Real-Time Optical Monitoring of Ligand-Mediated Internalization of 1b-Adrenoceptor with Green Fluorescent Protein

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Abstract
The study of G protein-coupled receptor signal transduction and behavior in living cells is techni- cally difficult because of a lack of useful biological reagents. We show here that a fully functional a1b- adrenoceptor tagged with the green fluorescent protein (a1bAR/GFP) can be used to determine the molecular mechanism of internalization of a1bAR/ GFP in living cells. In mouse aT3 cells, a1bAR/GFP demonstrates strong, diffuse fluorescence along the plasma membrane when observed by confocal laser scanning microscope. The fluorescent recep- tor binds agonist and antagonist and stimulates phosphatidylinositol/Ca21 signaling in a similar fashion to the wild receptor. In addition, a1bAR/ GFP can be internalized within minutes when ex- posed to agonist, and the subcellular redistribution of this receptor can be determined by measure- ment of endogenous fluorescence. The phospho- lipase C inhibitor U73,122, the protein kinase C activator PMA, and inhibitor staurosporine, and the Ca21-ATPase inhibitor thapsigargin were used to examine the mechanism of agonist-promoted a1bAR/GFP redistribution. Agonist-promoted inter- nalization of a1bAR/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 Ca21 concen- tration. This study demonstrated that real-time op- tical monitoring of the subcellular localization of a1bAR (as well as other G protein-coupled recep- tors) in living cells is feasible, and that this may provide a valuable system for further study of the biochemical mechanism(s) of agonist-induced re- ceptor endocytosis. (Molecular Endocrinology 12: 1099-1111, 1998)

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