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 they extend upstream of its intracellular tail. Here, using tandem MS, we identified Lys388 in the third loop and Lys484 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 Thr387/Thr392 and within the Ser489-Ser493 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 Thr387/Thr392 suppressed β-arrestin binding and inhibited PTHR ubiquitination, suggesting that PTHR phosphorylation and ubiquitination are interdependent. Of note, Lys-deficient PTHR mutants promoted normal cAMP formation, but exhibited differential mitogen-activated protein kinase (MAPK) signaling. Lys-deficient PTHR triggered early onset and delayed ERK1/2 signaling compared with wildtype PTHR. Moreover, ubiquitination of Lys388 and Lys484 in wildtype PTHR strongly decreased p38 signaling, whereas Lys-deficient PTHR retained signaling comparable to unstimulated wildtype PTHR. Lys-deficient, 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.
Highlights
G protein– coupled receptor (GPCR) signaling and trafficking are essential for cellular function and regulated by phosphorylation, -arrestin, and ubiquitination
Evidence established that ligand-induced parathyroid hormone receptor (PTHR) phosphorylation is required for internalization (9 –11) and that PTHR phosphorylated by G protein– coupled receptor kinases (GRK) [12] binds to -arrestin, thereby physically uncoupling the receptor from its associated heterotrimeric G proteins, leading to receptor desensitization
To identify the intracellular PTHR domains possessing candidate Lys residues that could be targeted for ubiquitination upon PTH stimulation, we generated four mutant receptor constructs by replacing cytosolic Lys (K) residues with Arg (R) as follows (Fig. 1A): 2K-PTHR; 3K-PTHR; CTK-PTHR (C-terminal Lys present; others converted to Arg); and 0K-PTHR (Lys-deficient construct), where all 11 Lys residues were replaced by Arg
Summary
Extensive evidence shows that multiple GPCRs, including the 2-adrenergic receptor (2AR), PAR1 and PAR2 protease-activated receptors, - and ␦-opioid receptors, CXCR chemokine receptors, V2R vasopressin receptor, D4 dopamine receptor, and PTHR [14], are ubiquitinated by the covalent addition of ubiquitin to intracellular Lys. Ubiquitination regulates internalization and trafficking of these receptors, often targeting receptor protein for degradation, either in an agonist-dependent or -independent manner [15]. Based on the prevailing view that ubiquitination was restricted to the intracellular C terminus of GPCRs [17], we tested the effect of mutating carboxyl tail Lys residues individually or in combination or truncating most of the intracellular PTHR C terminus. These interventions, failed to eliminate PTH-stimulated ubiquitination. PTHR ubiquitination did not alter receptor trafficking but significantly altered ERK and p38 signaling with attendant changes in cell proliferation and apoptosis
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
