Random mutagenesis of CSF-1 receptor (FMS) reveals multiple sites for activating mutations within the extracellular domain.

AUTOR(ES)

van Daalen Wetters, T

RESUMO

Retroviral vectors containing human FMS protooncogene cDNA were reconfigured to allow single-step excision and reinsertion of restriction fragments encoding short segments of the extracellular domain of the colony-stimulating factor 1 receptor (CSF-1R). Fragments ligated into M13 bacteriophages were subjected to random chemical mutagenesis on both strands and recloned into the parental vector to create libraries of FMS genes containing mutations restricted to predefined target cassettes. Transfection of retroviral vector libraries into NIH/3T3 cells gave rise to transformed foci from which cellular DNA was amplified by the polymerase chain reaction (PCR), using primers flanking the mutagenized target sequences. Amplified fragments from individual primary transformants were recloned into intact FMS vector plasmids, and those with transforming activity were subjected to nucleotide sequence analysis. Alternatively, retroviruses rescued from transformed cells by superinfection with helper virus were used to generate secondary transformants containing unique copies of proviral DNA, whose sequences were determined after PCR amplification. Novel activating mutations were identified within sequences separating the third and fourth immunoglobulin-like loops, as well as within non-covalently stabilized loop 4 of the CSF-1R extracellular domain. Thus, FMS mutations able to convert human CSF-1R to an active oncoprotein are not restricted to those previously identified at codon 301. This approach should be generally applicable for defining activating mutations in related growth factor receptors, including those for platelet-derived growth factor and Steel factor (KIT ligand), in which ligand-independent oncoprotein variants have not been identified.

Documentos Relacionados

• Synthesis of membrane-bound colony-stimulating factor 1 (CSF-1) and downmodulation of CSF-1 receptors in NIH 3T3 cells transformed by cotransfection of the human CSF-1 and c-fms (CSF-1 receptor) genes.
• Role of dimerization and modification of the CSF-1 receptor in its activation and internalization during the CSF-1 response.
• Colony-stimulating factor 1-mediated regulation of a chimeric c-fms/v-fms receptor containing the v-fms-encoded tyrosine kinase domain.
• Loss of both CSF1R (FMS) alleles in patients with myelodysplasia and a chromosome 5 deletion.
• Specific binding of the mononuclear phagocyte colony-stimulating factor CSF-1 to the product of the v-fms oncogene.