Characterization of Mammalian Stanniocalcin Receptors

Abstract

The polypeptide hormone stanniocalcin (STC) is widely expressed in mammalian tissues. STC acts locally in kidney and gut to modulate calcium and phosphate excretion, and its overexpression in mice results in high serum phosphate, dwarfism, and increased metabolic rate. STC has also been linked to cancer, pregnancy, lactation, angiogenesis, organogenesis, cerebral ischemia, and hypertonic stress. In this report we have characterized the STC receptor and the functional targeting of ligand and receptor to mitochondria. For receptor binding analysis, a stanniocalcin-alkaline phosphatase fusion protein was engineered. Subsequent binding assays using the fusion protein indicated that kidney and liver contained the highest number of binding sites with affinities of 0.8 and 0.25 nM, respectively. Intriguingly, purified mitochondria from both tissues yielded similar high affinity binding sites. Fractionation analysis revealed that the majority of binding sites were localized to the inner mitochondrial membrane. In further studies, we characterized the time course of STC-alkaline phosphatase fusion protein sequestration by intact mitochondria. In situ ligand binding also revealed discrete, displaceable binding to plasma membranes and mitochondria of nephron cells and liver hepatocytes. The existence of mitochondrial receptors prompted a similar search for the ligand. Immunogold electron microscopy revealed that STC was preferentially concentrated in the mitochondria of all nephron segments targeted by STC. Subcellular fractionation revealed that >90% of cellular STC immunoreactivity was mitochondrial, confined to the inner matrix, and similar in size to recombinant STC (50 kDa). In functional studies, recombinant STC had concentration-dependent stimulatory effects on electron transfer by sub-mitochondrial particles. Collectively the evidence implies a role for STC in cell metabolism.

Targeting of Big Stanniocalcin and Its Receptor to Lipid Storage Droplets of Ovarian Steroidogenic Cells

“…However, most of the STC was confined to the cytoplasm, and the majority of this was associated with the CLD fraction where it co-localized with perilipin, indicating that big STC is distinct both structurally and in its sub-cellular targeting. To determine whether this CLD targeting of the ligand was receptor-mediated, we analyzed the sub-cellular distribution of receptors by employing the same STP⅐AP fusion protein technology that was used to characterize mitochondrial receptors in rodent liver and kidney (2). The results revealed that this was indeed the case.…”

“…Interestingly, STC protein is found not only in TICs but also in oocytes and cells of the corpus luteum (1), suggesting that the STC is produced by TICs for targeting to oocytes and luteal cells. The fact that the protein is readily detectable in cells that do not make the hormone further suggests that, as in the case of STC 50 signaling in liver and kidney (2), ovarian STC is also sequestered by its target cells. An important question here is whether or not ovarian STC undergoes the same mitochondrial targeting as observed in liver and kidney.…”

“…In this regard, we recently used an STC⅐alkaline phosphatase (AP) fusion protein (STC⅐AP) to characterize receptors and delineate the mitochondrial targeting of STC 50 in liver and kidney (2). The objective of this study, therefore, was to use the same methodology to characterize STC receptors in ovarian luteal cells and thereby address the question as to whether or not big STC is similarly targeted.…”

“…Receptor Binding Assays/Receptor Localization-In situ ligand binding and receptor binding assays were performed on whole luteal cells and purified plasma membranes as described previously using an STC⅐AP fusion protein (2). Receptor binding studies were also performed on cholesterol/lipid storage droplet (CLD) isolates from bovine corpus luteum (9).…”

“…Following centrifugation, free and CLD-bound STC⅐AP fusion proteins were separated by aspiration of the infranatant with a 23-gauge needle. AP assay buffer was then added to the remaining supernatant containing the CLD fraction, and AP activity was detected as described previously (2).…”