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.

scholarly journals Structure/functional aspects of the human riboflavin transporter-3 (SLC52A3): role of the predicted glycosylation and substrate-interacting sites

2017 ◽  
Vol 313 (2) ◽  
pp. C228-C238 ◽  
Author(s):  
Veedamali S. Subramanian ◽  
Subrata Sabui ◽  
Trevor Teafatiller ◽  
Jennifer A. Bohl ◽  
Hamid M. Said
Keyword(s):  
Cell Surface ◽  
Surface Expression ◽  
Protein Docking ◽  
Significant Inhibition ◽  
Confocal Imaging ◽  
Cell Surface Expression ◽  
Molecular Approaches ◽  
Docking Model ◽  
Er Retention

The human riboflavin (RF) transporter-3 (hRFVT-3; product of the SLC52A3 gene) plays an essential role in the intestinal RF absorption process and is expressed exclusively at the apical membrane domain of polarized enterocytes. Previous studies have characterized different physiological/biological aspects of this transporter, but nothing is known about the glycosylation status of the hRFVT-3 protein and role of this modification in its physiology/biology. Additionally, little is known about the residues in the hRFVT-3 protein that interact with the ligand, RF. We addressed these issues using appropriate biochemical/molecular approaches, a protein-docking model, and established intestinal/renal epithelial cells. Our results showed that the hRFVT-3 protein is glycosylated and that glycosylation is important for its function. Mutating the predicted N-glycosylation sites at Asn94 and Asn168 led to a significant decrease in RF uptake; it also led to a marked intracellular (in the endoplasmic reticulum, ER) retention of the mutated proteins as shown by live-cell confocal imaging studies. The protein-docking model used in this study has identified a number of putative substrate-interacting sites: Ser16, Ile20, Trp24, Phe142, Thr314, and Asn315. Mutating these potential interacting sites was indeed found to lead to a significant inhibition in RF uptake and to intracellular (ER) retention of the mutated proteins (except for the Phe142 mutant). These results demonstrate that the hRFVT-3 protein is glycosylated and this glycosylation is important for its function and cell surface expression. This study also identified a number of residues in the hRFVT-3 polypeptide that are important for its function/cell surface expression.

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 Ezrin binding domain-deficient NHERF attenuates cAMP-mediated inhibition of Na+/H+ exchange in OK cells

2001 ◽  
Vol 281 (2) ◽  
pp. F374-F380 ◽  
Author(s):  
Edward J. Weinman ◽  
Deborah Steplock ◽  
James B. Wade ◽  
Shirish Shenolikar
Keyword(s):  
Epithelial Cells ◽  
Surface Expression ◽  
Inhibitory Effect ◽  
Cell Surface Expression ◽  
Wild Type ◽  
Opossum Kidney ◽  
Ok Cells ◽  
Proposed Model ◽  
Exchange Activity ◽  
In Cells

Na+/H+ exchanger regulatory factor (NHERF), an essential protein cofactor in cAMP-mediated inhibition of Na+/H+ exchange transporter 3 (NHE3), facilitates the formation of a signal complex of proteins that includes NHE3, NHERF, and ezrin. This model for NHE3 regulation was developed in fibroblasts and its applicability to epithelial cells remains to be established. Opossum kidney (OK) cells were transfected with either empty vector (control), full-length mouse (m) NHERF(1–355), or a truncated mNHERF(1–325) that lacked ezrin binding and had been demonstrated in fibroblasts to bind NHE3 but not mediate its cAMP-associated inhibition. 8-Bromoadenosine 3′,5′-cyclic monophosphate (8-BrcAMP) at 10−4 M inhibited Na+/H+ exchange activity in control and OK cells expressing wild-type mNHERF(1–355) by >60% but by <10% in cells expressing mNHERF(1–325). NHE3 coimmunoprecipitated with mNHERF(1–325), but cAMP phosphorylation of NHE3 was impaired in cells expressing mNHERF(1–325). The inhibitory effect of hyperosmolality on NHE3 activity and the uptake of 3- O-methyl-d-glucose was the same in all three cell lines. Cell surface expression of NHE3 was not changed by cAMP in any of the cells lines. These data indicate that disruption of the NHERF-ezrin signal complex attenuates the inhibitory effect of cAMP on NHE3 activity in OK cells and provides evidence supporting the proposed model of protein kinase A regulation of NHE3 in epithelial cells.

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 Two-gene control of the expression of a murine Ia antigen.

1978 ◽  
Vol 148 (4) ◽  
pp. 925-939 ◽  
Author(s):  
P P Jones ◽  
D B Murphy ◽  
H O McDevitt
Keyword(s):  
Cell Surface ◽  
Polypeptide Chain ◽  
Polyacrylamide Gel Electrophoresis ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Gene Control ◽  
Cell Surface Molecules ◽  
Surface Molecules ◽  
Ia Antigens ◽  
Spleen Lymphocytes

Two dimensional polyacrylamide gel electrophoresis of Non-Idet P-40 extracts and of specific Ia immunoprecipitates from [35S]methionine-labeled mouse spleen lymphocytes has revealed that the cell surface expression of some Ia antigens appears to be controlled by two genes. One locus, which maps in the I-A subregion, is probably the structural gene for an Ia polypeptide chain. The second locus, which maps between the I-J and H-2D regions, controls whether this I-A encoded molecule (Ae) remains in the cytoplasm or is modified and expressed on the cell surface. Complementation between these two loci allowing surface expression of Ae can occur in the cis or trans chromosomal position. Both the I-A molecule and a polypeptide chain coded for by a locus in I-E are coprecipitated by anti-I-E antibodies, suggesting that these two chains are associated with each other as a multisubunit complex in the cell. Because the ability to complement I-A for Ae expression is a property only of those strains which synthesize an I-E-encoded protein, it is likely that the I-E product itself is regulating the expression of Ae. These observations suggest several mechanisms by which interaction between two I region loci can generate new cell surface molecules. As a result, they may have important implications for understanding the molecular basis of two gene control of immune responsiveness and immune suppression.

scholarly journals Molecular determinants dictating cell surface expression of the human sodium-dependent vitamin C transporter-2 in human liver cells

2010 ◽  
Vol 298 (2) ◽  
pp. G267-G274 ◽  
Author(s):  
Veedamali S. Subramanian ◽  
Jonathan S. Marchant ◽  
Hamid M. Said
Keyword(s):  
Plasma Membrane ◽  
Vitamin C ◽  
Cell Surface ◽  
Hepg2 Cells ◽  
Surface Expression ◽  
Liver Cells ◽  
Confocal Imaging ◽  
Cell Surface Expression ◽  
Molecular Determinants ◽  
Sodium Dependent

The human sodium-dependent vitamin C transporter-2 (hSVCT2) plays an important role in cellular accumulation of ascorbic acid in liver cells. However, little is known about the molecular determinants that direct hSVCT2 to the cell surface in hepatocytes. We addressed this issue using live cell imaging methods to resolve the distribution and trafficking of truncated or mutated hSVCT2 constructs in a cellular model of human hepatocytes, HepG2 cells. Whereas a full-length hSVCT2-yellow fluorescent protein (YFP) fusion protein was functionally expressed at the cell surface in HepG2 cells, serial truncation and mutation analysis demonstrated an essential role for both NH2- and COOH-terminal sequence(s) for cell surface expression and function. Video-rate confocal imaging showed evidence of dynamic hSVCT2-YFP containing intracellular trafficking vesicles, the motility of which was impaired following disruption of microtubules using nocodazole. However, in a HepG2 cell line stably expressing hSVCT2-YFP at the cell surface, plasma membrane levels of hSVCT2 were unaffected by inhibition of microtubule-associated motor proteins; rather, surface expression of hSVCT2-YFP was increased following treatment with myosin inhibitors. Together, these results show that 1) both NH2- and COOH-terminal sequences are essential for proper localization of hSVCT2, 2) cell surface delivery is dependent on intact microtubules, and 3) peripheral microfilaments regulate insertion and retrieval of hSVCT2 into the plasma membrane.

scholarly journals Increased synthesis and expression of H-2 antigens on thymocytes as a result of radiation leukemia virus infection: a possible mechanism for H-2 linked control of virus-induced neoplasia.

1978 ◽  
Vol 147 (2) ◽  
pp. 470-487 ◽  
Author(s):  
D Meruelo ◽  
S H Nimelstein ◽  
P P Jones ◽  
M Lieberman ◽  
H O McDevitt
Keyword(s):  
Virus Infection ◽  
Cell Surface ◽  
Leukemia Virus ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Electrophoretic Analysis ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Altered Expression ◽  
Thymus Cells

Previous studies from this laboratory have mapped resistance and/or susceptibility to radiation-induced leukemia virus (RadLV)-induced neoplasia to the H-2D region. H-2 linked effects on virus replication can be detected subsequent to the initial virus infection, and clear-cut differences in numbers of virus infected thymus cells can be detected as early as 5 wk after RadLV inoculation. Rapid increases in cellular synthesis and cell surface expression of H-2 antigens are detectable immediately after virus inoculation. These changes have been studied by immunofluorescence, absorption, cell surface iodination followed by sodium dodecyl-sulfate-polyacrylamide gel electrophoresis, and two dimensional gel electrophoretic analysis of internally labeled lymphocyte proteins. Expression of H-2K molecules is significantly increased in cells of susceptible and resistant animals. However, significant increases in expression of H-2D antigens occurs only on thymus cells from resistant strains (H-2Dd). Transformed cells of resistant and susceptible H-2 haplotypes adapted to tissue culture lack detectable H-2 antigens as determined by serological absorption studies. It is argued that altered expression of H-2 antigens plays a very significant role in the mechanism of host defense to virus infection.

scholarly journals The rs35112940 CD33 Polymorphism Reduces CD33 Internalization and Efficacy of CD33-Directed Gemtuzumab Ozogamicin

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2247-2247
Author(s):  
Mohammed O Gbadamosi ◽  
Vivek M. Shastri ◽  
Soheil Meshinchi ◽  
Jatinder K. Lamba
Keyword(s):  
Flow Cytometry ◽  
Cell Surface ◽  
Conflicts Of Interest ◽  
Surface Expression ◽  
Gemtuzumab Ozogamicin ◽  
Cell Surface Expression ◽  
Specific Cell ◽  
Antibody Drug Conjugate ◽  
Critical Feature ◽  
The Impact

Abstract Background CD33 is a myeloid-specific cell surface protein widely expressed on acute myeloid leukemia (AML) cells making it an excellent immunotherapeutic target. Current CD33-directed immunotherapeutic treatment strategies include gemtuzumab ozogamicin (GO), an antibody-drug conjugate (ADC) which was approved for the treatment AML in 2017 and has demonstrated promising results thus far. The mechanism of action of GO begins with recognition of CD33 by the antibody portion of GO, followed by internalization of the CD33-GO complex, and finally delivery of free calicheamicin molecules to the cell to induce cellular apoptosis. As such, modifications that impact these steps on any level presumably impact the response and overall efficacy of GO. Indeed, previous studies from our group have identified germline variations in CD33 that are associated with differences in CD33 structure, CD33 cell surface expression levels, and clinical outcomes in response to GO. Among these germline variations is rs35112940 (G&gt;A; Arg304Gly), a missense polymorphism which is located in exon five of CD33 adjacent to the cytoplasmic immunoreceptor tyrosine-based inhibitory motif (ITIM) domain, a critical feature for CD33 internalization. While our previous work identified statistical associations between the A allele of rs35112940 and lower CD33 expression and reduced benefit from treatment using GO, these results are yet to be validated functionally. Additionally, it still remains unknown if the impact of the rs35112940 variation is due to reduced CD33 expression alone or if the rs35112940 variation also impacts CD33 internalization thereby modulating CD33 efficacy. Methods To functionally validate the effect of the rs35112940 variant, we used CRISPR/cas9 to knockout CD33 in HL60 cells and subsequently engineered the HL60-CD33 KO cells to express either wildtype CD33 (HL60-CD33 FL) or CD33 encoding the rs35112940 variant (HL60-CD33 FL-rs35112940). The engineered cells were then treated with GO for 48 hours to capture the impact of the rs35112940 variation on the efficacy of GO. To assess the impact of the rs35112940 variation on CD33 internalization, we performed a flow cytometry-based internalization assay using secondary antibodies to capture the remaining amounts of CD33 present on the cell surface after 4 hours allowing us to determine the internalization of CD33 over time. Results All engineered cells expressed CD33 with less than 1-log fold difference in median fluorescence intensity (MFI) (HL60-CD33 FL MFI vs HL60-CD33 FL-rs35112940 MFI: 22536 vs 24882, Figure 1) and thus we were able to characterize the impact of the rs35112940 variant independent of its impact on CD33 cell surface expression. After 48-hour treatment with 250 ng/mL of GO, we observed that HL60-CD33 FL-rs35112940 cells were more resistant to GO than HL60-CD33 FL cells (66.4% vs 46.5% cell viability, P = 0.02, Figure 2A). Similar results were observed at multiple concentrations of GO. Given the proximity of the rs35112940 loci to the ITIM domain of CD33, we hypothesized that the rs35112940 variation may impact CD33 internalization as well. In a flow cytometry-based internalization assay over a 4-hour window, we observed that that HL60-CD33 FL-rs35112940 cells had an approximate 10% reduction in CD33 internalization in comparison to HL60-CD33 FL cells (Figure 2B). Taken together these results provide insight into the effect of the rs35112940 variant on GO efficacy and CD33 biology, corroborating our previous findings, and support the use of CD33 polymorphisms to guide patient selection for treatment with GO. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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