Excessive Signal Transduction of Gain-of-Function Variants of the Calcium-Sensing Receptor (CaSR) Are Associated with Increased ER to Cytosol Calcium Gradient

In humans, gain-of-function mutations of the calcium-sensing receptor (CASR) gene are the cause of autosomal dominant hypocalcemia or type 5 Bartter syndrome characterized by an abnormality of calcium metabolism with low parathyroid hormone levels and excessive renal calcium excretion. Functional characterization of CaSR activating variants has been so far limited at demonstrating an increased sensitivity to external calcium leading to lower Ca-EC50. Here we combine high resolution fluorescence based techniques and provide evidence that for the efficiency of calcium signaling system, cells expressing gain-of-function variants of CaSR monitor cytosolic and ER calcium levels increasing the expression of the Sarco-Endoplasmic Reticulum Calcium-ATPase (SERCA) and reducing expression of Plasma Membrane Calcium-ATPase (PMCA). Wild-type CaSR (hCaSR-wt) and its gain-of-function (hCaSR-R990G; hCaSR-N124K) variants were transiently transfected in HEK-293 cells. Basal intracellular calcium concentration was significantly lower in cells expressing hCaSR-wt and its gain of function variants compared to mock. In line, FRET studies using the D1ER probe, which detects [Ca2+]ER directly, demonstrated significantly higher calcium accumulation in cells expressing the gain of function CaSR variants compared to hCaSR-wt. Consistently, cells expressing activating CaSR variants showed a significant increase in SERCA activity and expression and a reduced PMCA expression. This combined parallel regulation in protein expression increases the ER to cytosol calcium gradient explaining the higher sensitivity of CaSR gain-of-function variants to external calcium. This control principle provides a general explanation of how cells reliably connect (and exacerbate) receptor inputs to cell function.     

Hypotonicity causes actin reorganization and recruitment of the actin-binding ERM protein moesin in membrane protrusions in collecting duct principal cells

Hypotonicity-induced cell swelling is characterized by a modification in cell architecture associated with actin cytoskeleton remodeling. The ezrin/radixin/moesin (ERM) family proteins are important signal transducers during actin reorganization regulated by the monomeric G proteins of the Rho family. We report here that in collecting duct CD8 cells hypotonicity-induced cell swelling resulted in deep actin reorganization, consisting of loss of stress fibers and formation of F-actin patches in membrane protrusions where the ERM protein moesin was recruited. Cell swelling increased the interaction between actin and moesin and induced the transition of moesin from an oligomeric to a monomeric functional conformation, characterized by both the COOH- and NH₂-terminal domains being exposed. In this conformation, which is stabilized by phosphorylation of a conserved threonine in the COOH-terminal domain by PKC or Rho kinase, moesin can bind interacting proteins. Interestingly, hypotonic stress increased the amount of threonine-phosphorylated moesin, which was prevented by the PKC- inhibitor Gö-6976 (50 nM). In contrast, the Rho kinase inhibitor Y-27632 (1 µM) did not affect the hypotonicity-induced increase in phosphorylated moesin. The present data represent the first evidence that hypotonicity-induced actin remodeling is associated with phosphorylated moesin recruitment at the cell border and interaction with actin. ezrin/radixin/moesin; protein kinase C; Rho     

The V2 receptor antagonist tolvaptan raises cytosolic calcium and prevents AQP2 trafficking and function: an in vitro and in vivo assessment

Tolvaptan, a selective vasopressin V2 receptor antagonist, is a new generation diuretic. Its clinical efficacy is in principle due to impaired vasopressin‐regulated water reabsorption via aquaporin‐2 (AQP2). Nevertheless, no direct in vitro evidence that tolvaptan prevents AQP2‐mediated water transport, nor that this pathway is targeted in vivo in patients with syndrome of inappropriate antidiuresis (SIAD) has been provided. The effects of tolvaptan on the vasopressin–cAMP/PKA signalling cascade were investigated in MDCK cells expressing endogenous V2R and in mouse kidney. In MDCK, tolvaptan prevented dDAVP‐induced increase in ser256‐AQP2 and osmotic water permeability. A similar effect on ser256‐AQP2 was found in V1aR ?/? mice, thus confirming the V2R selectively. Of note, calcium calibration in MDCK showed that tolvaptan per se caused calcium mobilization from the endoplasmic reticulum resulting in a significant increase in basal intracellular calcium. This effect was only observed in cells expressing the V2R, indicating that it requires the tolvaptan–V2R interaction. Consistent with this finding, tolvaptan partially reduced the increase in ser256‐AQP2 and the water permeability in response to forskolin, a direct activator of adenylyl cyclase (AC), suggesting that the increase in intracellular calcium is associated with an inhibition of the calcium‐inhibitable AC type VI. Furthermore, tolvaptan treatment reduced AQP2 excretion in two SIAD patients and normalized plasma sodium concentration. These data represent the first detailed demonstration of the central role of AQP2 blockade in the aquaretic effect of tolvaptan and underscore a novel effect in raising intracellular calcium that can be of significant clinical relevance.     

Balancing Selection of a Frame-Shift Mutation in the MRC2 Gene Accounts for the Outbreak of the Crooked Tail Syndrome in Belgian Blue Cattle

We herein describe the positional identification of a 2-bp deletion in the open reading frame of the MRC2 receptor causing the recessive Crooked Tail Syndrome in cattle. The resulting frame-shift reveals a premature stop codon that causes nonsense-mediated decay of the mutant messenger RNA, and the virtual absence of functional Endo180 protein in affected animals. Cases exhibit skeletal anomalies thought to result from impaired extracellular matrix remodeling during ossification, and as of yet unexplained muscular symptoms. We demonstrate that carrier status is very significantly associated with desired characteristics in the general population, including enhanced muscular development, and that the resulting heterozygote advantage caused a selective sweep which explains the unexpectedly high frequency (25%) of carriers in the Belgian Blue Cattle Breed.     

IgD-receptor-positive human T lymphocytes. I. Modulation of receptor expression by oligomeric IgD and lymphokines

Studies with human myeloma-derived IgD have demonstrated the existence of IgD-R on peripheral blood T cells. These receptors, which are detected by rosetting with IgD-coated ox E (IgD-rosette-forming cells), are competitively inhibited by IgD, but not by IgM or IgG. Similar results were obtained with human T cell clones and T hybridomas derived from such clones either by rosetting assays or by staining with biotinylated-IgD. In agreement with studies of murine IgD-R+ cells, human IgD-R can be up-regulated by exposure of peripheral blood T cells, T cell clones, and hybridomas derived from such clones, to oligomeric IgD, but not monomeric IgD. Human IgD-R can also be induced by IL-2, IL-4, and IFN-gamma. In contrast with studies of murine IgD-R, which are expressed primarily by CD4+ cells, phenotyping studies show that both the CD4+ and CD8+ human T cell subsets are capable of expressing IgD-R.     

Ethylene and ethane release during tobacco protoplast isolation and protoplast survival potential in vitro

Studies on stress ethylene and ethane during protoplast isolation from water-stressed and waterlogged donor plants Nicotiana tabacum L. xanthi-nc, show a correlation between ethane, but not ethylene, release and protoplast survival in vitro. Ethane release shows a high negative correlation with protoplast survival potential from donor plants subjected to both stresses. Ethylene showed a high negative correlation with protoplast survival potential in tissues from water-stressed but not from long-term waterlogged plants. The absence of correlation in the latter may be related to decreased ability to produce ethylene in hyperstressed plants.
The results are discussed in relation to the use of stress ethane release as a parameter of the physiological status of the plant.     

Facilitated antigen presentation by B cells expressing IgD when responding T cells express IgD-receptors

In vitro studies have confirmed that cognate interactions between T and B cells are required to demonstrate enhanced helper activity using T cells with upregulated IgD-receptors (IgD-Rs). We studied the mechanism by which IgD-R+ T cells facilitate antibody responses by examining whether T cells also benefit from their expression of IgD-R. Experiments were designed to determine whether upregulation of IgD-R on T cells facilitates antigen presentation by IgD+ B cells. Goat Ig-primed splenic T cells from BALB/c mice were tested for their ability to respond to antigen-presenting B cells treated with goat anti-mouse (GAM) IgM or GAM IgD. T cell responses to GAM IgM and GAM IgD presented by B cells were significantly higher when goat Ig-primed cells were induced to express IgD-R by exposure to oligomeric IgD compared with goat Ig-primed control T cells. This effect was inhibited when monomeric IgD was added to the cultures. No differences in T and IgD-R+ T cell responses were seen using adherent cells as APCs. B cells from IgD-/- mice were also tested. Such B cells present antigen to IgD-R+ T cells without promoting enhanced responses compared with B cells from heterozygous IgD+/- mice. These studies suggest that IgD may play a costimulatory role during antigen presentation. We conclude that when T cells are induced to express IgD-R, these lectin-like receptors can ligate B cell membrane IgD during antigen presentation to facilitate responses of each of the cells engaged in cognate interaction, yielding enhanced antigen-specific T cell and B cell responses.     

IgD receptor-mediated signal transduction in T cells

Upregulation of immunoglobulin D-specific receptors (IgD-R) on CD4+ T cells may facilitate their interaction with specific carbohydrate moieties uniquely associated with membrane IgD on B cells. Previous studies have shown that upregulation of IgD-R facilitates cognate T-B cell interactions by mediating bidirectional signaling resulting in increased antibody responses and clonal expansion of antigen-specific T cells. Murine T hybridoma cells, 7C5, constitutively express IgD-R, as has been confirmed by staining with biotinylated IgD. Earlier studies have shown that inhibitors of protein tyrosine kinase (PTK) completely prevented upregulation of IgD-R in response to oligomeric IgD, suggesting that cross-linking of IgD-R may induce signal transduction and functional consequences through one or more PTK activation pathways, leading to upregulation of IgD-R. In the present study we show that cross-linking of IgD-R by oligomeric IgD indeed results in (a) T cell activation as seen by tyrosine phosphorylation of several intracellular proteins, (b) tyrosine phosphorylation of p56 Lck and PLC-gamma in 7C5 T hybridoma cells, and (c) phosphorylation of an approximately 29-kDa band that exhibits strong affinity for IgD. We analyzed tyrosine phosphorylation of p56 Lck and PLC-gamma in BALB/c splenic T cells that were exposed to oligomeric IgD both in vivo and in vitro. In vitro cross-linking as well as in vivo followed by in vitro cross-linking of IgD-R resulted in enhanced phosphorylation of p56 Lck and moderate tyrosine phosphorylation of PLC-gamma. These results suggest that interactions between IgD-R and IgD mediate signal transduction and support our previous findings that IgD-R+ T cells enhance cognate T cell-B cell interactions and antibody production.