Live Cell Imaging of Gs and the β2-Adrenergic Receptor Demonstrates That Both αs and β1γ7 Internalize upon Stimulation and Exhibit Similar Trafficking Patterns That Differ from That of the…

By T. Hynes, Stacy M. Mervine, E. Yost, Jonathan L. Sabo, C. Berlot. Journal of Biological Chemistry, 2004

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To visualize and investigate the regulation of the localization patterns of Gs and an associated receptor during cell signaling, we produced functional fluorescent fusion proteins and imaged them in HEK-293 cells. αs-CFP, with cyan fluorescent protein (CFP) inserted into an internal loop of αs, localized to the plasma membrane and exhibited similar receptor-mediated activity to that of αs. Functional fluorescent β1γ7 dimers were produced by fusing an amino-terminal yellow fluorescent protein (YFP) fragment to β1 (YFP-N-β1) and a carboxyl-terminal YFP fragment to γ7 (YFP-C-γ7). When expressed together, YFP-N-β1 and YFP-C-γ7 produced fluorescent signals in the plasma membrane that were not seen when the subunits were expressed separately. Isoproterenol stimulation of cells co-expressing αs-CFP, YFP-N-β1/YFP-C-γ7, and the β2-adrenergic receptor (β2AR) resulted in internalization of both fluorescent signals from the plasma membrane. Initially, αs-CFP and YFP-N-β1/YFP-C-γ7 stained the cytoplasm diffusely, and subsequently they co-localized on vesicles that exhibited minimal overlap with β2AR-labeled vesicles. Moreover, internalization of β2AR-GFP, but not αs-CFP or YFP-N-β1/YFP-C-γ7, was inhibited by a fluorescent dominant negative dynamin 1 mutant, Dyn1(K44A)-mRFP, indicating that the Gs subunits and β2AR utilize different internalization mechanisms. Subsequent trafficking of the Gs subunits and β2AR also differed in that vesicles labeled with the Gs subunits exhibited less overlap with RhoB-labeled endosomes and greater overlap with Rab11-labeled endosomes. Because Rab11 regulates traffic through recycling endosomes, co-localization of αs and β1γ7 on these endosomes may indicate a means of recycling specific αsβγ combinations to the plasma membrane.

Subfields: Biophysics, Molecular Biology

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