Fig 2: USP33 deubiquitinates the GCGR in quiescent and agonist-treated cells.A, HEK-293 cells with stable expression of GCGR-MYC-FLAG were transfected with siRNA targeting no mRNA (control) or USP33 mRNA. Cells were serum-starved for 1 h and stimulated with 200 nm GCG for 15 min, followed by immunoprecipitation with anti-FLAG agarose gel. Subsequent steps were as described in Fig. 1. B, the graph represents means ± S.E. (error bars) from three independent experiments. *, p < 0.05 versus no agonist, control siRNA; **, p <0.05 versus the rest; #, p < 0.05 versus GCG, control siRNA, two-way ANOVA and Holm–Sidak's multiple-comparison test. C, HEK-293 cells stably expressing GCGR-MYC-FLAG receptor were serum-starved for 1 h and stimulated with 200 nm GCG for the indicated times followed by immunoprecipitation with anti-FLAG agarose beads. The immunoprecipitates were resolved on 4–20% Tris-glycine gels and immunoblotted for the indicated proteins. D, scatter plot with bars summarizes quantification of USP33 normalized to receptor level from three independent experiments. *, p < 0.05 versus unstimulated (0 min) condition, one-way ANOVA, Bonferroni's multiple-comparison test. IP, immunoprecipitation; IB, immunoblotting; Ub, ubiquitin.

Fig 3: βarrestin1 isoform exclusively scaffolds p38 kinase cascade promoted by GCGR agonist-stimulation. HEK-293 cells stably expressing GCGR-WT (A–D) or GCGR-K333R (E–H) were transfected transiently with HA-tagged βarr1 or βarr2. Forty-eight hours post-transfection, cells were stimulated with 100 nM GCG for 15 min. Samples were immunoprecipitated using anti-HA affinity magnetic beads and both eluted proteins and cognate lysates were immunoblotted sequentially for the indicated proteins. Band signals for p-p38, p38, or MKK3 were normalized to respective βarr bands and presented in the scatter plots with bars in panels B, C, and D for GCGR-WT and panels F, G, and H for GCGR-K333R. ∗p < 0.05 between indicated samples, two-way ANOVA, and Tukey's multiple-comparison test. The mobility of molecular weight markers (kDa) is shown beside each blot panel. I, GCGR deubiquitination status potentiates bias toward βarr1-dependent p38 MAPK signaling. The left side of the schematic displays activation of GCGR-WT that retains ubiquitin tag before agonist activation. Glucagon stimulation promotes several immediate events: G protein activation, cAMP production, βarr recruitment, and internalization into early endosomes where the GCGR is rapidly deubiquitinated by USP33 and STAMBP (not shown). With the GCGR-WT, we observe equivalent potential to signal through Gs and βarr1-p38 signaling. However, our results suggest that Gs/PKA attenuates glucagon-induced p38 MAPK phosphorylation. The right side of the schematic displays activation of GCGR-K333R that is impaired in ubiquitination and retains a deubiquitinated state. Glucagon stimulation promotes similar trafficking itinerary as for the GCGR-WT but we observe enhanced mobilization of the receptor at early endosomes. The major differences from the WT pathway are not only a significantly decreased coupling to Gs leading to impaired cAMP production but also greater p38 MAPK scaffolding by βarr1 increasing the bias toward βarr1–dependent signal transduction. GCGR, glucagon receptor; MAPK, mitogen-activated protein kinase; STAMBP, signal transducing adaptor molecule–binding protein; USP33, ubiquitin-specific peptidase 33.

Fig 4: S-nitrosylation of pVHL at Cys77 alters binding to multiple pVHL regulatory proteins. (A) Schematic depicting regulatory proteins known or hypothesized to regulate pVHL ubiquitinylation and subsequent proteasomal degradation. (B) HEK293 cells were transfected with EV, WT pVHL, or pVHL–C77S, followed by treatment with GSNO (1 mM) for 4 h in the presence of MG-132 (10 µM). pVHL binding partners were assessed by immunoprecipitation using α-HA probe, followed by Western blot using antibodies directed at VDU1, HSP90, UBE2S, SRC, c-Cbl, WSB1, and pVHL in both the pulldown (Top) and input lysate (Bottom). (C–F) HEK293 cells were transfected with siRNA targeting HSP90 (C), VDU1 (D), UBE2S (E), c-Cbl (F), or control siRNA, together with pVHL, for 48 h, followed by GSNO treatment in (C, E, and F) [1 mM (C and E) or 1.5 mM (F)] for 18 h. Panel (A) was created in https://BioRender.com.