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. 1998 Apr 28;95(9):5057-60.
doi: 10.1073/pnas.95.9.5057.

Sequestration of the G protein beta gamma subunit complex inhibits receptor-mediated endocytosis

H C Lin  1 , J A Duncan, T Kozasa, A G Gilman
Affiliations

Sequestration of the G protein beta gamma subunit complex inhibits receptor-mediated endocytosis

H C Lin et al. Proc Natl Acad Sci U S A. .

Abstract

Cell surface receptors that mediate endocytosis cluster into clathrin-coated pits, which pinch off to form vesicles that transport the receptors and their ligands. This multi-step process requires the coordinated action of many factors, including GTP-hydrolyzing proteins such as dynamin and regulators of actin cytoskeleton assembly. We note herein that sequestration of heterotrimeric G protein beta gamma subunits in intact cells strongly inhibits clathrin-coated pit-mediated endocytosis and causes rearrangement of the actin cytoskeleton. Our results suggest that cells contain a pool of free beta gamma and that it functions constitutively to permit endocytosis.

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Figures

Figure 1
Figure 1
Overexpression of Giα1 inhibits receptor-mediated endocytosis. (A) Immunoblot of COS-7 cells infected with adenoviruses encoding Giα1, β1, γ2, and/or β-galactosidase. Antiserum P960 recognizes Giα1 and several other Gα proteins (31), whereas antiserum B600 recognizes β subunits (20). Hela cells (B) and CV1 cells (C and D) were grown in 35-mm 6-well culture dishes for 16 h before infection with the indicated viruses. Cell were exposed to 150 moi (multiplicity of infection) units of total virus (50 moi of virus encoding each indicated G protein subunit, the remainder encoding β-galactosidase). Cells were approximately 80% confluent at the time of infection. Cells were assayed 48 h later for uptake of 125I-transferrin (B) or 125I-LDL (C and D). Error bars represent standard deviations (n = 3).
Figure 2
Figure 2
Effects of injected G protein subunits on endocytosis. One hour after injection, cells were incubated with Texas Red-conjugated transferrin (25 μg/ml, Molecular Probes) for 5 min at 37°C. Cells were then chilled on ice, washed, and fixed with 3% paraformaldehyde. Panels A, C, E, G, and I show fluorescence of fluorescein-dextran and thus demonstrate which cell in the field was injected with buffer (A), myrGiα1 (C), myrG203AGiα1 (E), myrG203AGiα1 plus β1γ2 (G), or myrQ204LGiα1 (I). Panels B, D, F, H, and J show accumulation of Texas Red-transferrin by all cells in the same field. (Bar = 15 μm.)
Figure 3
Figure 3
Endocytosis of fluorescent ligands. Cells were injected with solutions containing 50 μM G protein subunits or 10 mM GTP[γS] as described in Fig. 2, except fluorescein-conjugated dextran was omitted. Two hours after injection, COS-7 cells and HEK-293 cells were incubated with 20 μg/ml FITC-transferrin, and CV1 cells were incubated with 5 μg/ml DiI-LDL for 4 min at 37°C. Quantitation of the internalized fluorescent ligands is described in detail under Materials and Methods.
Figure 4
Figure 4
F-actin staining of CV1 cells. Cells infected for 48 h with adenoviruses encoding β-galactosidase (A), Giα1 (B), β1γ2 (C), or Giα1β1γ2 (D) were fixed and stained with Oregon green phalloidin (Molecular Probes). (Bar = 15 μm.)
Figure 5
Figure 5
Giα1-mediated inhibition of endocytosis is partially reversed by C3 exoenzyme. CV1 cells infected with adenoviruses encoding Giα1 or β-galactosidase were scrape-loaded with C3 (10 μg/ml) 24 h after infection (32). 125I-LDL uptake was quantified 24 h after scrape loading, as described under Materials and Methods. Error bars represent standard deviations (n = 3).
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