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2-Adrenergic Receptor-Green Fluorescent Protein
Conjugate
Howard Hughes Medical Institute Laboratories (L.S.B., S.S.G.F.,
J.Z., M.G.C.),
Department of Cell Biology (L.S.B., S.S.G., J.Z., C.M.,
T.M., M.G.C.), and
Department of Medicine (M.G.C.), Duke University
Medical Center, Durham, North Carolina 27710
The 
2-adrenergic receptor (2AR) is
prototypic of the large family of G protein-coupled receptors (GPCRs)
whose desensitization and resensitization are regulated by
intracellular kinases, arrestin proteins, phosphatases, and ill-defined
components of the cellular endocytic machinery. The study of
2AR signal transduction and behavior in living cells is
technically difficult because of the relatively low cellular expression
of the receptor and a lack of useful biological reagents. Availability
of a functional 2AR tagged with the highly sensitive
Green Fluorescent Protein (GFP) could allow measurements of the various
properties of the 2AR. We demonstrate that a fully
functional 2AR/GFP can be engineered. In mammalian
cells, 2AR/S65T/GFP demonstrates strong, diffuse plasma
membrane fluorescence when observed with 480 nm excitation. The
fluorescent receptor binds agonist and antagonist, stimulates adenylyl
cyclase, undergoes phosphorylation, and is internalized in a manner
indistinguishable from wild-type receptor. We then show that its
internal trafficking and surface mobility can be determined by
measuring only the endogenous fluorescence of the conjugate.
2AR/S65T/GFP was found to be localized on endosomal membranes in living cells within minutes of agonist treatment, and
within 15 min it is observed in more complicated structures formed from
fusion of multiple endosomes. Finally, its free diffusion (diffusion
coefficient, 4.0-12 × 10
9 cm2/sec) was
assessed on living cells using photobleaching recovery measurements.
This approach and the fidelity of the biochemical properties of the
2AR/S65T/GFP demonstrate that real-time optical measurements of 2AR (as well as other GPCR) interactions
and dynamics on living cells are feasible.
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