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. 2011 Dec;26(12):2923-34.
doi: 10.1002/jbmr.494.

Ubiquitination-deubiquitination balance dictates ligand-stimulated PTHR sorting

Verónica Alonso  1 , Clara E Magyar, Bin Wang, Alessandro Bisello, Peter A Friedman
Affiliations

Ubiquitination-deubiquitination balance dictates ligand-stimulated PTHR sorting

Verónica Alonso et al. J Bone Miner Res. 2011 Dec.

Abstract

Parathyroid hormone receptors (PTHR) are promptly internalized upon stimulation by activating (PTH[1-84], PTH[1-34]) and non-activating (PTH[7-84], PTH[7-34]) ligands. Here, we characterized the mechanism regulating the sorting of internalized receptors between recycling and degradative pathways. PTHR recycles faster after challenge with PTH(1-34) than with PTH(7-34). PTHR recycling is complete by 2 h after PTH(1-34) stimulation, but incomplete at this time in cells treated with PTH(7-34). The slower and incomplete recycling induced by PTH(7-34) is due to proteasomal degradation. Both PTH(1-34) and PTH(7-34) induced PTHR polyubiquitination. Ubiquitination by PTH(1-34) was transient, whereas receptor ubiquitination after PTH(7-34) was sustained. PTH(1-34), but not PTH(7-34), induced expression of the PTHR-specific deubiquitinating enzyme USP2. Overexpression of USP2 prevented PTH(7-34)-induced PTHR degradation. We conclude that PTH(1-34) promotes coupled PTHR ubiquitination and deubiquitination, whereas PTH(7-34) activates only ubiquitination, thereby leading to PTHR downregulation. These findings may explain PTH resistance in diseases associated with elevated PTH(7-84) levels.

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Conflict of interest statement

Conflict of interests: No authors have conflicts of interest

Figures

Fig. 1
Fig. 1. PTHR recycling and abundance after stimulation by PTH(1–34) or PTH(7–34)
DCT cells were incubated with either (A) 100 nM PTH(1–34) or (B) 1 μM PTH(7–34) for 30 minutes at 37°C, rinsed, acid-washed to remove any residual bound ligand, and allowed to recycle for the times indicated. Receptor binding is shown as a function of recycling time and was measured by 125I-PTH(1–34) binding and expressed as the percent of specific binding relative to the total binding of radioligand in unstimulated cells. (C) HK-2 cells were treated with either 100 nM PTH(1–34) or 1 μM PTH(7–34) for 1–8 hours. Total lysates were extracted and immunoblotted as described in Materials and Methods. PTHR was detected using a specific primary human antibody (1:1000) and HRP-tagged antibody (1:1000). Average relative abundance of PTHR (shown as a percent of total receptor abundance in untreated HK-2 cells). Data are summarized as ± S.E. of 3 independent experiments. *p < 0.05 vs. 0 hr.
Fig. 2
Fig. 2. Proteasome inhibition leads to PTHR accumulation
CHO-N10-R3 (A), HK-2 (B) or ROS (C) cells were pre-treated with the proteasome inhibitor MG-132 at 25 μM for 1 hour before addition of either 100 nM PTH(1–84), 1 μM PTH(7–34) or 1 μM PTH(7–84) as indicated for an additional 6 hours at 37°C. Equal amounts of cell lysates were analyzed by immunoblot for HA-PTHR (A and C) or endogenous PTHR (B) and shown as the relative abundance as a percentage of total receptor abundance in untreated cells. Data illustrate 5 (A and B) or 4 (C) independent experiments. *p < 0.05; **p < 0.01 vs. corresponding vehicle condition.
Fig. 3
Fig. 3. PTHR downregulation requires antecedent internalization
CHO-N10-R3 (A, C) and HK-2 cells (transiently transfected with HA-PTHR) (E, F) were incubated with 100 nM PTH(1–84), 1 μM PTH(7–34), or 1 μM PTH(7–84) as indicated for 3 hours. Receptor internalization was assayed in triplicate by ELISA as described in Materials and Methods. PTHR abundance was detected by western blot (B, D) in CHO-N10-R3 cells after a 3-hour exposure to 1 μM PTH(7–34). Data are the mean of 3 independent experiments performed. *p < 0.05; **p < 0.01 vs. corresponding –NHERF or scrambled conditions.
Fig. 4
Fig. 4. PTHR ubiquitination
ROS cells transfected with HA-PTHR and Myc-ubiquitin were pre-treated with MG-132 (25 μM; 1 hour) before incubation with either 100 nM PTH(1–34) or 1 μM PTH(7–34) for 5–120 minutes at 37°C. Cell lysates were immunoprecipitated using monoclonal anti-HA beads. Typical immunoblot of immunoprecipitates probed with polyclonal ubiquitin antibody (A, C) or Lys48 –specific ubiquitin antibody (D) are shown. Representative images of 3 independent experiments.
Fig. 5
Fig. 5. PTHR is ubiquitinated at the plasma membrane
ROS cells were transfected with HA-PTHR. After 48 hours, the cells were treated with 100 nM PTH(1–34) or 1 μM PTH(7–34) for 5 minutes. Membrane proteins were isolated and immunoprecipitated using monoclonal anti-HA beads as described in Materials and Methods. Representative immunoblot of immunoprecipitate probed with polyclonal ubiquitin antibody. Values are mean ± SEM from 3 independent experiments.
Fig. 6
Fig. 6. PTH(1–34) increases USP2 expression
(A) ROS cells were transiently transfected with HA-PTHR and treated with 100 nM PTH(1–34) or 1 μM PTH(7–34) for 1 hour. USP2 expression was assayed by immunoblot. (B) ROS cells were transfected with HA-USP2 and Flag-PTHR. After 48 hours, cells were treated with agonist as indicated. PTHR was detected using an anti-Flag primary antibody (1:1000). Values are mean ± SEM from ≥ 3 independent experiments. *p<0.05 vs. PTH(7–34).
Fig. 7
Fig. 7. Silencing USP2 deubiquitinase promotes ubiquitinated PTHR accumulation and favors PTHR downregulation
ROS cells were transfected with HA-PTHR. After 24 hours cells were transfected with shRNA-USP2 and incubated for 48 hours. Cells were then treated with 100 nM PTH(1–34) for 30 or 120 minutes. Total lysates and immunoprecipitated protein were analyzed by SDS-polyacrylamide gels and transferred to Immobilon-P membranes. Representative autoradiograms of USP2 (A) Ubiquitinated PTHR (B) and total PTHR (C) are shown. Values are mean ± SEM from ≥ 3 independent experiments. *p<0.05; **p < 0.01 vs. corresponding scrambled shRNA value.
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