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Cell-Based Assays for G-Protein Independent Signaling Using Arrestin Binding and Endocytosis

Cell-Based Assays for G-Protein Independent Signaling Using Arrestin Binding and Endocytosis
Version:
MIPTEC0810_V1

File Name/Number:
MIPTEC 2010

Year:
2010

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GPCR activation results in G-protein dependent events such as second messenger signaling, as well as initiation of a host of cellular responses that regulate function and activate alternative pathways that are independent of G-protein activation.  Recent studies have highlighted the importance of G-protein independent signaling in normal and pathogenic tissue physiology.  Two of the well-characterized G-protein independent events are Arrestin recruitment and endocytosis.  Quantitatively examining these pathways in detail can aid in defining compound function and lead to the discovery of novel compounds with unique attributes.  Previously, DiscoveRx has developed a system to monitor protein-protein interactions in live cells using enzyme fragment complementation (EFC).  More recently, we have adapted this methodology to monitor the fate of activated and internalized GPCRs as they traffic through the endosomal compartment using a simple chemiluminescent detection method that is amenable to high-throughput screening.  Here we present the development of the system and the application to a wide range of GPCRs.  Case studies demonstrating compound-specific internalization and recycling patterns of multiple receptors including the human delta opioid receptor (OPRD1) will be discussed.