HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) Supplemental Data All Versions of this Article: fj.07-8777comv1 21/14/3827    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Google Scholar Articles by Hammer, M. M. Articles by Blau, H. M. PubMed PubMed Citation Articles by Hammer, M. M. Articles by Blau, H. M.

A novel enzyme complementation-based assay for monitoring G-protein-coupled receptor internalization

Baxter Laboratory in Genetic Pharmacology, Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California, USA

³Correspondence: Baxter Laboratory, CCSR 4215, 269 Campus Dr., Stanford, CA 94305-5175, USA. E-mail: hblau{at}stanford.edu

G-protein-coupled receptor (GPCR) signaling is involved in a wide range of physiological processes and diseases, and around one-half of currently used drugs target GPCRs. Assays for the signaling of GPCRs have suffered from drawbacks, including low signal-to-noise, temporally transient signals, and difficulty in applying a single assay to a wide range of GPCRs. We have developed a set of assays for G-protein-coupled receptor signaling based on β-galactosidase enzyme complementation in live mammalian cells. We previously described an assay for GPCR activation by monitoring the binding of β-arrestin to the receptor. Here we describe a second assay that monitors the internalization of GPCRs to endosomes, an event that follows receptor activation and is critical in desensitizing and resensitizing the receptor. We show that both assays display high signal-to-noise ratios with low variability and are quantitative for a wide range of GPCRs. EC₅₀s obtained with these assays closely match results reported in the literature. Finally, we show that these assays are readily adapted to high-throughput chemical screens. Thus, these two assays for monitoring G-protein-coupled receptor activation and internalization should prove valuable in basic biological studies as well as in high-throughput screens.—Hammer, M. M., Wehrman, T. S., Blau, H. M. A novel enzyme complementation-based assay for monitoring G-protein-coupled receptor internalization.

Key Words: GPCR signaling • ligand binding • high-throughput screening