Functional characterization of a loss-of-function mutant I324M of arginine vasopressin receptor 2 in X-linked nephrogenic diabetes insipidus

Abstract

X-linked nephrogenic diabetes insipidus (X-linked NDI) is a rare inherited disease mainly caused by lost-of-function mutations in human AVPR2 gene encoding arginine vasopressin receptor 2 (V2R). Our focus of the current study is on exploration of the functional and biochemical properties of Ile324Met (I324M) mutation identified in a pedigree showing as typical recessive X-linked NDI. We demonstrated that I324M mutation interfered with the conformation of complex glycosylation of V2R. Moreover, almost all of the I324M-V2R failed to express on the cell surface due to being captured by the endoplasmic reticulum control system. We further examined the signaling activity of DDAVP-medicated cAMP and ERK1/2 pathways and the results revealed that the mutant receptor lost the ability in response to DDAVP stimulation contributed to the failure of accumulation of cAMP and phosphorylated ERK1/2. Based on the characteristics of molecular defects of I324M mutant, we selected two reagents (SR49059 and alvespimycin) to determine whether the functions of I324M-V2R can be restored and we found that both compounds can significantly "rescue" I324M mutation. Our findings may provide further insights for understanding the pathogenic mechanism of AVPR2 gene mutations and may offer some implications on development of promising treatments for patients with X-linked NDI.

Figure 1

HEK293T cells expressing the I324M-V2R fail to produce mature receptors, which was in the form of immature precursors without complex glycosylated groups. HEK293T cells transiently transfected with cDNAs encoding the pcDNA3.1 (Ctr), wild type-V2R (WT) and I324M-V2R (I324M). 48 h after transfection, the cells lysate was treated with or without EndoH, PNGase.F, or O-glycosidase & Sialidase and detected by western blotting using anti-Myc rabbit polyclonal antibody. (A) Lanes 1 was used as negative control. Lanes 2–3 and 4–5 showed the EndoH sensitivity of WT group and I324M group respectively. (B) Lanes 1–2 showed the PNGase.F sensitivity of WT group; lanes 3–4 showed the PNGase.F insensitivity of I324M group. (C) Lane 1–3 showed the PNGase.F and O-glycosidase & Sialidase sensitivity of WT group; lanes 4–6 showed the PNGase.F and O-glycosidase & Sialidase insensitivity of I324M group. EndoH: endoglycosidase H; PNGase.F: peptide-N-glycosidase F. Representative of 3 separate experiments.

Figure 2

The V2R proteins carrying I324M mutant lost the ability expressed on the plasma membrane and almost entirely retained in the ER. COS7 cells were transiently expressed Myc-tagged-WT-V2R (WT) or Myc-tagged-I324M-V2R (I324M), respectively. (A) The fluorescence imaging of ER location of WT and I324M co-stained with PDI respectively using anti-Myc rabbit polyclonal antibody and anti–PDI mouse monoclonal antibody. (B) The fluorescence imaging of Golgi location of WT and I324M co-stained with GM130 respectively using anti-Myc rabbit polyclonal antibody and anti–GM130 mouse monoclonal antibody. V2R was shown in green; PDI and GM130 were shown in red; The superposition of V2R and PDI or GM130 was shown in orange. PDI: protein disulfide isomerase, a marker of endoplasmic reticulum (ER); GM130: a marker of Golgi apparatus; DAPI: a marker of nucleus. Representative of 3 separate experiments.

Figure 3

The I324M mutant have no obvious influence on V2R protein’s production and stability compared with WT. (A) HEK293T cells transiently expressing either WT-V2R (WT) or I324M-V2R (I324M), 48 h after transfection, cells were incubated with 20 μg/ml cycloheximide (CHX) for 0 ,4 ,8 and 12 h before detected by western blotting using anti-Myc rabbit polyclonal antibody. (B) Density of degraded protein bands were normalized against endogenous HSP90 and quantified by ImageJ 1.49 (https://imagej.nih.gov/ij/) The data was from four independent experiments.

Figure 4

There is no increasement in cAMP accumulation of cells expressing I324M mutant under the stimulation of DDAVP. HEK293T cells were transiently transfected with pcDNA3.1 (Ctr), WT-V2R (WT) or I324M-V2R (I324M). cAMP measurements were performed after treated with or without various concentrations of DDAVP (1 nM, 100 nM, 1 μM or 10 μM) for 15 min (as described in “Methods”). (A) Results were shown as bar graph and data represent mean ± SEM using Two-way ANOVA were obtained from three independent experiments. ****P < 0.0001. (B) V2R expression of WT and I324M groups in the absence of DDAVP. Representative of 3 separate experiments.

Figure 5

The I324M mutant did not show the activity for initiating the phosphorylation of ERK1/2 signaling pathway. (A) HEK293T cells were transiently transfected with plasmid encoding pcDNA3.1 (Ctr), WT-V2R (WT) or I324M-V2R (I324M) and preincubated with or without 1μΜ DDAVP for 15 min before total or phosphorylated ERK1/2 determination was performed by western blotting using anti-ERK1/2 and antiphospho-ERK1/2 rabbit polyclonal antibody, respectively. (B) Data was shown as the percentage of p-ERK1/2/t-ERK1/2 resulted from the intensity analysis of protein bands using ImageJ 1.49 and drafted in the GraphPad Prism 7.0. Values were mean ± SEM using One-way ANOVA. ***P < 0.001 (n = 3).

Figure 6

The impaired maturity and cAMP signaling activity of I324M mutant were partially restored by SR49059 and alvespimycin. HEK293T cells were transiently transfected with plasmid encoding pcDNA3.1 (Ctr), WT-V2R (WT) or I324M-V2R (I324M). 24 h after transfection, the cells were incubated with or without SR49059 (1 μM) for 16 h and alvespimycin (1 μM) for 20 h before treated with or without DDAVP (100 nM). (A) Results were shown as bar graph and data represent the mean ± SEM using Two-way ANOVA were obtained from three independent experiments. ****P < 0.0001, **P < 0.01. (B) V2R expression of WT and I324M groups in the stimulation with or without SR49059 (1 μM) and alvespimycin (1 μM). Representative of 3 separate experiments.