Molecular basis for arrestin-mediated signaling

PROJECT SUMMARY Arrestin proteins are master regulators of G protein coupled receptor (GPCR) signaling, and act in two ways. First, arrestins terminate the coupling of G proteins to cognate receptor by physically blocking the G protein coupling site. Second, arrestins can support G protein independent signaling. The best studied arrestin-mediated signaling pathways include the activation of mitogen activated protein (MAP) kinases and Src family kinases. Arrestins also have complex functions beyond the modulation of GPCR signaling. An increasing body of evidence suggests that arrestins may interact with some receptor tyrosine kinases (RTKs) and may also link RTKs to GPCR during some types of transactivation. In our last grant cycle, we expanded what is known about the function of arrestin in signaling, developed working assays for following arrestin functions in cells, and we developed a range of biochemical tools that can help to decipher how arrestins might link signaling cascades. Preliminary data from these advances include an arrestin-dependent signal initiation complex for MAP kinase activation. We leverage these findings, tools, and assays with three aims that explore the influence of arrestins on linking signal cascades, in three aims. In Aim 1, we combine in vitro structural and biophysical techniques (array assays, X-ray crystallography, and binding measurements) with functional measurements in cells to investigate how arrestins interact with RTKs. In Aim 2, we combine in cell assays with mutagenesis and functional analysis to reveal the requirements for arrestin to link RTKs with GPCRs during transactivation. In Aim 3, we use structural approaches to investigate arrestin-effector interactions.