Transgenic Overexpression of the Extra-Large Gsα Variant XLαs Enhances Gsα-Mediated Responses in the Mouse Renal Proximal Tubule in Vivo

Abstract

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 unaltered, protein kinase A activity and 25-hydroxyvitamin D 1-α-hydroxylase (Cyp27b1) mRNA levels were significantly higher in renal proximal tubules of rptXLαs 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 rptXLαs 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 rptXLαs mice than in controls (152 ± 16 vs. 222 ± 41 pg/ml; P < 0.05). After crossing the rptXLαs mice to mice with ablation of maternal Gnas exon 1 (E1(m-/+)), male offspring carrying both the XLαs transgene and maternal Gnas exon 1 ablation (rptXLαs/E1(m-/+)) were significantly less hypocalcemic than gender-matched E1(m-/+) littermates. Both E1(m-/+) and rptXLαs/E1(m-/+) offspring had higher serum PTH than wild-type littermates, but the degree of secondary hyperparathyroidism tended to be lower in rptXLαs/E1(m-/+) mice. Hence, transgenic XLαs expression in the proximal tubule enhanced Gsα-mediated responses, indicating that XLαs can mimic Gsα in vivo.

Loss of XLαs (extra-large αs) imprinting results in early postnatal hypoglycemia and lethality in a mouse model of pseudohypoparathyroidism Ib

“…This explanation would be consistent with previous reports that XLαs can mimic Gαs actions (49,50), and the absence of hypocalcemia and hyperphosphatemia in adult Gnasxl m+/p-mice may indicate that the contribution of XLαs protein to mediating the renal effects of PTH may decline with age (50). Conversely, the calcemic response to PTH is blunted in adult Gnasxl m+/p− mice (Fig.…”

“…Isolation of the renal proximal tubules and extraction of total RNA were as described (50). Total RNA from 2-d-old mouse whole kidney was extracted by using the Qiagen RNeasy Mini kit.…”

“…Blood ionized calcium, plasma phosphorus, and plasma PTH in P10 and adult mice were measured as described (50). In assays to determine PTH responsiveness in vivo, blood and urine were collected from mice before and after s.c. injection of 50 nmol/kg human PTH .…”

“…In assays to determine PTH responsiveness in vivo, blood and urine were collected from mice before and after s.c. injection of 50 nmol/kg human PTH . cAMP was quantified by using a RIA (50), and urinary cAMP values were normalized to urinary creatinine measured by using a STANBIO kit.…”

Imprinted genes and hypothalamic function

“…XLas stimulates adenylyl cyclase in response to receptor activation in a similar manner to Gsa (Bastepe et al 2002), although it may interact more avidly with the plasma membrane and be sensitive to lower ligand concentrations (Kaya et al 2009). In some circumstances in vivo XLas can replace Gsa (Liu et al 2011). In contrast to similar biochemical properties, at a physiological level Gsa and XLas appear to act in opposing pathways.…”