scholarly journals Impact of Clinically Relevant Mutations on the Pharmacoregulation and Signaling Bias of the Calcium-Sensing Receptor by Positive and Negative Allosteric Modulators

Endocrinology ◽  
2013 ◽  
Vol 154 (3) ◽  
pp. 1105-1116 ◽  
Author(s):  
Katie Leach ◽  
Adriel Wen ◽  
Anna E. Cook ◽  
Patrick M. Sexton ◽  
Arthur D. Conigrave ◽  
...  
Keyword(s):  
Cell Surface ◽  
Intracellular Signaling ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Allosteric Modulators ◽  
Loss Of Function ◽  
Functional Impairments ◽  
Calcium Sensing Receptor ◽  
Calcium Sensing ◽  
The Impact

Abstract Cinacalcet is predominantly used to treat secondary hyperparathyroidism due to end-stage renal failure, but, more recently, its potential clinical efficacy in treating patients with loss-of-function mutations in the calcium-sensing receptor (CaSR) has been recognized. Many clinically relevant CaSR mutations are located in the heptahelical membrane spanning and extracellular loop regions of the receptor, where allosteric modulators are predicted to bind. The aim of the present study was to investigate the impact of such mutations on the pharmacoregulation of the CaSR by the positive and negative allosteric modulators, cinacalcet and NPS-2143, respectively. Both cinacalcet and NPS-2143 effectively rescued mutants whose cell surface expression was substantially impaired, suggesting that both classes of drug can stabilize a receptor conformation that is trafficked more effectively to the cell surface. In addition, functional impairments in almost all mutant CaSRs were rescued by either cinacalcet or NPS-2143 via restoration of intracellular signaling. There was a significantly greater ability of both compounds to modulate agonist-stimulated intracellular Ca2+ mobilization than ERK1/2 phosphorylation, indicating that the allosteric modulators engender bias in agonist-stimulated CaSR signaling to different pathways. Three mutations (G670R, P748R, and L773R) altered the binding affinity of allosteric modulators to the CaSR, and 3 mutations (V817I, L773R, and E767K) altered the cooperativity between the allosteric modulator and Ca2+o. These findings have important implications for the treatment of diseases associated with CaSR mutations using allosteric CaSR modulators and for analyzing the effects of mutations on the function and pharmacoregulation of the CaSR.

scholarly journals Loss-of-Function and Gain-of-Function Mutations of Calcium-Sensing Receptor: Functional Analysis and the Effect of Allosteric Modulators NPS R-568 and NPS 2143

2013 ◽  
Vol 98 (10) ◽  
pp. E1692-E1701 ◽  
Author(s):  
Akie Nakamura ◽  
Tomoyuki Hotsubo ◽  
Keiji Kobayashi ◽  
Hiroshi Mochizuki ◽  
Katsura Ishizu ◽  
...  
Keyword(s):  
Cell Surface ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Allosteric Modulators ◽  
Familial Hypocalciuric Hypercalcemia ◽  
Calcium Sensing Receptor ◽  
Activating Mutations ◽  
Casr Gene ◽  
Calcium Sensing ◽  
Inactivating Mutations

Abstract Objective: Activating mutations in the calcium-sensing receptor (CASR) gene cause autosomal dominant hypoparathyroidism, and heterozygous inactivating CASR mutations cause familial hypocalciuric hypercalcemia. Recently, there has been a focus on the use of allosteric modulators to restore the functional activity of mutant CASRs. In this study, the effect of allosteric modulators NPS R-568 and NPS 2143 on CASR mutants was studied in vitro. Methods: DNA sequence analysis of the CASR gene was undertaken in autosomal dominant hypoparathyroidism and familial hypocalciuric hypercalcemia Japanese patients, and the functional consequences for the Gi-MAPK pathway and cell surface expression of CASR were determined. Furthermore, we studied the effect of NPS R-568 and NPS 2143 on the signal transduction activity and cell surface expression of each mutant CASR. Results: We identified 3 activating mutations (S122C, P569H, and I839T) and 2 inactivating mutations (A110T and R172G) in patients. The activating and inactivating mutations caused leftward and rightward shifts, respectively, in the dose-response curves of the signaling pathway. NPS R-568 rescued the signal transduction capacity of 2 inactivating mutants without increasing cell surface expression levels. NPS 2143 suppressed the enhanced activity of the activating mutants without altering cell surface expression levels, although A843E, which is a constitutively active mutant, was suppressed to a lesser degree. Conclusions: We have identified 4 novel mutations of CASR. Moreover, our results indicate that allosteric modulators can restore the activity of the loss- and gain-of-function mutant CASRs, identified in this study.

scholarly journals Calcium-sensing Receptor Decreases Cell Surface Expression of the Inwardly Rectifying K+Channel Kir4.1

2010 ◽  
Vol 286 (3) ◽  
pp. 1828-1835 ◽  
Author(s):  
Seung-Kuy Cha ◽  
Chunfa Huang ◽  
Yaxian Ding ◽  
Xiaoping Qi ◽  
Chou-Long Huang ◽  
...  
Keyword(s):  
Cell Surface ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
K Channel ◽  
Calcium Sensing Receptor ◽  
Calcium Sensing ◽  
Inwardly Rectifying

Heterozygous Mutation (Q459R) in the Calcium-Sensing Receptor Gene Causes Familial Hypocalciuric Hypercalcemia 1 (FHH1)

2019 ◽  
Vol 105 (4) ◽  
pp. e1322-e1330 ◽  
Author(s):  
Ida Marie Boisen ◽  
Iris Mos ◽  
Eva Merete Lerche-Black ◽  
Anders Juul ◽  
Hans Bräuner-Osborne ◽  
...  
Keyword(s):  
Receptor Gene ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Familial Hypocalciuric Hypercalcemia ◽  
Loss Of Function ◽  
Wild Type ◽  
Calcium Sensing Receptor ◽  
Calcium Sensing ◽  
Calcium Sensing Receptor Gene

Abstract Context Several heterozygous loss-of-function mutations in the calcium-sensing receptor gene (CASR) leading to elevated ionized serum calcium and familial hypocalciuric hypercalcemia 1 (FHH1) have been characterized. Few mutations are not pathogenic, and previous studies suggested that the Q459R mutation does not result in an FHH1 phenotype. Objective We identified a family with a heterozygous CASR Q459R mutation and characterized their calcium homeostasis and the pathophysiological mechanisms of a homozygous and heterozygous Q459R mutation in vitro. Design The index patient and her family had clinical, biochemical, and genetic analyses performed. In vitro functional characterization of homozygous and heterozygous (Q459R) mutations was conducted by determining CaSR cell-surface expression and inositol monophosphate (IP1) signaling in transiently transfected human embryonic kidney 293A (HEK293A) cells. Results All 3 heterozygous carriers had mild asymptomatic hypercalcemia, hypocalciuria, and 2 had elevated serum parathyroid hormone (PTH). In vitro characterization in HEK293A cells revealed that CASR Q459R is a loss-of-function mutation with no impact on cell-surface expression. Cells with the homozygous Q459R genotype had significantly reduced calcium potency of IP1 signaling compared to wild type, whereas the heterozygous Q459R also had lower calcium potency albeit not significantly different from wild type. Conclusion A loss-of-function Q459R mutation in CASR in a family caused FHH1 characterized by elevated ionized calcium and PTH and low calcium excretion. The marked presence of CaSR at the membrane and inhibition of IP1 signaling in vitro suggest that calcimimetics may be functional in patients with this mutation, which seems to be a mild loss-of-function mutation associated with autosomal dominant transmission of FHH1.

scholarly journals Functional Analysis of the Murine Cytomegalovirus Chemokine Receptor Homologue M33: Ablation of Constitutive Signaling Is Associated with an Attenuated Phenotype In Vivo

2007 ◽  
Vol 82 (4) ◽  
pp. 1884-1898 ◽  
Author(s):  
Ruth Case ◽  
Emma Sharp ◽  
Tau Benned-Jensen ◽  
Mette M. Rosenkilde ◽  
Nicholas Davis-Poynter ◽  
...  
Keyword(s):  
Cell Surface ◽  
Salivary Glands ◽  
Surface Expression ◽  
Receptor Activation ◽  
Murine Cytomegalovirus ◽  
Cell Surface Expression ◽  
Transmembrane Receptors ◽  
C Terminus ◽  
The Impact

ABSTRACT The murine cytomegalovirus (MCMV) M33 gene is conserved among all betaherpesviruses and encodes a homologue of seven-transmembrane receptors (7TMR) with the capacity for constitutive signaling. Previous studies have demonstrated that M33 is important for MCMV dissemination to or replication within the salivary glands. In this study, we probed N- and C-terminal regions of M33 as well as known 7TMR signature motifs in transmembrane (TM) II and TM III to determine the impact on cell surface expression, constitutive signaling, and in vivo phenotype. The region between amino acids R340 and A353 of the C terminus was found to be important for CREB- and NFAT-mediated signaling, although not essential for phosphatidylinositol turnover. Tagging or truncation of the N terminus of M33 resulted in loss of cell surface expression. Within TM II, an F79D mutation abolished constitutive signaling, demonstrating a role, as in other cellular and viral 7TMR, of TM II in receptor activation. In TM III, the arginine (but not the asparagine) residue of the NRY motif (the counterpart of the common DRY motif in cellular 7TMR) was found to be essential for constitutive signaling. Selected mutations incorporated into recombinant MCMV showed that disruption of constitutive signaling for a viral 7TMR homologue resulted in a reduced capacity to disseminate to or replicate in the salivary glands. In addition, HCMV UL33 was found to partially compensate for the lack of M33 in vivo, suggesting conserved biological roles of the UL33 gene family.

scholarly journals The CD28-transmembrane domain mediates chimeric antigen receptor heterodimerization with CD28

2020 ◽  
Author(s):  
Yannick D. Muller ◽  
Duy P. Nguyen ◽  
Leonardo M.R. Ferreira ◽  
Patrick Ho ◽  
Caroline Raffin ◽  
...  
Keyword(s):  
T Cells ◽  
Chimeric Antigen Receptor ◽  
Intracellular Signaling ◽  
Transmembrane Domain ◽  
Antigen Receptor ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Human T Cells ◽  
Engineered T Cells ◽  
The Impact

AbstractAnti-CD19 chimeric antigen receptor (CD19-CAR)-engineered T cells are approved therapeutics for malignancies. The impact of the hinge (HD) and transmembrane (TMD) domains between the extracellular antigen-targeting and the intracellular signaling modalities of CARs has not been systemically studied. Here, a series of CD19-CARs differing only by their HD (CD8/CD28/IgG4) and TMD (CD8/CD28) was generated. CARs containing a CD28-TMD, but not a CD8-TMD, formed heterodimers with the endogenous CD28 in human T cells, as shown by co-immunoprecipitation and CAR-dependent proliferation to anti-CD28 stimulation. This dimerization depended on polar amino-acids in the CD28-TMD. CD28-CAR heterodimerization was more efficient in CARs containing a CD8-HD or CD28-HD as compared to an IgG4-HD. CD28-CAR heterodimers did not respond to CD80 and CD86 stimulation but led to a significant reduction of CD28 cell-surface expression. These data unveil a new property of the CD28-TMD and suggest that TMDs can modulate CAR T-cell activities by engaging endogenous partners.Abstract Figure

Processing and stability of type IIc sodium-dependent phosphate cotransporter mutations in patients with hereditary hypophosphatemic rickets with hypercalciuria

2012 ◽  
Vol 302 (9) ◽  
pp. C1316-C1330 ◽  
Author(s):  
Sakiko Haito-Sugino ◽  
Mikiko Ito ◽  
Akiko Ohi ◽  
Yuji Shiozaki ◽  
Natsumi Kangawa ◽  
...  
Keyword(s):  
Cell Surface ◽  
Polyacrylamide Gel Electrophoresis ◽  
Surface Expression ◽  
Confocal Imaging ◽  
Hypophosphatemic Rickets ◽  
Cell Surface Expression ◽  
Opossum Kidney ◽  
Ok Cells ◽  
Er Retention ◽  
The Impact

Mutations in the apically located Na+-dependent phosphate (NaPi) cotransporter, SLC34A3 (NaPi-IIc), are a cause of hereditary hypophosphatemic rickets with hypercalciuria (HHRH). We have characterized the impact of several HHRH mutations on the processing and stability of human NaPi-IIc. Mutations S138F, G196R, R468W, R564C, and c.228delC in human NaPi-IIc significantly decreased the levels of NaPi cotransport activities in Xenopus oocytes. In S138F and R564C mutant proteins, this reduction is a result of a decrease in the Vmax for Pi, but not the Km. G196R, R468W, and c.228delC mutants were not localized to oocyte membranes. In opossum kidney (OK) cells, cell surface labeling, microscopic confocal imaging, and pulse-chase experiments showed that G196R and R468W mutations resulted in an absence of cell surface expression owing to endoplasmic reticulum (ER) retention. G196R and R468W mutants could be partially stabilized by low temperature. In blue native-polyacrylamide gel electrophoresis analysis, G196R and R468W mutants were either denatured or present in an aggregation complex. In contrast, S138F and R564C mutants were trafficked to the cell surface, but more rapidly degraded than WT protein. The c.228delC mutant did not affect endogenous NaPi uptake in OK cells. Thus, G196R and R468W mutations cause ER retention, while S138F and R564C mutations stimulate degradation of human NaPi-IIc in renal epithelial cells. Together, these data suggest that the NaPi-IIc mutants in HHRH show defective processing and stability.

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