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Internal trafficking and surface mobility of a functionally intact beta2-adrenergic receptor-green fluorescent protein conjugate.

Publication ,  Journal Article
Barak, LS; Ferguson, SS; Zhang, J; Martenson, C; Meyer, T; Caron, MG
Published in: Mol Pharmacol
February 1997

The beta2-adrenergic receptor (beta2AR) 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 beta2AR 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 beta2AR tagged with the highly sensitive Green Fluorescent Protein (GFP) could allow measurements of the various properties of the beta2AR. We demonstrate that a fully functional beta2AR/GFP can be engineered. In mammalian cells, beta2AR/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. beta2AR/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 x 10(-9) cm2/sec) was assessed on living cells using photobleaching recovery measurements. This approach and the fidelity of the biochemical properties of the beta2AR/S65T/GFP demonstrate that real-time optical measurements of beta2AR (as well as other GPCR) interactions and dynamics on living cells are feasible.

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Published In

Mol Pharmacol

DOI

ISSN

0026-895X

Publication Date

February 1997

Volume

51

Issue

2

Start / End Page

177 / 184

Location

United States

Related Subject Headings

  • Receptors, Adrenergic, beta-2
  • Phosphorylation
  • Pharmacology & Pharmacy
  • Isoproterenol
  • GTP-Binding Proteins
  • Fluorescence
  • Cells, Cultured
  • Cell Membrane
  • Animals
  • Adenylyl Cyclases
 

Citation

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Barak, L. S., Ferguson, S. S., Zhang, J., Martenson, C., Meyer, T., & Caron, M. G. (1997). Internal trafficking and surface mobility of a functionally intact beta2-adrenergic receptor-green fluorescent protein conjugate. Mol Pharmacol, 51(2), 177–184. https://doi.org/10.1124/mol.51.2.177
Barak, L. S., S. S. Ferguson, J. Zhang, C. Martenson, T. Meyer, and M. G. Caron. “Internal trafficking and surface mobility of a functionally intact beta2-adrenergic receptor-green fluorescent protein conjugate.Mol Pharmacol 51, no. 2 (February 1997): 177–84. https://doi.org/10.1124/mol.51.2.177.
Barak LS, Ferguson SS, Zhang J, Martenson C, Meyer T, Caron MG. Internal trafficking and surface mobility of a functionally intact beta2-adrenergic receptor-green fluorescent protein conjugate. Mol Pharmacol. 1997 Feb;51(2):177–84.
Barak, L. S., et al. “Internal trafficking and surface mobility of a functionally intact beta2-adrenergic receptor-green fluorescent protein conjugate.Mol Pharmacol, vol. 51, no. 2, Feb. 1997, pp. 177–84. Pubmed, doi:10.1124/mol.51.2.177.
Barak LS, Ferguson SS, Zhang J, Martenson C, Meyer T, Caron MG. Internal trafficking and surface mobility of a functionally intact beta2-adrenergic receptor-green fluorescent protein conjugate. Mol Pharmacol. 1997 Feb;51(2):177–184.

Published In

Mol Pharmacol

DOI

ISSN

0026-895X

Publication Date

February 1997

Volume

51

Issue

2

Start / End Page

177 / 184

Location

United States

Related Subject Headings

  • Receptors, Adrenergic, beta-2
  • Phosphorylation
  • Pharmacology & Pharmacy
  • Isoproterenol
  • GTP-Binding Proteins
  • Fluorescence
  • Cells, Cultured
  • Cell Membrane
  • Animals
  • Adenylyl Cyclases