Fig 1.
Map of the human arginine-vasopressin 2 receptor showing mutants associated with nephrogenic diabetes insipidus in black.
The location of L83Q and Y128S, the mutants used in the present study, are shown. The amino acid sequence of the naturally-occurring ligand, vasopressin, is shown at the top. When there are multiple mutations at a single site, the number of mutations is noted in a triangle. The mutants in this study are a blue circle so they stand out. Adapted and modified from [36].
Fig 2.
IP production following transient transfection of HeLa cells for selected GPCRs and V2R mutants.
Transiently transfected HeLa cells containing a total of 100 ng cDNA/ 0.125 ml of hWT V2R or the mutant hL83Q were used to compare Gq (IP) coupling with the vasopressin receptor, endogenous muscarinic M3 subtype and histamine H1 receptors with or without 10 μM SR121463B pharmacoperone (rescues the L83Q mutant), DMSO (vehicle) is a negative control. Cells were stimulated with various doses of vasopressin for 2 h, acetylcholine and histamine dihydrochloride for 30 minutes. The results shown in the figure are from at least 3 independent experiments performed in quadruplicate (n = 3 ± SEM), p value < 0.05 is considered significant.
Fig 3.
IP production from HeLa cells stably expressing WT V2R, mutant L83Q or tTA.
Stably transfected HeLa cells containing tTA + hWTV2R or tTA + L83Q mutant were used to compare Gq (IP) coupling (a) with the vasopressin receptor with or without 10 μM SR121463B pharmacoperone (rescues the L83Q mutant), DMSO (vehicle) is a negative control. (b) Stably transfected HeLa cells with the WT V2R or tTA alone, were used to show if the oxytocin, V1a or V1b receptors are being stimulated with vasopressin to produce IP. Cells were stimulated with various doses of vasopressin for 2 h because the HeLa cells produce low amounts of total IP with the addition of 5 mM LiCl. The results shown in the figure are from at least 3 independent experiments performed in quadruplicate (n = 3 ± SEM), p value < 0.05 is considered significant.
Fig 4.
IP or cAMP production from HeLa cells transiently transfected with WT V2R in the presence of increasing amounts of mutant.
Cells were transiently transfected with an increasing amount of cDNA for the L83Q mutant in the presence of hWT V2R, keeping the WT at 5 ng and keeping the total amount of cDNA at 20 ng by supplementing the difference with empty vector pcDNA3.1. The cells were stimulated with 1 μM vasopressin and IP production (a) or cAMP (b) was measured. The dashed line shows the response to the empty vector pcDNA3.1. The results shown in the figure are from at least 3 independent experiments performed in quadruplicate (n = 3 ± SEM), p values < 0.05 is considered significant. *a, p < 0.05 compared with vector and WT V2R IP or cAMP production, *b, p < 0.05 compared with WT V2R and WT V2R + L83Q (10 and 15ng) IP or cAMP production.
Fig 5.
IP production by HeLa cells, with or without cDNA for Gq, with or without rescue by pharmacoperone SR121463B.
HeLa cells were transiently co-transfected with L83Q mutant + 10ng Gq (a) or hWT V2R + 10ng Gq (b) to assess IP response with with or without rescue. Cells were either treated with SR121463B or not (in which case an equivalent amount of DMSO was present). Cells were stimulated with 1 μM vasopressin for 2 hours. The results shown in the figure are from at least 3 independent experiments performed in quadruplicate (n = 3 ± SEM), p values < 0.05 is considered significant.
Fig 6.
Pharmacoperones used in this study, showing that compounds in many different structural classes rescue the mutant L83Q.
The 14 compounds shown have been grouped into 7 structure classes.
Fig 7.
Rescue of IP production by WT and mutant-expressing HeLa cells by various pharmacoperones.
Transiently transfected HeLa cells containing a total of 100 ng cDNA / 0.125 ml of hWT V2R or the L83Q mutant were treated with different pharmacoperones (10 μM, or #50 utilized at 1 μM) to show if the rescued L83Q mutant is coupled to Gq (IP). The cells were incubated with the pharmacoperones for 18 h with 4 μCi/ml 3H-inositol for “preloading” IP pathway, then washed and stimulated with 1 μM vasopressin for 2 h and IP response was measured. DMSO (vehicle) is a negative control that does not rescue the mutant and SR121463B is a known pharmacoperone for the L83Q mutant. The results shown in the figure are from at least 3 independent experiments performed in quadruplicate (n = 3 ± SEM).
Fig 8.
Rescue of cAMP production by WT and mutant-expressing HeLa cells by various pharmacoperones.
Transiently transfected HeLa cells containing a total of 100 ng cDNA / 0.125 ml of hWT V2R or the L83Q mutant were treated with different pharmacoperones (10 μM or #50 was 1 μM) to show if the rescued L83Q mutant is coupled to Gs (cAMP). The cells were incubated with the pharmacoperones for 16 h, then washed and stimulated with 1 μM vasopressin containing 0.2 mM IBMX for 30 minutes and the cAMP response was measured. DMSO (vehicle) is a negative control that does not rescue the mutant and SR121463B is a known pharmacoperone for the L83Q mutant. The results shown in the figure are from at least 3 independent experiments performed in quadruplicate (n = 3 ± SEM).
Fig 9.
Constitutive or stimulated activity of V2R mutants with or without rescue.
Transiently transfected HeLa cells containing a total of 100ng cDNA / 0.125 ml of hWT V2R, L83Q mutant and empty vector were used to assess if there is constitutive activity (CA) with the IP pathway, 9a, or with the cAMP pathway, 9b. Figure 9c shows that Y128S, is coupled to Gq/11 in the presence of agonist (1 μM vasopressin for 2 hours), but does not show constitutive activity (no agonist). Figure 9d shows constitutive activity of Y128S for the cAMP pathway. Cells were stimulated for 30 minutes with 1 μM vasopressin. The results shown in the figure are from at least 3 independent experiments performed in quadruplicate (n = 3 ± SEM), p values < 0.05 is considered significant.
Fig 10.
Scatchard assay of stably transfected HeLa cells with WT V2R or mutant.
Stably transfected HeLa cells containing tTA + hWT V2R (a) or tTA + L83Q mutants (b) were used to determine the number of receptors present at the membrane using the scatchard assay. Six concentrations of 3H-AVP (specific activity = 66.3 Ci/mmol) was used ranging from 1.56 to 50 nM with serial dilutions of 1:2. The averaged receptors per cell, Bmax and Kd for each of the 3 experiments is shown in the graph for the mutant and the WT V2R.