Template‐Directed Self‐Assembly Enhances RTK Catalytic Domain Function

Abstract

Receptor Tyrosine Kinases (RTKs) comprise a large family of single‐pass transmembrane proteins. Test‐tube phosphorylation assays routinely employ cloned RTK catalytic domain fragments, but these are removed from the membrane environment and thus cannot exhibit full function. Consequently, we developed template‐directed self‐assembly of RTK catalytic domains to recreate the membrane environment with the objective of restoring native activity and function. Autophosphorylation and substrate phosphorylation of template‐assembled catalytic domains from the Insulin and EphB2 receptors (IR and EphB2R, resp.) were substantially larger than corresponding activities in solution. Robust IR domain autophosphorylation was found on templates, where only a background level was observed with domains in the absence of template. EphB2R domain autophosphorylation increased ~4‐fold by with template. Template assembly increased the total (auto‐ plus substrate) phosphorylation exhibited by the IR and EphB2R domains 20‐fold and 8‐fold, respectively. Results with catalytic domains from other RTKs support the conclusion that the effect is general. Although we used templates to serve as platforms for assembling receptor domains, we also note that domains from co‐receptors, adaptor proteins and various other signaling components may also be used to permit easy and effective reassembly of several required components. These results and the underlying similarity in organization of transmembrane signaling systems suggest that template‐directed assembly of signaling pathways will provide widespread benefits to basic and applied signal transduction research.