REAL-TIME OPTICAL MONITORING OF LIGAND-MEDIATED INTERNALIZATION OF ALPHA(1B)-ADRENOCEPTOR WITH GREEN FLUORESCENT PROTEIN

The study of G protein-coupled receptor signal transduction and behavi or in living cells is technically difficult because of a lack of usefu l biological reagents. We show here that a fully functional alpha(1b)- adrenoceptor tagged with the green fluorescent protein (alpha(1b)AR/GF P) can be used to determine the molecular mechanism of internalization of alpha(1b)AR/GFP in living cells. In mouse alpha T3 cells, alpha(1b )AR/GFP demonstrates strong, diffuse fluorescence along the plasma mem brane when observed by confocal laser scanning microscope. The fluores cent receptor binds agonist and antagonist and stimulates phosphatidyl inositol/Ca2+ signaling in a similar fashion to the wild receptor. In addition, alpha(1b)AR/GFP can be internalized within minutes when expo sed to agonist, and the subcellular redistribution of this receptor ca n be determined by measurement of endogenous fluorescence. The phospho lipase C inhibitor U73,122, the protein kinase C activator PMA, and in hibitor staurosporine, and the Ca2+-ATPase inhibitor thapsigargin were used to examine the mechanism of agonist-promoted alpha(1b)AR/GFP red istribution. Agonist-promoted internalization of alpha(1b)AR/GFP was c losely linked to phospholipase C activation and was dependent on prote in kinase C activation, but was independent of the increase in intrace llular free Ca2+ concentration. This study demonstrated that real-time optical monitoring of the subcellular localization of alpha(1b)AR (as well as other G protein-coupled receptors) in living cells is feasibl e, and that this may provide a valuable system for further study of th e biochemical mechanism(s) of agonist-induced receptor endocytosis.