Nuclear targeting of stanniocalcin to mammary gland alveolar cells during pregnancy and lactation

In most mammalian tissues, the stanniocalcin-1 gene (STC-1) produces a 50-kDa polypeptide hormone known as STC50. Within the ovaries, however, the STC-1 gene generates three higher-molecular-mass variants known as big STC. Big STC is targeted locally to corpus luteal cells to block progesterone release. During pregnancy and lactation, however, ovarian big STC production increases markedly, and the hormone is released into the serum. During lactation, this increase in hormone production is dependent on a suckling stimulus, suggesting that ovarian big STC may have regulatory effects on the lactating mammary gland. In this report, we have addressed this possibility. Our results revealed that virgin mammary tissue contained large numbers of membrane- and mitochondrial-associated STC receptors. However, as pregnancy progressed into lactation, there was a decline in receptor densities on both organelles and a corresponding rise in nuclear receptor density, most of which were on milk-producing, alveolar cells. This was accompanied by nuclear sequestration of the ligand. Sequestered STC resolved as one approximately 135-kDa band in the native state and therefore had the appearance of a big STC variant. However, chemical reduction collapsed this one band into six closely spaced, lower-molecular-mass species (28-41 kDa). Mammary gland STC production also underwent a dramatic shift during pregnancy and lactation. High levels of STC gene expression were observed in mammary tissue from virgin and pregnant rats. However, gene expression then fell to nearly undetectable levels during lactation, coinciding with the rise in nuclear targeting. These findings have thus shown that the mammary glands are indeed targeted by STC, even in the virgin state. They have further shown that there are marked changes in this targeting pathway during pregnancy and lactation, accompanied by a switch in ligand source (endogenous to exogenous). They also represent the first example of nuclear targeting by STC.     

Characterization of big stanniocalcin variants in mammalian adipocytes and adrenocortical cells

The hormone stanniocalcin (STC) is widely distributed, and in rodents the highest levels of expression are in the ovaries. In both cows and rodents, ovarian STC consists of three high-molecular-weight variants collectively known as big STC. In the ovary, big STC is made by theca cells and interstitial cells and is targeted to lipid storage droplets of nearby luteal cells to inhibit progesterone release. An endocrine pathway is operative during pregnancy and lactation. Whether or not big STC is made by tissues other than ovary has never been addressed. Therefore, the purpose of this study was to determine via a detailed characterization of adrenal glands and adipocytes whether big STC is present in other cells that store and release lipids. The results showed that STC was made in bovine and mouse adrenals, mainly in steroidogenic, adrenocortical cells. The majority of ligand and receptor were likewise confined to cortical zone cells. As in luteal cells, high levels of ligand and receptor were found in the adrenocortical cell lipid droplet fraction. However, adrenals made only the largest (135 kDa) of the three big STC variants. Nonetheless, adrenal STC had much greater receptor affinity than a mixture of the three big STC variants. Adipocytes contained all three big STC variants, and both ligand and receptor were heavily concentrated on the lipid droplets. Moreover, adipocyte lipid storage droplets had 50-fold more receptors than those in steroidogenic cells, indicating that big STC is heavily targeted to adipose cells. The findings collectively support the hypothesis that big STC is not unique to ovarian steroidogenic cells but is in fact common to cells with a role in lipid storage and release.     

斯钙素的研究进展

斯钙素(stanniocalcin,STC)是一种糖蛋白激素,最早在硬骨鱼中发现,起着调节钙/磷平衡的作用.近年来在人和其它哺乳动物中发现也存在STC,先后分别命名为STC1和STC2.STC1基因可以产生两种形式的STC:一个是分子量为50kD的多肽,被称作STC50;另一种是一组分子量较大的不同形式的STC,被统称为big STC.STC1和STC2均可广泛表达于各种组织.STC成为一种新的肿瘤标志物,并且在心血管疾病、炎症细胞迁移、胚泡着床和子宫的蜕膜化等多方面都起重要作用.     

Evidence for stanniocalcin and a related receptor in annelids

Stanniocalcin (STC) is a prime example of a hormone whose discovery in fish led to its subsequent discovery in mammals. STC is considered to be first and foremost a vertebrate polypeptide hormone with regulatory effects on ion transport, mitochondrial function and steroid hormone synthesis. The gene is widely expressed in both fishes and mammals, and the hormone can operate via both local and endocrine signaling pathways. In spite of the growing catalogue of vertebrate hormones and receptors with homologues in invertebrates, the notion that there might be an invertebrate STC homolog has received scant attention to date. In the present study, we have provided evidence for STC in annelid worms (freshwater leeches). Western blot analysis revealed the presence of two STC immunoreactive (STCir) proteins in leech tissue extracts of 100 and 193 kDa. These same extracts significantly lowered the rate of gill calcium transport upon injection into fish. Similarly, fish STC increased the rate of whole body calcium uptake when administered to leeches, and STC receptors of high affinity were identified on isolated leech plasma membranes. Two discrete populations of STC-positive cells were also identified in leeches using antibodies to fish STC and fish STC cRNA probes. One of the cell types was confined to the skin. The second cell type was confined to the coelomic cavity and identified as an adipose cell, which in leeches is a major repository of fat. Collectively, the data constitutes compelling evidence for the existence of STC-related proteins and receptors in annelids that share structural and functional similarities with those in vertebrates.     

Characterization of mammalian stanniocalcin receptors. Mitochondrial targeting of ligand and receptor for regulation of cellular metabolism

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.     

Absence of specific luteinizing hormone releasing hormone receptors in ovine, bovine and porcine ovaries

Crude and membrane-enriched homogenates of unfrozen follicular and luteal tissue from cows, ewes and sows were assayed for the presence of specific luteinizing hormone releasing hormone (LHRH) receptors by one-point saturation analysis using [D-Ser-(TBU)6, des-Gly-NH2(10)] LHRH-EA as the labeled and unlabeled ligand. Pituitaries from cows, ewes, sows and rats, and rat ovaries served as positive controls and were assayed with each ovarian tissue assay. Scatchard analysis was used to determine binding affinity of pools of ovarian and pituitary tissue. Specific high-affinity LHRH receptors were found in the pituitaries of cows, ewes, sows and rats and in the rat ovary. In contrast, no specific LHRH binding was detected in follicular or luteal tissue of cows, ewes or sows. Thus, unlike the rat ovary which contains LHRH receptors, ovaries from these domestic species lack specific LHRH receptors.     

A silent H-bond can be mutationally activated for high-affinity interaction of BMP-2 and activin type IIB receptor

Background  

Bone morphogenetic proteins (BMPs) are key regulators in the embryonic development and postnatal tissue homeostasis in all animals. Loss of function or dysregulation of BMPs results in severe diseases or even lethality. Like transforming growth factors β (TGF-βs), activins, growth and differentiation factors (GDFs) and other members of the TGF-β superfamily, BMPs signal by assembling two types of serine/threonine-kinase receptor chains to form a hetero-oligomeric ligand-receptor complex. BMP ligand receptor interaction is highly promiscuous, i.e. BMPs bind more than one receptor of each subtype, and a receptor bind various ligands. The activin type II receptors are of particular interest, since they bind a large number of diverse ligands. In addition they act as high-affinity receptors for activins but are also low-affinity receptors for BMPs. ActR-II and ActR-IIB therefore represent an interesting example how affinity and specificity might be generated in a promiscuous background.     

A theoretical model for adhesion between cells mediated by multivalent ligands

A theoretical model is developed for cell-to-cell binding by bivalent ligands that can bind to mobile receptors on the cell surfaces. Monovalent inhibitors that can bind either to receptors or ligands are also included. For symmetrical ligands, that is, ligands in which both binding sites are the same, it is shown that crosslinking of receptors on each cell will interfere with intercellular bridge formation. At equilibrium, such interference is not drastic, but if the crosslinks can form before the cells are brought into contact, crosslinking may greatly impede the rate of intercellular binding. Comparison is made with experiments, and the importance of receptor mobility is discussed. It is noted that ligands can also bind a cell to itself or to a surface.     

A theoretical model for adhesion between cells mediated by multivalent ligands.

A theoretical model is developed for cell-to-cell binding by bivalent ligands that can bind to mobile receptors on the cell surfaces. Monovalent inhibitors that can bind either to receptors or ligands are also included. For symmetrical ligands, that is, ligands in which both binding sites are the same, it is shown that crosslinking of receptors on each cell will interfere with intercellular bridge formation. At equilibrium, such interference is not drastic, but if the crosslinks can form before the cells are brought into contact, crosslinking may greatly impede the rate of intercellular binding. Comparison is made with experiments, and the importance of receptor mobility is discussed. It is noted that ligands can also bind a cell to itself or to a surface.     

Model of glycoprotein hormone receptor ligand binding and signaling

Studies described here were initiated to develop a model of glycoprotein hormone receptor structure and function. We found that the region that links the lutropin receptor leucine-rich repeat domain (LRD) to its transmembrane domain (TMD) has substantial roles in ligand binding and signaling, hence we term it the signaling specificity domain (SSD). Theoretical considerations indicated the short SSDs in marmoset lutropin and salmon follitropin receptors have KH domain folds. We assembled models of lutropin, follitropin, and thyrotropin receptors by aligning models of their LRD, TMD, and shortened SSD in a manner that explains how substitutions in follitropin and thyrotropin receptors distant from their apparent ligand binding sites enable them to recognize lutropins. In these models, the SSD is parallel to the concave surface of the LRD and makes extensive contacts with TMD outer loops 1 and 2. The LRD appears to contact TMD outer loop 3 and a few residues in helices 1, 5, 6, and 7. We propose that signaling results from contacts of the ligands with the SSD and LRD that alter the LRD, which then moves TMD helices 6 and 7. The positions of the LRD and SSD support the notion that the receptor can be activated by hormones that dock with these domains in either of two different orientations. This would account for the abilities of some ligands and ligand chimeras to bind multiple receptors and for some receptors to bind multiple ligands. This property of the receptor may have contributed significantly to ligand-receptor co-evolution.     

Influence of targeting ligand flexibility on receptor binding of particulate drug delivery systems

Receptor-mediated drug targeting via nanoengineered particulate delivery systems is an emerging field. However, little is known about how such magic bullets should be assembled to yield optimal targeting efficiency. Here we investigated the influence of targeting ligand flexibility on binding of ligand-coated microparticles to cell surface receptors. Using the ganglioside G(M1)-binding B subunit of cholera toxin as ligand and fluorescent microparticles as a model delivery system, conjugates with different numbers of linkages between ligand and particle were prepared and tested for their efficiency to bind to live fibroblast monolayers. Our results show that multiple bonds between ligand and particle reduce the targeting rate by up to 50% compared to constructs where ligands are attached via single aliphatic chains. Thus, for maximum performance, targeted particulate drug delivery systems should be assembled such that ligands are attached via single sigma bonds only, allowing the ligand molecules to adopt an optimal binding conformation.     

Genuine monovalent ligands of TrkA nerve growth factor receptors reveal a novel pharmacological mechanism of action

Developing small molecule agonistic ligands for tyrosine kinase receptors has been difficult, and it is generally thought that such ligands require bivalency. Moreover, multisubunit receptors are difficult to target, because each subunit contributes to ligand affinity, and each subunit may have distinct and sometimes opposing functions. Here, the nerve growth factor receptor subunits p75 and the tyrosine kinase TrkA were studied using artificial ligands that bind specifically to their extracellular domain. Bivalent TrkA ligands afford robust signals. However, genuine monomeric and monovalent TrkA ligands afford partial agonism, activate the tyrosine kinase activity, cause receptor internalization, and induce survival and differentiation in cell lines and primary neurons. Monomeric and monovalent TrkA ligands can synergize with ligands that bind the p75 subunit. However, the p75 ligands used in this study must be bivalent, and monovalent p75 ligands have no effect. These findings will be useful in designing and developing screens of small molecules selective for tyrosine kinase receptors and indicate that strategies for designing agonists of multisubunit receptors require consideration of the role of each subunit. Last, the strategy of using anti-receptor mAbs and small molecule hormone mimics as receptor ligands could be applied to the study of many other heteromeric cell surface receptors.     

Glucocorticoid hormone receptors in rat pancreas

Although glucocorticoiods influence pancreatic function, it has not been established whether they act directly at the level of the pancreas, or indirectly by causing metabolic changes in other target tissues. As a step in elucidating the actions of glucocorticoids on the pancreas, a search was conducted for glucocorticoid hormone receptors in this tissue. Uptake and binding studies indicated that there were glucocorticoid hormone receptors in the high-speed cytosolic extract of rat pancreas. These receptors appear to be similar to other rat glucocorticoid receptors: they bind glucocorticoids rapidly in a reversible manner at 0°C, competitive binding analysis studies show that they have a preference for glucocorticoids and, like receptors, bind the synthetic steroids triamcinolone acetonide and dexamethasone with a higher affinity than corticosterone. Scatchard analysis demonstrated that there are 1.37 · 10^?13 mol glucocorticoid-binding sites/mg cytosolic protein. This demonstration of a glucocorticoid hormone receptor in pancreatic cytosol suggests that some of the effects glucocorticoids exert on pancreatic function are a consequence of their direct actions on this target tissue.     

A low molecular weight agonist signals by binding to the transmembrane domain of thyroid-stimulating hormone receptor (TSHR) and luteinizing hormone/chorionic gonadotropin receptor (LHCGR)

Many cognate low molecular weight (LMW) agonists bind to seven transmembrane-spanning receptors within their transmembrane helices (TMHs). The thienopyrimidine org41841 was identified previously as an agonist for the luteinizing hormone/chorionic gonadotropin receptor (LHCGR) and suggested to bind within its TMHs because it did not compete for LH binding to the LHCGR ectodomain. Because of its high homology with LHCGR, we predicted that thyroid-stimulating hormone receptor (TSHR) might be activated by org41841 also. We show that org41841 is a partial agonist for TSHR but with lower potency than for LHCGR. Analysis of three-dimensional molecular models of TSHR and LHCGR predicted a binding pocket for org41841 in common clefts between TMHs 3, 4, 5, 6, and 7 and extracellular loop 2 in both receptors. Evidence for this binding pocket was obtained in signaling studies with chimeric receptors that exhibited improved responses to org41841. Furthermore, a key receptor-ligand interaction between the highly conserved negatively charged E3.37 and the amino group of org41841 predicted by docking of the ligand into the three-dimensional TSHR model was experimentally confirmed. These findings provide the first evidence that, in contrast to the ectodomain binding of cognate ligands, a LMW agonist can bind to and activate glycoprotein hormone receptors via interaction with their transmembrane domain.