PubMed · 6049072
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F L Bygrave. 1967-05-13. The ionic environment and metabolic control.. https://doi.org/10.1038/214667a0
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The low density lipoprotein receptor-related protein (LRP) is a cysteine-rich, multifunctional receptor that binds and endocytoses a diverse array of ligands. Recent studies have shown that a 39-kDa receptor-associated protein (RAP) facilitates the proper folding and subsequent trafficking of LRP within the early secretory pathway. In the current study, we have examined the potential role of Ca2+ and its relationship to RAP during LRP folding. We found that depletion of Ca2+ following either ionomycin or thapsigargin treatment significantly disrupts the folding process of LRP. The misfolded LRP molecules migrate as high molecular weight aggregates under nonreducing SDS-polyacrylamide gel electrophoresis, suggesting the formation of intermolecular disulfide bonds. This misfolding is reversible because misfolded LRP can be re-folded into functional receptor molecules upon Ca2+ restoration. Using an LRP minireceptor representing the fourth ligand binding domain of LRP, we also observed significant variation in the conformation of monomeric receptor upon Ca2+ depletion. The role of Ca2+ in LRP folding is independent from that of RAP because RAP remains bound to LRP and its minireceptor following Ca2+ depletion. Furthermore, Ca2+ depletion-induced LRP misfolding occurs in RAP-deficient cells. Taken together, these results clearly demonstrate that Ca2+ and RAP independently participate in LRP folding.
We previously showed that 6-trans isomers of leukotriene B4 but not leukotriene B4 itself are converted to dihydro metabolites by human neutrophils. The first step in the formation of these metabolites is oxidation of the 5-hydroxyl group by 5-hydroxyeicosanoid dehydrogenase. The objective of the present investigation was to characterize the second step in the formation of the dihydro metabolites, reduction of an olefinic double bond. We found that the olefin reductase reduces the 6,7-double bond of 5-oxoeicosanoids, is localized in the cytosolic fraction of neutrophils, and requires NADPH as a cofactor. Neutrophil cytosol converts a variety of both 5-oxo- and 15-oxoeicosanoids to dihydro products. However, conversion of 5-oxoeicosanoids to their 6,7-dihydro metabolites is inhibited by EGTA and a calmodulin antagonist and stimulated by the addition of calcium and calmodulin, whereas the reduction of 15-oxoeicosanoids to their 13,14-dihydro metabolites is slightly inhibited by calcium. Furthermore, eicosanoid Delta6- and Delta13-reductases could be separated by chromatography on DEAE-Sepharose. 5-Oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) is converted by the Delta6-reductase to 6,7-dihydro-5-oxo-ETE, which is 1000 times less potent than 5-oxo-ETE in mobilizing calcium in neutrophils. We conclude that neutrophils contain both 5-oxoeicosanoid Delta6-reductase and prostaglandin Delta13-reductase. Metabolism of 5-oxo-ETE by the Delta6-reductase results in loss of its biological activity.
Previous studies in parathyroid cells, which express the G protein-coupled, extracellular calcium-sensing receptor (CaR), showed that activation of protein kinase C (PKC) blunts high extracellular calcium (Ca2+o)-evoked stimulation of phospholipase C and the associated increases in cytosolic calcium (Ca2+i), suggesting that PKC may directly modulate the coupling of the CaR to intracellular signaling systems. In this study, we examined the role of PKC in regulating the coupling of the CaR to Ca2+i dynamics in fura-2-loaded human embryonic kidney cells (HEK293 cells) transiently transfected with the human parathyroid CaR. We demonstrate that several PKC activators exert inhibitory effects on CaR-mediated increases in Ca2+i due to release of Ca2+ from intracellular stores. Consistent with the effect being mediated by activation of PKC, the inhibitory effect of PKC activators on Ca2+ release can be blocked by a PKC inhibitor. The use of site-directed mutagenesis reveals that threonine at amino acid position 888 is the major PKC site that mediates the inhibitory effect of PKC activators on Ca2+ mobilization. The effect of PKC activation can be maximally blocked by mutating three PKC sites (Thr888, Ser895, and Ser915) or all five PKC sites. In vitro phosphorylation shows that Thr888 is readily phosphorylated by PKC. Our results suggest that phosphorylation of the CaR is the molecular basis for the previously described effect of PKC activation on Ca2+o-evoked changes in Ca2+i dynamics in parathyroid cells.