Biomedical subjects

Daniela Rotin

Publications and source records attributed to Daniela Rotin.

21 records · Page 2Linked to original sources

PubMed2002

Direct binding of the beta1 adrenergic receptor to the cyclic AMP-dependent guanine nucleotide exchange factor CNrasGEF leads to Ras activation.

G-protein-coupled receptors (GPCRs) can indirectly activate Ras primarily through the betagamma subunits of G proteins, which recruit c-Src, phosphatidylinositol 3-kinase, and Grb2-SOS. However, a direct interaction between a Ras activator (guanine nucleotide exchange factor [GEF]) and GPCRs that leads to Ras activation has never been demonstrated. We report here a novel mechanism for a direct GPCR-mediated Ras activation. The beta1 adrenergic receptor (beta1-AR) binds to the PDZ domain of the cyclic AMP (cAMP)-dependent Ras exchange factor, CNrasGEF, via its C-terminal SkV motif. In cells heterologously expressing beta1-AR and CNrasGEF, Ras is activated by the beta1-AR agonist isoproterenol, and this activation is abolished in beta1-AR mutants that cannot bind CNrasGEF or in CNrasGEF mutants lacking the catalytic CDC25 domain or cAMP-binding domain. Moreover, the activation is transduced via Gsalpha and not via Gbetagamma. In contrast to beta1-AR, the beta2-AR neither binds CNrasGEF nor activates Ras via CNrasGEF after agonist stimulation. These results suggest a model whereby the physical interaction between the beta1-AR and CNrasGEF facilitates the transduction of Gsalpha-induced cAMP signal into the activation of Ras. The present study provides the first demonstration of direct physical association between a Ras activator and a GPCR, leading to agonist-induced Ras activation

Animals↗

PubMed2002

Pituitary, pancreatic and gut neuroendocrine defects in protein tyrosine phosphatase-sigma-deficient mice.

The expression of receptor protein tyrosine phosphatase sigma (PTPfinal sigma) is developmentally regulated in neuronal and neuroendocrine tissues. We have previously shown that mice deficient in PTPfinal sigma demonstrate nervous system abnormalities, pituitary hypoplasia, increased neonatal mortality (60%), and death from a wasting syndrome at 2-3 wk of age (38%). We have now examined the role of PTPfinal sigma on pituitary, pancreas and enteroendocrine cytodifferentiation, hormone production, and development. The adenohypophyses of PTPfinal sigma(-/-) mice were small and exhibited reduced GH and PRL immunoreactivity. Cells containing TSH, LH, FSH, ACTH, pituitary-specific POU homeodomain factor (Pit-1), ER, and steroidogenic factor 1 were found in normal proportions and distributions. The diminished expression of GH and PRL was not associated with apoptosis of somatotrophs or lactotrophs. Pit-1-positive TSH-negative cells were detected, suggesting that impaired GH and PRL synthesis was not attributable to Pit-1 deficiency. In the knockout mice, pancreatic islets were hypoplastic with reduced insulin immunoreactivity, and there was also variable expression of gut hormones. Functionally, the GH deficiency was associated with hypoglycemia and death in the PTPfinal sigma(-/-) neonate and accordingly, ip administration of GH rescued the PTPfinal sigma(-/-) neonate and normalized the blood glucose. These data indicate that PTPfinal sigma plays a major role in differentiation and development of the neuroendocrine system.

Animals↗

PubMed2001

Trafficking and cell surface stability of the epithelial Na+ channel expressed in epithelial Madin-Darby canine kidney cells.

The apically located epithelial Na(+) channel (alphabetagamma-ENaC) plays a key role in the regulation of salt and fluid transport in the kidney and other epithelia, yet its mode of trafficking to the plasma membrane and its cell surface stability in mammalian cells are poorly understood. Because the expression of ENaC in native tissues/cells is very low, we generated epithelial Madin-Darby canine kidney (MDCK) cells stably expressing alphabetagamma-ENaC, where each subunit is tagged differentially at the intracellular C terminus and the beta-subunit is also Myc-tagged at the ectodomain (alpha(HA)beta(Myc,T7)gamma(FLAG)). ENaC expression in these cells was verified by immunoblotting with antibodies to the tags, and patch clamp analysis has confirmed that the tagged channel is functional. Moreover, using electron microscopy, we demonstrated apical, but not basal, membrane localization of ENaC in these cells. The glycosylation pattern of the intracellular pool of ENaC revealed peptide N-glycosidase F and endoglycosidase H sensitivity. Surprisingly, the cell surface pool of ENaC, analyzed by surface biotinylation, was also core glycosylated and lacked detectable endoglycosidase H-resistant channels. Extraction of the channel from cells in Triton X-100 demonstrated that both intracellular and cell surface pools of ENaC are largely soluble. Moreover, floatation assays to analyze the presence of ENaC in lipid rafts showed that both intracellular and cell surface pools of this channel are not associated with rafts. We have shown previously that the total cellular pool of ENaC is turned over rapidly (t(1/2) approximately 1-2 h). Using cycloheximide treatment and surface biotinylation we now demonstrate that the cell surface pool of ENaC has a similarly short half-life (t(1/2) approximately 1 h), unlike the long half-life reported recently for the Xenopus A6 cells. Collectively, these results help elucidate key aspects of ENaC trafficking and turnover rates in mammalian kidney epithelial cells.

Animals↗