Surface to intracellular fluorescence ratio for cell expressing WT/mutant AVPR2 with or without SCTR co-expression.

<p>The presence of SCTR increased the amount of fluorescence on the cell surface in cell expressing R137H AVPR2 tagged with YFP, indicating a rescue of the mutant receptor. The data were mean±SEM from three independent experiments from 5–6 ROIs per sample.</p

Oligo-complex formation rescues the constitutively endocytosed mAVPR2 mutant R137H.

<p>The constitutively β-arrestin bound R137H mutant of mAVPR2 is largely endocytosed. With co-expression of mSCTR, however, an increase in net BRET signal suggests rescue of the R137H mutant to the cell surface by heterocomplex formation. The mutations of A89P or Q174R, which lead to improper folded, non-surface reaching receptors, cannot be rescued by the formation of heteromer. Significance level was calculated against the mAVPR1b control. The data were mean±SEM from three to five independent experiments in triplicate. ***, P<0.001.</p

Comparable transcript levels of mSCTR and mAVPR2 in CHOK1 cells transiently transfected with combinations of mSCTR and mAVPR2 by quantitative real-time PCR.

<p>The total receptor gene transcript levels were compared to the internal house-keeping control GAPDH by the 2 ΔΔ ct method. There is no significant difference among the groups. mSCTR/pcDNA3.1, 1ug mSCTR plasmid with 1ug pcDNA 3.1 empty vector; mSCTR/mAVPR2, 1ug receptor plasmid each; pcDNA3.1, 2ug pcDNA3.1; mAVPR2/pcDNA3.1, 1ug mAVPR2 plasmid with 1ug pcDNA 3.1. Data are presented as means ± SEM from three independent experiments in duplicate. ns, not significant.</p

Surface expression of mAVPR1a, mAVPR1b, mAVPR2 and mSCTR are similar.

<p>Shown are representative images of CHOK1 cells expressing mAVPR1a/mAVPR1b/mAVPR2 or mSCTR constructs. Surface to intracellular fluorescence ratios were similar for these four types of cells. The data were mean±SEM from three independent experiments with 5–6 ROIs per sample. Scale bar, 10μM.</p

mSCTR specifically oligomerizes with mAVPR2, and mAVPR1a, but not mAVPR1b.

<p>Shown are the net BRET ratios for CHO-K1 cells expressing a combination of mSCTR-Rlu donor and mAVPR-YFP acceptor constructs. Saturable curves from BRET assays were obtained for mAVPR2 and mAVPR1a, but not for mAVPR1b. The data were mean±SEM from three to five independent experiments in triplicate. ***, P<0.001. **, P<0.01. *, P<0.05.</p

Rescue of R137H mutant by SCTR as reflected by reduced affinity to β-arrestin.

<p>The affinity between WT or mutant AVPR2 with β-arrestin were determined by BRET, using AVRP2 tagged with Rlu and β-arrestin tagged with YFP. A) In the native state, the R137H mutant shows significantly higher affinity to β-arrestin than the WT AVPR2. While the two other mutants, A89P and Q174R, showed slightly higher BRET than the WT receptor, but the increase was not significant. Upon co-expression of SCTR, β-arrestin affinity of R137H was significantly reduced compared to the scenario when no SCTR was present. B) BRET was measured at 10 min after addition of 1μM Vp to stimulate receptor internalization. WT AVPR2 showed increase affinity to β-arrestin, but not the R137H mutant without SCTR co-expression. With SCTR, R137H demonstrated increased β-arrestin binding, suggesting functional rescue of the receptor. Data are presented as means±SEM from three independent experiments in duplicate. ***, P<0.001, **, P<0.01.</p

Rescue of the constitutively endocytosed R137H mAVPR2 mutant upon co-expression of mSCTR.

<p>Representative confocal images indicating the cellular location of mAVPR2-YFP receptors. The WT mAVPR2 is evenly distributed in the cell surface regardless of mSCTR co-expression. The R137H mutant is predominantly located in the intracellular endocytotic vesicles. Vesicular retention is not observed when mSCTR is co-transfected. The A89P and Q174R mutants cannot be rescued by mSCTR co-expression and remain intracellular. Scale bar, 10μM.</p

Signaling modification as a specific consequence of SCTR-AVPR2 oligomerization.

<p>Percentage changes in maximum cellular cAMP levels when cells were expressing a combination of mSCTR and/or mAVPR constructs, comparing to cAMP responses in cells bearing only the native receptor to the ligand. A) SCT stimulated a marked shift in E_max and potency in mSCTR in the presence of mAVPR2. No significant changes were observed when the non-interacting mAVPR1b replaced mAVPR2. B) Similarly, Vp stimulated a notable reduction in E_max and potency in mAVPR2 in the presence of mSCTR. The data were mean±SEM from three to five independent experiments in triplicate. ***, P<0.001. **, P<0.01. *, P<0.05. 10pM to 1μM SCT (panel C) or Vp (panel D) were treated to cells with mSCTR and/or mAVPR2. Calcium response curves are presented as percentages of maximal changes in RFU of cells expressing mSCTR or mAVPR2 only and stimulated with 1μM peptide. Except for cells with mSCTR only, a marked increase in cellular calcium response can only be seen at 1μM peptide concentration. Data were obtained from three individual experiments with 5–6 ROIs per dose.</p

Lrp5 physically interacts with GCGR.

<p>A). HEK293 cells cultured in 6-well plate were transfected with 2000 ng of a control vector (GFP), v5-tagged Lrp5, HA-tagged GCGR, or both for 24 h and then were treated with or without 50 nM GCG1-29 for 1 h. Cells were harvested and lysed, and equal amounts of protein were used for western blot analysis. The blot was first probed with v5 antibody and then stripped and reprobed with HA antibody. The β-actin blot was used as a loading control. B). HEK293 cells were transfected and treated same as in A. The cells were harvested and lysed, and equal amounts of lysate were immunoprecipitated with the indicated antibody. For HA antibody, the antibody complex was pulled down by protein G beads. For v5 antibody, it was a single-step pull-down because the antibody was directly conjugated to the agarose beads. After pull-down, the beads were washed three times with 1× TBST and then incubated in 1× SDS sample buffer to release the bound proteins. The lysates were used for western blot analysis and probed with the indicated antibody. C–E). BRET data for Rlu-tagged Lrp5 and YFP-tagged GCGR expressed on COS-1 cells. Shown are the static BRET signals (C), saturation BRET analysis (D), and effect of natural agonist ligand binding on the BRET signals (E). Shaded area represents the background signal of 0.12 determined using Rlu-tagged Lrp5 with soluble YFP as noted. Coexpression of untagged GCGR or Lrp5 competitively reduced the BRET signals obtained between Lrp5-Rlu and GCGR-YFP (black bars). Saturation BRET analysis supported the specific interaction of Lrp5 and GCGR. The non-specific bystander type BRET signal (linear) was observed when the non-structurally-related CCK1 receptors were co-expressed with Lrp5. Incubation with the natural agonist peptide ligand, glucagon (up to 1 µM), did not significantly change the Lrp5 and GCGR BRET signal. Data are represented as the means ± S.E.M. from four to six independent experiments performed in triplicate. Data marked with ** were significantly different from background signals at the level of p<0.01.</p

Blocking Lrp5/6 inhibited glucagon-induced β-catenin signaling.

<p>A). 293STF cells cultured in 24-well plate were transfected with a combination of GCGR (100 ng), Lrp5 (100 ng), Lrp5ECD (200 ng) and TKRlu (5 ng) plasmids as indicated on day 1, and then were treated with or without 50 nM GCG1-29 on day 2. Cells were harvested on day 3 to measure the TCF-mediated luciferase activity. Triplicate samples were used for each treatment. *p<0.005 compared with the non-treated (NT) group. ^#p<0.005 compared with the group without the Lrp5ECD transfection. B). DKK1 protein inhibits glucagon-induced β-catenin signaling. 293STF cells cultured in 24-well plate were transfected with pcDNA3.1 and GCGR plasmids (100 ng each) or GCGR and Lrp5 plasmids (100 ng each) along with 5 ng TKRlu plasmid on day 1, and then were treated with 50 nM GCG1-29±2 µg/ml DKK1 on day 2. Cells were harvested on day 3 to measure the luciferase activity. Duplicate samples were used for each treatment. *p<0.02 compared with the GCG1-29-treated group.</p