Role of endocytosis in the activation of the extracellular signal-regulated kinase cascade by sequestering and nonsequestering G protein-coupled receptors - PubMed (original) (raw)

Role of endocytosis in the activation of the extracellular signal-regulated kinase cascade by sequestering and nonsequestering G protein-coupled receptors

K L Pierce et al. Proc Natl Acad Sci U S A. 2000.

Abstract

Acting through a number of distinct pathways, many G protein-coupled receptors (GPCRs) activate the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) cascade. Recently, it has been shown that in some cases, clathrin-mediated endocytosis is required for GPCR activation of the ERK/MAPK cascade, whereas in others it is not. Accordingly, we compared ERK activation mediated by a GPCR that does not undergo agonist-stimulated endocytosis, the alpha(2A) adrenergic receptor (alpha(2A) AR), with ERK activation mediated by the beta(2) adrenergic receptor (beta(2) AR), which is endocytosed. Surprisingly, we found that in COS-7 cells, ERK activation by the alpha(2A) AR, like that mediated by both the beta(2) AR and the epidermal growth factor receptor (EGFR), is sensitive to mechanistically distinct inhibitors of clathrin-mediated endocytosis, including monodansylcadaverine, a mutant dynamin I, and a mutant beta-arrestin 1. Moreover, we determined that, as has been shown for many other GPCRs, both alpha(2A) and beta(2) AR-mediated ERK activation involves transactivation of the EGFR. Using confocal immunofluorescence microscopy, we found that stimulation of the beta(2) AR, the alpha(2A) AR, or the EGFR each results in internalization of a green fluorescent protein-tagged EGFR. Although beta(2) AR stimulation leads to redistribution of both the beta(2) AR and EGFR, activation of the alpha(2A) AR leads to redistribution of the EGFR but the alpha(2A) AR remains on the plasma membrane. These findings separate GPCR endocytosis from the requirement for clathrin-mediated endocytosis in EGFR transactivation-mediated ERK activation and suggest that it is the receptor tyrosine kinase or another downstream effector that must engage the endocytic machinery.

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Figures

Figure 1

Agonist-promoted α2A AR and β2 AR sequestration and ERK 1/2 phosphorylation in COS-7 cells. (A) COS-7 cells transiently expressing either HA-epitope tagged α2A ARs or Flag epitope-tagged β2 ARs were serum-starved overnight and exposed to UK14304 (10 μM) or isoproterenol (10 μM), respectively, for 30 min at 37°C. Cell-surface receptors were labeled with an 12CA5 monoclonal antibody or an M2 Flag monoclonal antibody, by using FITC-conjugated goat anti-mouse IgG as the secondary antibody. Receptor sequestration, quantified as the percent loss of cell-surface fluorescence in agonist-treated cells, was measured by using flow cytometry. The data are expressed as the mean ± SEM of four independent experiments performed in triplicate. (B) Appropriately transfected COS-7 cells were serum-starved overnight in the presence or absence of pertussis toxin (100 ng/ml) before stimulation with 1 μM UK14304 (Upper) or 1 μM isoproterenol (Lower) for 5 min. Aliquots of whole-cell lysate (approximately 30 μg of protein per lane) were resolved by SDS/PAGE, and ERK 1/2 phosphorylation was detected by protein immunoblotting by using rabbit polyclonal phospho-MAPK-specific IgG. Data are expressed as the fold ERK 1/2 phosphorylation over the basal value in appropriately transfected cells. The data are expressed as the mean ± SEM of three independent experiments.

Figure 2

The effect of chemical and transfectable inhibitors of clathrin-mediated endocytosis on α2A AR- and β2 AR-mediated ERK 1/2 phosphorylation. (A) Cells transiently expressing the α2A AR-, the β2 AR-, or vector-transfected cells were pretreated with 300 μM MDC before a 5-minute stimulation with 1 μM UK14304 (Left), 1 μM isoproterenol (Center), or 10 ng/ml EGF (Right). Aliquots of whole-cell lysate (approximately 30 μg of protein per lane) were resolved by SDS/PAGE, and ERK 1/2 phosphorylation was detected by protein immunoblotting by using rabbit polyclonal phospho-MAP kinase-specific IgG. Data are expressed as the fold ERK 1/2 phosphorylation over the basal value in appropriately transfected cells. The data shown are the mean ± SEM of four independent experiments. (B) Cells in 100-mm dishes were transiently transfected with a HA-tagged ERK-1 plasmid (0.5 μg) together with the α2A AR (2 μg, Left), the β2 AR (2 μg, Center), or pCDNA3 (Right) alone or with either dynamin I Y231F/Y597F (7.5 μg) or β-arrestin 1 318–419 (7.5 μg). One day after transfection, cells were split into two 100-mm dishes and serum-starved overnight. After stimulation for 5 minutes with either 100 nM UK14304 (α2A AR), 1 μM isoproterenol (β2 AR), or 1 ng/ml EGF (EGFR), cell lysates were prepared, and the HA–ERK-1 was immunoprecipitated. Immunoblots were probed with both an anti-phospho-ERK 1/2 and a total ERK 1/2 antibody. Under each condition, data are expressed as the fold ERK 1/2 phosphorylation over the unstimulated. Data shown are the mean ± SEM of three independent experiments.

Figure 3

UK14304, Isoproterenol- and EGF-stimulated tyrosine phosphorylation of the EGFR and the effect of the EGFR-specific tyrphostin, AG1478, on α2A AR- and β2 AR-mediated ERK 1/2 phosphorylation. (A) Serum-starved COS-7 cells transiently expressing the α2A AR or β2 AR or pCDNA3 were stimulated with 1 μM UK14304, 1 μM isoproterenol, or 10 ng/ml EGF for 2 min. Monolayers were lysed in glycerol lysis buffer, and endogenous EGFRs were immunoprecipitated by using a sheep anti-human EGFR polyclonal antiserum. Immunoprecipitates were resolved by SDS/PAGE, and EGFR tyrosine phosphorylation was determined by immunoblotting by using a horseradish peroxidase-conjugated anti-phosphotyrosine monoclonal antiserum as described in Materials and Methods. (B) Cells transiently overexpressing the α2A AR-, the β2 AR-, or vector-transfected cells were preincubated for 15 min with tyrphostin AG1478 (125 nM) before stimulation with isoproterenol (1 μM), UK14304 (1 μM), or EGF (10 ng/ml) for 5 min. ERK 1/2 phosphorylation was determined from whole-cell lysates as described in Materials and Methods. Data shown are the mean ± SEM of four independent experiments and are normalized to the level of ERK 1/2 phosphorylation in untreated cells.

Figure 4

The effect of isoproterenol and EGF on the cellular distribution of epitope-tagged β2 ARs and EGFR–GFP. Confocal microscopic images depicting the cellular distribution of HA-tagged β2 AR (a, d, and g), and EGFR–GFP (23) (b, e, and h) before (NS; a_–_c) and after 30 min exposure to isoproterenol (d_–_f) or EGF (g, h, and i) in 293 cells. In the absence of agonist, both β2 AR and EGFR-GFP staining was predominantly confined to the plasma membrane (c). After exposure to isoproterenol, a portion of both receptor pools redistributed to an intracellular compartment (f). After exposure to EGF, redistribution of the EGFR–GFP, but not the β2 AR, was observed (i). Qualitatively similar results have been obtained in COS-7 cells.

Figure 5

The effect of UK14304 and EGF on the cellular distribution of epitope-tagged α2A ARs, and GFP–EGFR proteins. Confocal microscopic images depicting the cellular distribution of HA-tagged α2A AR (a, d, and g), and EGFR–GFP (b, e, and h) before (NS; a, b, and c) and after 30-min exposure to UK14304 (d_–_f) or EGF (g, h, and i) in 293 cells. In the absence of agonist, both α2A AR and GFP–EGFR staining was predominantly confined to the plasma membrane (c). After exposure to UK14304, the EGFR–GFP, but not the α2A AR redistributed to an intracellular compartment (f). A qualitatively similar pattern was observed after exposure to EGF, with redistribution of EGFR–GFP but not the α2A AR (i). Qualitatively similar results have been obtained in COS-7 cells.

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Role of endocytosis in the activation of the extracellular signal-regulated kinase cascade by sequestering and nonsequestering G protein-coupled receptors - PubMed (original) (raw)