Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2007 Jul 15;313(12):2586-96.
doi: 10.1016/j.yexcr.2007.04.034. Epub 2007 May 22.

Inhibitors of phosphoinositide 3-kinase cause defects in the postendocytic sorting of beta2-adrenergic receptors

Hibah O Awwad  1 , Varsha Iyer, Jennifer L Rosenfeld, Ellen E Millman, Estrella Foster, Robert H Moore, Brian J Knoll
Affiliations

Inhibitors of phosphoinositide 3-kinase cause defects in the postendocytic sorting of beta2-adrenergic receptors

Hibah O Awwad et al. Exp Cell Res. .

Abstract

Phosphatidylinositol 3-kinase inhibitors have been shown to affect endocytosis or subsequent intracellular sorting in various receptor systems. Agonist-activated beta(2)-adrenergic receptors undergo desensitization by mechanisms that include the phosphorylation, endocytosis and degradation of receptors. Following endocytosis, most internalized receptors are sorted to the cell surface, but some proportion is sorted to lysosomes for degradation. It is not known what governs the ratio of receptors that recycle versus receptors that undergo degradation. To determine if phosphatidylinositol 3-kinases regulate beta(2)-adrenergic receptor trafficking, HEK293 cells stably expressing these receptors were treated with the phosphatidylinositol 3-kinase inhibitors LY294002 or wortmannin. We then studied agonist-induced receptor endocytosis and postendocytic sorting, including recycling and degradation of the internalized receptors. Both inhibitors amplified the internalization of receptors after exposure to the beta-agonist isoproterenol, which was attributable to the sorting of a significant fraction of receptors to an intracellular compartment from which receptor recycling did not occur. The initial rate of beta(2)-adrenergic receptor endocytosis and the default rate of receptor recycling were not significantly altered. During prolonged exposure to agonist, LY294002 slowed the degradation rate of beta(2)-adrenergic receptors and caused the accumulation of receptors within rab7-positive vesicles. These results suggest that phosphatidylinositol 3-kinase inhibitors (1) cause a misrouting of beta(2)-adrenergic receptors into vesicles that are neither able to efficiently recycle to the surface nor sort to lysosomes, and (2) delays the movement of receptors from late endosomes to lysosomes.

PubMed Disclaimer

Figures

Figure 1
Figure 1. LY294002 augments the decrease in the number of surface β2ARs caused by exposure to agonist
(A). 12β6 cells were treated with varying concentrations of LY294002 for 30 min, then with 10 μM ISO for 20 min followed by chilling to 4°C. Surface receptor levels were measured by binding to [3H]CGP12177 as described in the Materials and Methods. (*) Significantly different from vehicle control (P < 0.05; N=3), one-way ANOVA, Dunnett’s multiple comparisons post-test. A one-way ANOVA with a post-test for linear trends is also significant (P < 0.0001). Inset. 12β6 cells were treated for 30 min with LY294002 (LY- 50 μM), wortmannin (WT- 100nM), ethanol vehicle (EtOH – 0.075%), DMSO vehicle (DMSO – 0.1%), or not treated, then chilled and incubated on ice with [3H]CGP 12177 as described in the Materials and Methods. Compared to the untreated control, the percentage of bound radioligand remaining on the surface was LY, 93.3 ± 3.0; WT, 86.12 ± 8.32; Eth, 91.7 ± 3.49; DMSO, 89.32 ± 5.61. No pair of values was significantly different (P > 0.05, one-way ANOVA, Tukey's Multiple Comparison Test). (B). 12β6 cells were treated with 50 μM LY294002 or vehicle for 30 min, then with 10 μM ISO for 20 min followed by fixation with 4% paraformaldehyde and labeling with antibodies against the β2AR HA-tag. The cells were imaged by deconvolution immunofluorescence microscopy. Scalebar = 10 μm.
Figure 2
Figure 2. LY294002 does not affect the initial rate of β2AR endocytosis
12β6 cells were treated with 50 μM LY294002 or vehicle for 30 min, then for varying times with 10 μM ISO followed by chilling to 4°C. Surface receptor levels were measured by binding to [3H]CGP12177 as described in the Materials and Methods. (A) Time-course of internalization, modeled according to Morrison et al [19]. The calculated plateau values, which represent the maximal fraction of receptors left on the cell surface, were 0.48 ± 0.01 for vehicle-treated control cells, and 0.32 ± 0.03 for LY294002-treated cells (P = 0.0097; N=3); (B) A plot of the natural logarithm of the surface receptor fraction remaining during the first three min after addition of agonist. (■) Vehicle; (□) LY294002. For vehicle treated cells, the slope of the line is −0.15 ± 0.02, and for LY295002-treated cells, − 0.15 ± 0.01, representing first order rate constants of 0.15 min−1 for both vehicle and LY294002-treated cells.
Figure 3
Figure 3. LY294002 treatment inhibits the recycling of β2ARs
(A). 12β6 cells were treated with 50 μM LY294002 or vehicle for 30 min, then with 10 μM ISO for 20 min. The cells were washed extensively and returned to medium for varying times in the presence of 50 μM LY294002 or vehicle. After chilling on ice, the cells were incubated with [3H]CGP12177 to selectively label surface receptors. The fractional increases in surface receptors as a function of time after agonist removal were transformed to allow the plotting of the loss of internal receptors. The plots were then modeled using a first-order exponential function as described in Morrison et al [19] to obtain a plateau value for the number of receptors that fail to recycle, and the first order recycling rate constant. (■) Vehicle; (□) LY294002. The plateau values of the recycling curves were 40.0 ± 2.5% for LY294002-treated cells and 23.8 ± 2.7% for vehicle-treated control cells (P = 0.047; N=34). The recycling rate constants were 0.107 ± 0.014 min−1 for vehicle-treated cells and 0.085 ± 0.012 min−1 for LY294002-treated cells (the rates are not significantly different). (B). Recycling was plotted as a time course showing the return of receptors to the cell surface. (C). Immunofluorescence imaging of recycled β2ARs. Veh: 12β6 cells were treated with vehicle alone for 30 min, followed by 10 μM ISO for 20 min. The cells were then washed and incubated at 37°C in the presence of vehicle. Recycling was stopped after 40 min by fixation with 4% formaldehyde. LY: 12β6 cells were treated with 50 μM LY294002 for 30 min, followed by 10 μM ISO for 20 min. The cells were then washed and incubated at 37°C in the continuing presence of 50 μM LY294002. Recycling was stopped after 40 min by fixation with 4% formaldehyde. The fixed cells were labeled with antibodies against the β2AR C-terminus and imaged by conventional epifluorescence microscopy. Scalebar, 10 μm.
Figure 4
Figure 4. The effect of LY294002 treatment on the recycling of transferrin
12β6 cells were treated with 50 μM LY294002 or vehicle for 60 min in DMEM + 0.1% BSA at 37°C. ISO (10μM) and 50 μg/ml Alexa 594-holotransferrin were then added for 30 min at 37°C. The medium was removed and the cells were washed twice with PBS, and then were incubated for 40 min at 37°C with 1 μM propranolol and 50 μg/ml holotransferrin with either vehicle or 50 μM LY294002. The cells were fixed and then imaged using deconvolution fluorescence microscopy. The experiment was performed twice with similar results.
Figure 5
Figure 5. LY294002 delays agonist-induced degradation of β2ARs
12β6 cells were incubated for 30 min with 50 μM LY294002 or vehicle, then surface biotinylated as described in Materials and Methods. The cells were incubated up to 8 h in medium with or without 10 μM ISO and with 50 μM LY294002 or vehicle. The cells were lysed and biotinylated proteins were recovered with streptavidin agarose beads. After elution from the beads, the proteins were deglycosylated and immunoblotted with antibodies to the β2AR C-terminus and to the cell-surface Na+,K+ATPase (ATPase). Blots were imaged and quantified using a CCD camera system as described in Materials and Methods. (A). Representative blots, showing deglycosylated β2ARs migrating at ∼35 kDa, and Na+,K+ ATPase migrating at ∼100 kDa. (B). Plot of the density of the receptor bands corrected for incubation after the same time period with no agonist present, and normalized to the Na+,K+ ATPase signal. The error bars show the range of two determinations.
Figure 6
Figure 6. LY294002 increases β2AR and EGFP-rab7 colocalization after prolonged agonist treatment
12β6 cells were transfected with a plasmid expressing EGFP-rab7 for 24 h, then treated with 50 μM LY294002 or vehicle for 30 min prior to the addition of 10μM ISO + AT. (A) After 1–4 h of agonist exposure, the cells were fixed and then labeled with antibodies to the β2AR C-terminus (Alexa 594 secondary). Red, β2AR; Green, EGFP-rab7. The labeled cells were imaged by deconvolution microscopy as described in Materials and Methods. Insets: Two-fold magnifications of boxed areas in the panels. Scale bar = 10μm. (B) Quantification of β2AR and EGFP-rab7 overlap during prolonged agonist exposure. The overlap between β2AR and EGFP-rab7 (red over green) at various times following the addition of AT alone or AT + ISO was quantified using Metamorph as described in the Materials and Methods. The Y-axis is the percentage of β2AR labeling that overlaps with EGFP-rab7. (*) Significantly different from vehicle control: 1 h, P<0.0001; 2 h, P< 0.0007; 4 h, P< 0.005. (C) The overlap between EGFP-rab5a and β2ARs after 2 h of agonist exposure. LY, LY294002; Veh, vehicle. 12β6 cells were transfected, treated and imaged as described above in (A), except that anti-HA antibody mHA.11 was used to detect receptors.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. Tran TM, Friedman J, Qunaibi E, Baameur F, Moore RH, Clark RB. Characterization of agonist stimulation of cAMP-dependent protein kinase and G protein-coupled receptor kinase phosphorylation of the ®2-adrenergic receptor using phosphoserine-specific antibodies. Mol Pharmacol. 2004;65:196–206. - PubMed
    1. Benovic JL, Strasser RH, Caron MG, Lefkowitz RJ. ®-adrenergic receptor kinase: identification of a novel protein kinase that phosphorylates the agonist-occupied form of the receptor. Proc Natl Acad Sci U S A. 1986;83:2797–2801. - PMC - PubMed
    1. Yuan N, Friedman J, Whaley BS, Clark RB. cAMP-dependent protein kinase and protein kinase C consensus site mutations of the ®-adrenergic receptor. Effect on desensitization and stimulation of adenylyl cyclase. J Biol Chem. 1994;269:23032–23038. - PubMed
    1. Lohse MJ, Benovic JL, Codina J, Caron MG, Lefkowitz RJ. ®-arrestin: a protein that regulates ®-adrenergic function. Science. 1990;248:1547–1550. - PubMed
    1. Goodman OB, Jr, Krupnick JG, Santini F, Gurevich VV, Penn RB, Gagnon AW, Keen JH, Benovic JL. ®-arrestin acts as a clathrin adaptor in endocytosis of the ®2-adrenergic receptor. Nature. 1996;383:447–450. - PubMed

Publication types

MeSH terms