β-adrenergic receptor stimulation promotes Gαs internalization through lipid rafts: A study in living cells

John Allen, Jiang Z. Yu, Robert J. Donati, Mark M. Rasenick

Research output: Contribution to journalArticle

76 Citations (Scopus)

Abstract

Upon binding hormones or drugs, many G protein-coupled receptors are internalized, leading to receptor recycling, receptor desensitization, and down-regulation. Much less understood is whether heterotrimeric G proteins also undergo agonist-induced endocytosis. To investigate the intracellular trafficking of Gαs, we developed a functional Gas-green fluorescent protein (GFP) fusion protein that can be visualized in living cells during signal transduction. C6 and MCF-7 cells expressing Gαs-GFP were treated with 10 μM isoproterenol, and trafficking was assessed with fluorescence microscopy. Upon isoproterenol stimulation, Gαs-GFP was removed from the plasma membrane and internalized into vesicles. Vesicles containing Gαs-GFP did not colocalize with markers for early endosomes or late endosomes/lysosomes, revealing that Gαs does not traffic through common endocytic pathways. Furthermore, Gαs-GFP did not colocalize with internalized β2-adrenergic receptors, suggesting that Gαs and receptors are removed from the plasma membrane by distinct endocytic pathways. Nonetheless, activated Gαs-GFP did colocalize in vesicles labeled with fluorescent cholera toxin B, a lipid raft marker. Agonist significantly increased Gαs protein in Triton X-100-insoluble membrane fractions, suggesting that Gas moves into lipid rafts/caveolae after activation. Disruption of rafts/caveolae by treatment with cyclodextrin prevented agonist-induced internalization of Gαs-GFP, as did overexpression of a dominant-negative dynamin. Taken together, these results suggest that receptor-activated Gαs moves into lipid rafts and is internalized from these membrane microdomains. It is suggested that agonist-induced internalization of Gαs plays a specific role in G protein-coupled receptor-mediated signaling and could enable Gαs to traffic into the cellular interior to regulate effectors at multiple cellular sites.

Original languageEnglish (US)
Pages (from-to)1493-1504
Number of pages12
JournalMolecular Pharmacology
Volume67
Issue number5
DOIs
StatePublished - May 2005
Externally publishedYes

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Green Fluorescent Proteins
Adrenergic Receptors
Lipids
Caveolae
Endosomes
G-Protein-Coupled Receptors
Isoproterenol
Gases
Cell Membrane
Membrane Microdomains
Dynamins
Heterotrimeric GTP-Binding Proteins
Cholera Toxin
MCF-7 Cells
Octoxynol
Cyclodextrins
Endocytosis
Lysosomes
Fluorescence Microscopy
Signal Transduction

ASJC Scopus subject areas

  • Pharmacology

Cite this

β-adrenergic receptor stimulation promotes Gαs internalization through lipid rafts : A study in living cells. / Allen, John; Yu, Jiang Z.; Donati, Robert J.; Rasenick, Mark M.

In: Molecular Pharmacology, Vol. 67, No. 5, 05.2005, p. 1493-1504.

Research output: Contribution to journalArticle

Allen, John ; Yu, Jiang Z. ; Donati, Robert J. ; Rasenick, Mark M. / β-adrenergic receptor stimulation promotes Gαs internalization through lipid rafts : A study in living cells. In: Molecular Pharmacology. 2005 ; Vol. 67, No. 5. pp. 1493-1504.
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