THE KINASE DOMAIN AND MEMBRANE LOCALIZATION DETERMINE INTRACELLULAR INTERACTIONS BETWEEN EPIDERMAL GROWTH-FACTOR RECEPTORS

Receptor tyrosine kinases play a central role in cellular growth, differentiation, and oncogenesis. Ah of these responses are triggered by growth factors interacting with the extracellular domain of transmembrane-spanning receptors, leading to dimerization and activation of an intrinsic tyrosine specific kinase activity by an allosteric mechanism. Precise mechanisms of receptor dimerization remain poorly understood, and current models suggest that the ligand binding domain plays a major determining role. To examine the role of the in tracellular domain in the association of juxtaposing receptor molecules, the full-length epidermal growth factor receptor was transiently co-expressed in human 293 fibroblasts with a truncated receptor that lacks the extracellular domain. After metabolic labeling with [S-35]methionine, the association of these receptor constructs was monitored by co-immunoprecipitation with an extracellular domain-specific antibody. Specific interactions found between these receptors were independent of ligand binding or an intact ATP-binding site. Truncated receptors that had sequences necessary for membrane localization, and that were capable of interacting with full-length receptor tyrosine kinase, also displayed constitutive kinase activity as well as the capacity to transphosphorylate kinase-negative receptors. Receptor co-immunoprecipitation occurred between constructs that comprise the intracellular domains of the epidermal growth factor and beta-platelet-derived growth factor receptors, and HER-2. Subsequent deletion analysis has identified the major region of epidermal growth factor receptor intracellular interaction to be within the kinase domain.