Analysis of Receptor Tyrosine Kinase and G-Protein Coupled Receptor Signaling Dynamics on Micro-Structured Surfaces

Abstract

Membrane-localized proteins are essential to transmit signals into the cell. An important issue is the interaction of these proteins with cytosolic proteins. To quantify such often short-lived interactions we have introduced a method based on the combination of micro-structured surfaces and TIRF microscopy.(1,2) We have used the assay to validate the efficacy of medically relevant receptor tyrosine kinase (RTK) modulators.(3) Bait epidermal growth factor (EGF) receptor molecules were forced into microscopic domains, while monitoring co-recruitment of fluorescent intracellular prey Grb2 molecules. Pretreatment with pharmacologically active ingredients used for the treatment of human cancers significantly reduced this interaction. A similar approach was used for the quantitative analysis of the interaction between different insulin receptor substrate (IRS) proteins and the insulin/IGF-I receptor.(4) The micro-patterning technique enabled the measurement of equilibrium associations and interaction dynamics of these molecules with high specificity. We revealed that several domains of IRS critically determine the turnover rate of the receptors. Furthermore, we found significant differences among IRS proteins in the strength and kinetic stability of the interaction with the receptors, which could account for the diverse IRS functions. Finally, we studied the interaction of the GPCR s2-adrenoceptor (s2AR) with arrestin-3.(5) By measuring arrestin-3 recruitment and the stability of arrestin-3-receptor complexes in real time using FRAP analysis on micro-patterned surfaces, we could demonstrate that arrestin-3 dissociates quickly and almost completely from the β2AR. Recently, we have implemented micro-structured and functionalized multi-well plates. This development step sets a milestone in terms of throughput rates of our methodology.