Misfolding of Mutated Vasopressin Causes ER-Retention and Activation of ER-Stress Markers in Neuro-2a Cells

Abstract

Summary Arginine-vasopressin (AVP) is a peptide hormone normally secreted from neuroendocrine cells via the regulated secretory pathway. In Familial Neurohypophyseal Diabetes Insipidus (FNDI), an autosomal dominant form of central diabetes insipidus, mutations of pro-vasopressin appear to accumulate in the endoplasmic reticulum (ER) causing a lack of biologically active AVP in the blood. To investigate the effect of pro-vasopressin mutations regarding intracellular functions of protein targeting and secretion, we created two FNDI-associated amino acid substitution mutants, e.g., G14R, and G17V in frame with green fluorescent protein (GFP) and pro-vasopressin (VP) in frame with red fluorescent protein (VP-RFP). Fluorescence microscopy of Neuro-2a cells expressing these constructs revealed co-localization of VP-GFP and VP-RFP to punctate granules along the length and accumulating at the tips of neurites, characteristic of regulated secretory granules. In contrast, the two FNDI-associated amino acid substitution mutants, e.g., G14R-GFP, and G17VGFP, were localized to a perinuclear region of the Neuro-2a cells characteristic of the endoplasmic reticulum. Co-expression of these mutants with VP-RFP showed VP-RFP was retained in the ER, co-localized with the mutants suggesting the formation of heterodimers as found in FNDI. Stimulated secretion experiments indicated that VP-GFP was secreted in an inducible manner whereas, G14R-GFP and G17V-GFP were retained to nearly 100% within the cells. Analysis by western blotting and semi-quantitative RT-PCR indicated an increased protein and mRNA expression for an ER resident molecular chaperone, BiP. Further analysis of ER-storage disease-associated proteins such as caspase 12 and CHOP showed an increase in these as well. The results suggest that G14R-GFP and G17V-GFP are retained in the ER of Neuro-2a cells, resulting in up-regulation of the molecular chaperone BiP, and activation of the ER-storage disease-associated caspase cascade system.

Loss-of-function PCSK9 mutants evade the unfolded protein response sensor GRP78 and fail to induce endoplasmic reticulum stress when retained

“…To assess whether the accumulation of misfolded proteins does indeed cause UPR activation in hepatocytes, HuH7 cells transfected with either PCSK9 WT or its ER-retention variant PCSK9 Q152H were compared with cells transfected with two VP variants, VP G14R and VP G17V , known to induce ER stress in N2a cells (20,21). Consistent with previous reports, our data demonstrate that ER retention of VP leads to UPR activation as determined by immunoblotting for GRP78 and IRE1␣ ( Fig.…”

“…A well-characterized ERSD occurring in liver results from a number of established mutations in ␣ 1 -antitrypsin Z that induce ER stress, liver cirrhosis, and hepatocellular carcinoma (35,36). Additional examples affecting other tissues include arginine vasopressin mutations (G14R and G17V) in familial neurohypophyseal diabetes insipidus (20,21,37), cystic fibrosis transmembrane conductance regulator ⌬F508 in cystic fibrosis (38), thyroglobulin mutations in congenital goiter and hyperthyroidism (39), and mutations in the Notch receptor that lead to cerebral autosomal-dominant arteriopathy and leukoencephalopathy (40).…”

“…A well-described form of ERSD occurs as a result of ER arginine vasopressin (VP) retention arising from mutations such as VP G14R and VP G17V . These naturally occurring mutations play a central role in the development of familial neurohypophyseal diabetes insipidus in a manner dependent on ER stress and subsequent UPR activation (20,21). The UPR signaling cascade consists of three major transducers located at the ER membrane that are activated upon dissociation of GRP78 from their intra-ER domains.…”

“…C and D, cell viability assays were carried out on transfected HK-2 and HuH7 cells using trypan blue stain and lactate dehydrogenase assays, respectively. E, a lactate dehydrogenase assay was also carried out on cells transfected with VP G17V , a mutant known to induce ER stress as a result of ER retention (21). F, freshly isolated mouse primary hepatocytes were transfected with PCSK9 variants PCSK9 Q152H , PCSK9 Q152D , and PCSK9 S386A and examined for the apoptosis markers caspase-3 and PUMA via real-time PCR.…”

“…The plasmids used in these studies included the bicistronic pIRES2-EGFP plasmids encoding V5-PCSK9 WT and its variants PCSK9 Q152S , PCSK9 Q152H , PCSK9 Q152D , and PCSK9 FS . VP WT and its variants VP G14R and VP G17V were cloned into pEGFP-N1 as described previously (21), and PCSK9 S386A was cloned into a pcDNA3.1 plasmid.…”