Site-specific polyubiquitination differentially regulates parathyroid hormone receptor–initiated MAPK signaling and cell proliferation

Abstract

G protein-coupled receptor (GPCR) signaling and trafficking are essential for cellular function and regulated by phosphorylation, β-arrestin, and ubiquitination. The GPCR parathyroid hormone receptor (PTHR) exhibits time-dependent reversible ubiquitination. The exact ubiquitination sites in PTHR are unknown, but extend upstream of its intracellular tail. Here, using tandem MS, we identified Lys-388 in the third loop and Lys-484 in the C-terminal tail as primary ubiquitination sites in PTHR. We found that PTHR ubiquitination requires β-arrestin and does not display a preference for β-arrestin1 or 2. PTH stimulated PTHR phosphorylation at Thr-387/Thr-392 and within the Ser-489/Ser-493 region. Such phosphorylation events may recruit β-arrestin, and we observed that chemically or genetically blocking PTHR phosphorylation inhibits its ubiquitination. Specifically, Ala replacement at Thr-387/Thr-392 suppressed β-arrestin binding and inhibited PTHR ubiquitination, suggesting that PTHR phosphorylation and ubiquitination are interdependent. Of note, Lysdeficient PTHR mutants promoted normal cAMP formation, but exhibited differential mitogenactivated protein kinase (MAPK) signaling. Lysdeficient PTHR triggered early onset and delayed ERK1/2 signaling compared with wild-type PTHR. Moreover, ubiquitination of Lys-388 and Lys-484 in wild-type PTHR strongly decreased p38 signaling, whereas Lys-deficient PTHR retained signaling comparable to unstimulated wild-type PTHR. Lysdeficient, ubiquitination-refractory PTHR reduced cell proliferation and increased apoptosis. However, elimination of all 11 Lys residues in PTHR did not affect its internalization and recycling. These results pinpoint the ubiquitinated Lys residues in PTHR controlling MAPK signaling and cell proliferation and survival. Our findings suggest new opportunities for targeting PTHR ubiquitination to regulate MAPK signaling or manage PTHR-related disorders.

Parathyroid hormone initiates dynamic NHERF1 phosphorylation cycling and conformational changes that regulate NPT2A-dependent phosphate transport

“…In the absence of NHERF1, GnTI Ϫ cells express relatively little NPT2A (Fig. 6B) due to constitutive NPT2A internalization and down-regulation (11), as described previously (42). Remarkably, in both fluorescence anisotropy and coimmunoprecipitation experiments, NPT2A displayed somewhat higher binding to WT-NHERF1, essentially equivalent to the phosphomimic S290D, and much stronger than to the phosphoresistant S290A as reflected by the higher K D of 3.2 M in fluorescence anisotropy measurements ( Fig.…”

“…PTHR possesses four N-glycosylation sites, and the absence of GnTI restricts PTHR glycosylation to a single Man 5 GlcNAc 2 . This residual glycosylation, however, is sufficient for PTHR function (42). PTH(1-34) elicited increases of intracellular cAMP ( Fig.…”

Post-Translational Modifications of G Protein–Coupled Receptors Control Cellular Signaling Dynamics in Space and Time

“…In this study, Leclair et al mapped the site of CXCR2 ubiquitination to a single C-tail localized lysine K 327 residue and showed that an ubiquitinationdeficient CXCR2 mutant with a K 327 to arginine (R) conversion failed to recruit b-arrestin-2 at the plasma membrane and blocked intracellular signaling including ERK1/2 phosphorylation (Leclair et al, 2014), suggesting that receptor ubiquitination is necessary for triggering signaling cascades. Agonist-induced ubiquitination of the parathyroid hormone receptor (PTHR) also requires phosphorylation and b-arrestin binding, but here ubiquitination appears to function selectively in promoting p38 MAP kinase activation and not cAMP accumulation (Zhang et al, 2018). The chemokine receptor CXCR4 has also been shown to mediate ubiquitin-dependent signaling; however, in this case the effect is indirect via modulation of signal transducing adapter molecule (STAM)-1, an ESCRT-0 component (Malik et al, 2012).…”

“…4C). A more rigorous approach to identify the precise sites of GPCR ubiquitination has been determined by mass spectrometry and was shown recently for the b 2 -adrenoreceptor and PTHR (Xiao and Shenoy, 2011;Zhang et al, 2018). Ubiquitination of multiple GPCRs, including PAR1, have also been detected by curation of high-throughput proteomic mass spectrometry and low-throughput data sources and published on PhosphoSitePlus (Hornbeck et al, 2015).…”

ACE2 interaction with cytoplasmic PDZ protein enhances SARS-CoV-2 invasion

“…(E) Surface biotinylation of wild-type or mutant ACE2 receptor expressed in HEK293T cells. Receptor biotinylation was performed using cleavable EZ-Link sulfo-NHS-SS-biotin as described before (Zhang et al, 2018). After unreacted biotin is quenched by ice-cold PBS containing 100 mM glycine, cells were extensively washed with PBS and lysed.…”