000483:Alleles; D000818:Animals; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D019281:Dimerization; D006360:Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008648:Mesoderm / Q000187:drug effects; D020794:Receptor Protein-Tyrosine Kinases / Q000235:genetics / Q000502:physiology*; D011972:Receptor, Insulin / Q000502:physiology*; D020797:Receptor, Platelet-Derived Growth Factor beta; D017479:Receptors, Platelet-Derived Growth Factor / Q000235:genetics / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction; D016559:Tacrolimus / Q000031:analogs & derivatives / Q000494:pharmacology; D022021:Tacrolimus Binding Proteins; D014981:Xenopus
chemical_list: D002352:Carrier Proteins; D004268:DNA-Binding Proteins; C084260:FK 1012; D006360:Heat-Shock Proteins; D011993:Recombinant Fusion Proteins; D020794:Receptor Protein-Tyrosine Kinases; D011972:Receptor, Insulin; D020797:Receptor, Platelet-Derived Growth Factor beta; D017479:Receptors, Platelet-Derived Growth Factor; D022021:Tacrolimus Binding Proteins; D016559:Tacrolimus