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Real-Time Optical Monitoring of Ligand-Mediated Internalization of _1b-Adrenoceptor with Green Fluorescent Protein

Department of Molecular and Cell Pharmacology National Children’s Medical Research Center Setagaya-ku, Tokyo, 154 Japan

The study of G protein-coupled receptor signal transduction and behavior in living cells is technically difficult because of a lack of useful biological reagents. We show here that a fully functional _1b-adrenoceptor tagged with the green fluorescent protein (_1bAR/GFP) can be used to determine the molecular mechanism of internalization of _1bAR/GFP in living cells. In mouse T3 cells, _1bAR/GFP demonstrates strong, diffuse fluorescence along the plasma membrane when observed by confocal laser scanning microscope. The fluorescent receptor binds agonist and antagonist and stimulates phosphatidylinositol/Ca²⁺ signaling in a similar fashion to the wild receptor. In addition, _1bAR/GFP can be internalized within minutes when exposed to agonist, and the subcellular redistribution of this receptor can be determined by measurement of endogenous fluorescence. The phospholipase C inhibitor U73,122, the protein kinase C activator PMA, and inhibitor staurosporine, and the Ca²⁺-ATPase inhibitor thapsigargin were used to examine the mechanism of agonist-promoted _1bAR/GFP redistribution. Agonist-promoted internalization of _1bAR/GFP was closely linked to phospholipase C activation and was dependent on protein kinase C activation, but was independent of the increase in intracellular free Ca²⁺ concentration. This study demonstrated that real-time optical monitoring of the subcellular localization of _1bAR (as well as other G protein-coupled receptors) in living cells is feasible, and that this may provide a valuable system for further study of the biochemical mechanism(s) of agonist-induced receptor endocytosis.

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