A single amino acid substitution in a WW-like domain of diverse members of the PDGF receptor subfamily of tyrosine kinases causes constitutive receptor activation

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Abstract
Platelet‐derived growth factor β receptor (PDGFβR) is a transmembrane receptor tyrosine kinase involved in a variety of cellular functions. We have generated a constitutively activated murine PDGFβR containing a valine to alanine substitution at residue 536, located in the cytoplasmic juxtamembrane domain. When this mutant receptor (PR‐V536A) was expressed in Ba/F3 cells, it allowed the cells to survive and proliferate in the absence of IL‐3 or PDGF, and tyrosine phosphorylation of PR‐V536A was increased markedly compared with that of the wild‐type PDGFβR in the absence of ligand and similar to that observed in ligand‐activated PDGFβR. PR‐V536A displayed increased tyrosine kinase activity in vitro toward an exogenous substrate, and the tyrosine kinase activity of the receptor was required for the constitutive activation of the mutant. This valine to alanine substitution also activated a PDGFβR mutant unable to bind PDGF. Alanine substitutions at positions homologous to V536 of the murine PDGFβR also activated other members of the PDGF receptor subfamily. The amino acid sequence of this region revealed a strong similarity to WW domains present in other signal transduction proteins. Furthermore, GST fusion proteins containing the juxtamembrane region of the PDGFR specifically associated with peptides containing the WW domain consensus recognition sequence PPXY. The results suggest that the cytoplasmic juxtamembrane domain plays a role in the regulation of receptor activity and function, perhaps by participating in protein–protein interactions.