40 research outputs found

    Functional characterization of lfGHRHR and xGHRHR<sub>2</sub>.

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    <p>Intracellular cAMP accumulation ([cAMP]<sub>i</sub>) in response to 100 nM of GHRH and related peptides on CHO-K1 cells transfected with (A) lfGHRHR and (D) xGHRHR<sub>2</sub> (*** indicates <i>P</i><0.001, ** indicates <i>P</i><0.01, and * indicates <i>P</i><0.05). Effects of GHRH and related peptides on graded concentrations of peptides on [cAMP]<sub>i</sub> (B) lfGHRHR- and (E) xGHRHR<sub>2</sub>-expressing cells. The intracellular calcium mobilization ([Ca<sup>2+</sup>]<sub>i</sub>) assays of (C) lfGHRHR- and (F) xGHRHR<sub>2</sub>-expressing cells. For [cAMP]<sub>i</sub>, values represent mean ± SEM (n = 4). For ([Ca<sup>2+</sup>]<sub>i</sub>, data were expressed in ΔRFU value (maximum changes in the fluorescence signals from baseline) and converted to percentage of the maximum of xGHRH-induced [Ca<sup>2+</sup>]<sub>i</sub> elevation. Results are expressed as mean ± SEM from at least 10 independent experiments, cell number = 20 to 50. Peptide species: h, human; x, <i>X. laevis</i>, zf, zebrafish <i>D. rerio</i>; gf, goldfish <i>C. auratus</i>.</p

    Evolutionary analysis of the osteichthyans secretin GPCR family.

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    <p>The maximum likelihood (ML) optimal tree topology is presented and was constructed with MEGA5. ML bootstrap values higher than 50% are indicated at nodes. To facilitate interpretation, PTHR was used as an outgroup based on the proposed models for secretin GPCR family evolution <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053482#pone.0053482-Cardoso1" target="_blank">[7]</a>. The tree supported the identities of lfGHRHR and xGHRHR<sub>2</sub> as the orthologs of mammalian GHRHR and chicken GHRHR<sub>2</sub> respectively. Accession numbers of the sequences used were listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053482#pone.0053482.s009" target="_blank">Table S2</a>.</p

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

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    <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.

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    <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.

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    <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

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

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    <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