Evidence that downregulation of the M-CSF receptor is not dependent upon receptor kinase activity.

The downregulation of tyrosine kinase receptors attenuates signalling and is thought to be dependent upon intrinsic receptor kinase activity, largely because down-regulation is inhibited by a kinase-inactivating mutation of an invariant lysine residue of the receptors for EGF, insulin, M-CSF and PDGF. We confirmed that this mutation inhibited the degradation of the M-CSF receptor. However, two different kinase inactivating mutations of the invariant amino acids Gly 591 and Glu 633 did not prevent M-CSF-induced receptor degradation, so demonstrating that receptor kinase activity is not essential for this process. Three other kinase-inactivating mutations were found to cause constitutive receptor degradation in the absence of M-CSF, most probably by disrupting the structure of the activating loop of the kinase domain. It is known that extensive movement of the A-loop is necessary for kinase activation and is normally induced by ligand-binding. It is therefore suggested that some aspect or consequence of the change in structure of the A-loop caused by ligand binding also activates receptor downregulation, so ensuring that downregulation is coupled to but is not necessarily dependent upon receptor kinase activity.

Cell specific transformation by c-fms activating loop mutations is attributable to constitutive receptor degradation.

Expression of a receptor for human macrophage-colony stimulating factor (M-CSF or CSF-1), containing a point mutation which changes an aspartate to a valine at position 802 of the activating loop of the kinase domain, potently transforms the haemopoietic cell line FDC-P1 yet prevents Rat-2 fibroblast transformation. In order to understand this apparent paradox, aspartate 802 was changed by cassette mutagenesis to each of the other 19 amino acids. All hydrophobic amino acid substitutions were transforming when tested in FDC-P1 cells yet inactivating when tested in Rat-2 fibroblasts. These same amino acid substitutions also activated receptor degradation, strongly suggesting a causal relationship between receptor degradation and inactivation in fibroblasts. Point mutations or small deletions of Y708 within the kinase insert region of the mutant D802V receptor partly inhibited receptor degradation. The more stable D802V receptor derivatives were able to transform both FDC-P1 cells and Rat-2 fibroblasts, so establishing that the cell specific effect of the c-fmsD802V activating loop mutation is attributable to receptor degradation which accompanies kinase activation and prevents the transformation of Rat-2 but not of FDC-P1 cells.