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Transgenic overexpression of the extra-large Gs? variant XL?s enhances Gs?-mediated responses in the mouse renal proximal tubule in vivo.

ABSTRACT: XL?s, a variant of the stimulatory G protein ?-subunit (Gs?), can mediate receptor-activated cAMP generation and, thus, mimic the actions of Gs? in transfected cells. However, it remains unknown whether XL?s can act in a similar manner in vivo. We have now generated mice with ectopic transgenic expression of rat XL?s in the renal proximal tubule (rptXL?s mice), where Gs? mediates most actions of PTH. Western blots and quantitative RT-PCR showed that, while Gs? and type-1 PTH receptor levels were unaltered, protein kinase A activity and 25-hydroxyvitamin D 1-?-hydroxylase (Cyp27b1) mRNA levels were significantly higher in renal proximal tubules of rptXL?s mice than wild-type littermates. Immunohistochemical analysis of kidney sections showed that the sodium-phosphate cotransporter type 2a was modestly reduced in brush border membranes of male rptXL?s mice compared to gender-matched controls. Serum calcium, phosphorus, and 1,25 dihydroxyvitamin D were within the normal range, but serum PTH was ?30% lower in rptXL?s mice than in controls (152 ± 16 vs. 222 ± 41 pg/ml; P < 0.05). After crossing the rptXL?s mice to mice with ablation of maternal Gnas exon 1 (E1(m-/+)), male offspring carrying both the XL?s transgene and maternal Gnas exon 1 ablation (rptXL?s/E1(m-/+)) were significantly less hypocalcemic than gender-matched E1(m-/+) littermates. Both E1(m-/+) and rptXL?s/E1(m-/+) offspring had higher serum PTH than wild-type littermates, but the degree of secondary hyperparathyroidism tended to be lower in rptXL?s/E1(m-/+) mice. Hence, transgenic XL?s expression in the proximal tubule enhanced Gs?-mediated responses, indicating that XL?s can mimic Gs? in vivo.

SUBMITTER: Liu Z

PROVIDER: S-EPMC3060637 | biostudies-literature | 2011 Apr

REPOSITORIES: biostudies-literature

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Transgenic overexpression of the extra-large Gsα variant XLαs enhances Gsα-mediated responses in the mouse renal proximal tubule in vivo.

Liu Zun Z Segawa Hiroko H Aydin Cumhur C Reyes Monica M Erben Reinhold G RG Weinstein Lee S LS Chen Min M Marshansky Vladimir V Fröhlich Leopold F LF Bastepe Murat M

Endocrinology 20110208 4

XLαs, a variant of the stimulatory G protein α-subunit (Gsα), can mediate receptor-activated cAMP generation and, thus, mimic the actions of Gsα in transfected cells. However, it remains unknown whether XLαs can act in a similar manner in vivo. We have now generated mice with ectopic transgenic expression of rat XLαs in the renal proximal tubule (rptXLαs mice), where Gsα mediates most actions of PTH. Western blots and quantitative RT-PCR showed that, while Gsα and type-1 PTH receptor levels were ...[more]

PMID: 21303955

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Project description:Circadian variability in kidney function is well recognized but is often ignored as a potential confounding variable in physiological experiments. Here, we have created a data resource consisting of expression levels for mRNA transcripts in microdissected proximal tubule segments from mice as a function of the time of day. Small-sample RNA sequencing was applied to microdissected S1 proximal convoluted tubules and S2 proximal straight tubules. After stringent filtering, the data were analyzed using JTK-Cycle to detect periodicity. The data set is provided as a user-friendly webpage at https://esbl.nhlbi.nih.gov/Databases/Circadian-Prox2/. In proximal convoluted tubules, 234 transcripts varied in a circadian manner (4.0% of the total). In proximal straight tubules, 334 transcripts varied in a circadian manner (5.3%). Transcripts previously known to be associated with corticosteroid action and with increased flow were found to be overrepresented among circadian transcripts peaking during the "dark" portion of the day [zeitgeber time (ZT)14-22], corresponding to peak levels of corticosterone and glomerular filtration rate in mice. To ask whether there is a time-of-day dependence of protein abundances in the kidney, we carried out LC-MS/MS-based proteomics in whole mouse kidneys at ZT12 and ZT0. The full data set (n = 6,546 proteins) is available at https://esbl.nhlbi.nih.gov/Databases/Circadian-Proteome/. Overall, 293 proteins were differentially expressed between ZT12 and ZT0 (197 proteins greater at ZT12 and 96 proteins greater at ZT0). Among the regulated proteins, only nine proteins were found to be periodic in the RNA-sequencing analysis, suggesting a high level of posttranscriptional regulation of protein abundances.NEW & NOTEWORTHY Circadian variation in gene expression can be an important determinant in the regulation of kidney function. The authors used RNA-sequencing transcriptomics and LC-MS/MS-based proteomics to identify gene products expressed in a periodic manner. The data were used to construct user-friendly web resources.

Dataset Information

Transgenic overexpression of the extra-large Gs? variant XL?s enhances Gs?-mediated responses in the mouse renal proximal tubule in vivo.

Publications

Transgenic overexpression of the extra-large Gsα variant XLαs enhances Gsα-mediated responses in the mouse renal proximal tubule in vivo.

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