49 research outputs found

    Gene linkage comparisons of GHRHRs in the Osteichthyes lineage.

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    <p>Genes in vicinity of GHRHR were mapped, and syntenic genes were linked by straight lines. Size of the chromosomal region analyzed was given underneath based on the current edition of Ensembl databases. Syntenic genes encoding other secretin GPCR receptors were drawn in grey boxes, and the conserved flanking genes of GHRHR were drawn in closed boxes. (A) Gene environment of GHRHR in the Sarcopterygii lineage represented by human, mouse, lizard, chicken, frog and coelacanth was compared. Despite the syntenic genomic locations of GHRHR and neighbouring genes from human to avians, GHRHR was not located in frog. (B) Gene environment of GHRHR in the Actinopterygii lineage represented by fugu, tetraodon, stickleback, medaka, and zebrafish. Apart from the less conserved genomic region of zebrafish GHRHR, genes in proximity of other teleost GHRHRs were highly syntenic. However, they displayed an entirely different gene environment when compared to the sarcopterygian GHRHRs. (C) Genomic location analysis of xGHRHR characterized in present work and GHRHR<sub>2</sub> in zebrafish and chicken. Gene synteny could neither be identified inter-species nor between the two GHRHR genes in the same species. The figures were not drawn to scale.</p

    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

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

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    <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.

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    <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

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

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    <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.

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    <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<sub>max</sub> 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<sub>max</sub> 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
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