Modulation of mannose receptor activity by proteolysis.

The binding, internalization and degradation of 200 pM monoiodinated human atrial natriuretic factor-(99-126) (125I-hANF) by cultured bovine aortic endothelial cells (BAECs) were studied at 37 degrees C. 125I-hANF was rapidly cleared from the extracellular medium (t1/2 approximately 10 min), whereas preincubation of the cells in the presence of 20 mM-NH4Cl or 0.2 mM-chloroquine resulted in a significant inhibition of this process. The BAECs rapidly produce three major degradation products of 125I-hANF, namely [125I]iodotyrosine 126 (125I-Y), Arg125-[125I]iodotyrosine126 (125I-RY) and Phe124-Arg125-[125I]iodotyrosine126(125I-FRY), which were detected in the extracellular medium. NH4Cl and chloroquine acted to inhibit the generation of 125I-Y and 125I-RY, but not that of 125I-FRY. Furthermore, excess unlabelled hANF (300 nM) completely blocked the rapid production of 125I-Y and 125I-RY in the first 5 min, but only partially (49%) inhibited the generation of 125I-FRY. Thus, in contrast with our previous findings with cultured smooth-muscle cells [Johnson, Arik & Foster (1989) J. Biol. Chem. 264, 11637-11642], BAECs bind, internalize and rapidly degrade 125I-hANF, resulting in the release of 125I-Y and 125I-RY into the extracellular medium. Similarly to smooth-muscle cells, the BAECs generate 125I-FRY from 125I-hANF via an extracellular proteolytic event. The rapidity of the receptor-mediated process and its sensitivity to NH4Cl and chloroquine suggest that the 125I-hANF is proteolytically processed in the endosomes of BAECs and that its receptors cycle between the cell surface and intracellular stores.     

Activin-A up-regulates type I activin receptor mRNA levels in human immortalized extravillous trophoblast cells.

Activin is known to play an important regulatory role in reproduction, including pregnancy. To further examine the role and signaling mechanism of activin in regulating placental function, the steady-state level of activin type I receptor (ActRI) mRNA in immortalized extravillous trophoblasts (IEVT) cells was measured using competitive PCR (cPCR). An internal standard of ActRI cDNA for cPCR was constructed for the quantification of ActRI mRNA levels in IEVT cells. ActRI mRNA levels were increased in a dose-dependent manner by activin-A with the maximal effect observed at the dose of 10 ng/ml. Time course studies revealed that activin-A had maximal effects on ActRI mRNA levels at 6 hours after treatment. The effects of activin-A on ActRI mRNA levels was blocked by follistatin, an activin binding protein, in a dose-dependent manner. In addition, inhibin-A inhibited basal, as well as activin-A-induced ActRI mRNA levels. These findings provide evidence, for the first time, that activin-A modulates ActRI mRNA levels in human trophoblast cells.     

Pulmonary surfactant protein A up-regulates activity of the mannose receptor,a pattern recognition receptor expressed on human macrophages

Inhaled particulates and microbes are continually cleared by a complex array of lung innate immune determinants, including alveolar macrophages (AMs). AMs are unique cells with an enhanced capacity for phagocytosis that is due, in part, to increased activity of the macrophage mannose receptor (MR), a pattern recognition receptor for various microorganisms. The local factors that "shape" AM function are not well understood. Surfactant protein A (SP-A), a major component of lung surfactant, participates in the innate immune response and can enhance phagocytosis. Here we show that SP-A selectively enhances MR expression on human monocyte-derived macrophages, a process involving both the attached sugars and collagen-like domain of SP-A. The newly expressed MR is functional. Monocyte-derived macrophages on an SP-A substrate demonstrated enhanced pinocytosis of mannose BSA and phagocytosis of Mycobacterium tuberculosis lipoarabinomannan-coated microspheres. The newly expressed MR likely came from intracellular pools because: 1) up-regulation of the MR by SP-A occurred by 1 h, 2) new protein synthesis was not necessary for MR up-regulation, and 3) pinocytosis of mannose BSA via MR recycling was increased. AMs from SP-A(-/-) mice have reduced MR expression relative to SP-A(+/+). SP-A up-regulation of MR activity provides a mechanism for enhanced phagocytosis of microbes by AMs, thereby enhancing lung host defense against extracellular pathogens or, paradoxically, enhancing the potential for intracellular pathogens to enter their intracellular niche. SP-A contributes to the alternative activation state of the AM in the lung.     

Dexamethasone up-regulates type II IL-1 receptor in mouse primary activated astrocytes

Brain astrocytes play a pivotal role in the brain response to inflammation. They express IL-1 receptors including the type I IL-1 receptor (IL-1RI) that transduces IL-1 signals in cooperation with the IL-1 receptor accessory protein (IL-1RAcP) and the type II IL-1 receptor (IL-1RII) that functions as a decoy receptor. As glucocorticoid receptors are expressed on astrocytes, we hypothesized that glucocorticoids regulate IL-1 receptors expression. IL-1beta-activated mouse primary astrocytes were treated with 10(-6) M dexamethasone, and IL-1 receptors were studied at the mRNA and protein levels. Using RT-PCR, IL-1RI and IL-1RII but not IL-1RAcP mRNAs were found to be up-regulated by dexamethasone in a time-dependent manner. Dexamethasone (Dex), but not progesterone, had no effect on IL-1RI but strongly increased IL-1RII mRNA expression. Binding studies revealed an increase in the number of IL-1RII binding sites under the effect of Dex, but no change in affinity. These findings support the concept that glucocorticoids have important regulatory effect on the response of astrocytes to IL-1.     

Ligand recognition by purified human mannose receptor.

In this work we examine the carbohydrate binding properties of human placental mannose receptor (HMR) using a rapid and sensitive enzyme-linked immunosorbent microplate assay. The assay is based on the inhibition of binding of highly purified receptor to yeast mannan-coated 96-well plates. The specificity of ligand binding was inferred from the potency of different saccharides in blocking HMR binding to the mannan-coated wells. The relative inhibitory potency of monosaccharides was L-Fuc greater than D-Man greater than D-Glc greater than D-GlcNAc greater than Man-6-P much greater than D-Gal much greater than L-Rha much greater than GalNAc. The inhibitory potency of mannose increased by two orders of magnitude when linear oligomers were used. Oligomers containing alpha-1-3- and alpha-1-6-linked mannose residues were more inhibitory than those containing alpha-1-2- and alpha-1-4-linked mannoses. Linear or branched oligomannosides larger than three units did not have a significant influence on their inhibitory potency; rather, potency was found to decrease in comparison with oligomannosides with three units. Compared to linear oligomers, inhibition of binding was the best using branched mannose oligosaccharides, alpha-D-Man-bovine serum albumin conjugates, or mannan. A model is discussed in which branched ligand is bound to spatially distinct sites on the HMR.     

Dexamethasone and estrogen regulate Xenopus laevis albumin mRNA levels

The regulation of albumin mRNA levels by steroid hormones was examined in a primary Xenopus liver culture system. In the absence of exogenous steroid hormone, albumin mRNA levels declined rapidly in culture. Dexamethasone is required for preservation of albumin mRNA levels in culture and effects a greater than 10 fold induction of albumin mRNA in cultures maintained in steroid hormone-free medium. Estrogen can override the effect of dexamethasone and elicits a decline of greater than 80% in albumin mRNA levels. Our cDNA clones of the mRNAs encoding the two 74,000 dalton and one 72,000 dalton cellular albumins allowed us to show that all three albumin mRNAs were down regulated by estrogen.     

Calcitonin gene-related peptide and muscle activity regulate acetylcholine receptor alpha-subunit mRNA levels by distinct intracellular pathways

In cultured chicken myotubes, calcitonin gene-related peptide (CGRP), a peptide present in spinal cord motoneurons, increased by 1.5-fold the number of surface acetylcholine receptors (AChRs) and by threefold AChR alpha-subunit mRNA level without affecting the level of muscular alpha-actin mRNA. Cholera toxin (CT), an activator of adenylate cyclase, produced a similar effect, which did not add up with that of CGRP. In contrast, tetrodotoxin, a blocker of voltage-sensitive Na+ channels, elevated the level of AChR alpha-subunit mRNA on top of the increase caused by either CGRP or CT. 12-O-Tetradecanoyl phorbol-13-acetate (TPA), an activator of protein kinase C, markedly decreased the cell surface and total content of [125I]alpha BGT-binding sites and reduced the rate of appearance of AChR at the surface of the myotubes without reducing the level of AChR alpha-subunit mRNA. Moreover, TPA inhibited the increase of AChR alpha-subunit mRNA caused by tetrodotoxin without affecting that produced by CGRP or CT. Under the same conditions, TPA decreased the level of muscular alpha-actin mRNA and increased that of nonmuscular beta- and gamma-actins mRNA. These data suggest that distinct second messengers are involved in the regulation of AChR biosynthesis by CGRP and muscle activity and that these two pathways may contribute to the development of different patterns of AChR gene expression in junctional and extrajunctional areas of the muscle fiber.     

Developmental regulation of insulin-like growth factor-II/mannose 6-phosphate receptor mRNA in the rat.

This study examined levels of insulin-like growth factor-II/mannose 6-phosphate receptor (IGF-II/M6PR) mRNA in tissues of rats at different stages of growth. Northern blot analysis of total RNA from tissues of rats aged 2, 9, 21 and 42 days and from 21 day fetal rats was carried out using a cDNA probe to the IGF-II/M6PR. Northern blots showed this probe hybridized to a single 9kb band in all tissues tested. Highest hybridization signals were detected in fetal and neonatal tissues with levels rapidly decreasing after birth. For all age groups tested the highest signal was obtained with heart followed by muscle, lung, and kidney, with liver and brain showing lower levels of message. These results indicate that IGF-II/M6PR mRNA is developmentally regulated, and suggest a role for the IGF-II/M6PR in fetal and neonatal growth.     

Structural basis for recognition of phosphorylated high mannose oligosaccharides by the cation-dependent mannose 6-phosphate receptor.

Mannose 6-phosphate receptors (MPRs) play an important role in the targeting of newly synthesized soluble acid hydrolases to the lysosome in higher eukaryotic cells. These acid hydrolases carry mannose 6-phosphate recognition markers on their N-linked oligosaccharides that are recognized by two distinct MPRs: the cation-dependent mannose 6-phosphate receptor and the insulin-like growth factor II/cation-independent mannose 6-phosphate receptor. Although much has been learned about the MPRs, it is unclear how these receptors interact with the highly diverse population of lysosomal enzymes. It is known that the terminal mannose 6-phosphate is essential for receptor binding. However, the results from several studies using synthetic oligosaccharides indicate that the binding site encompasses at least two sugars of the oligosaccharide. We now report the structure of the soluble extracytoplasmic domain of a glycosylation-deficient form of the bovine cation-dependent mannose 6-phosphate receptor complexed to pentamannosyl phosphate. This construct consists of the amino-terminal 154 amino acids (excluding the signal sequence) with glutamine substituted for asparagine at positions 31, 57, 68, and 87. The binding site of the receptor encompasses the phosphate group plus three of the five mannose rings of pentamannosyl phosphate. Receptor specificity for mannose arises from protein contacts with the 2-hydroxyl on the terminal mannose ring adjacent to the phosphate group. Glycosidic linkage preference originates from the minimization of unfavorable interactions between the ligand and receptor.     

Acetylcholine receptor alpha-, beta-, gamma-, and delta-subunit mRNA levels are regulated by muscle activity

Denervation of adult skeletal muscle results in increased sensitivity to acetylcholine in extrajunctional regions of the muscle fiber. This increase in acetylcholine sensitivity is accompanied by a large increase in the level of mRNAs coding for the alpha-, beta-, gamma-, and delta-subunits of the acetylcholine receptor. To determine whether muscle activity is sufficient to regulate expression of extrajunctional acetylcholine receptor mRNA levels, denervated muscles were stimulated with extracellular electrodes. Direct stimulation of denervated muscle suppresses both the increase in extrajunctional acetylcholine sensitivity and the expression of mRNA encoding the alpha-, beta-, gamma-, and delta-subunits of the acetylcholine receptor. These results show that muscle activity regulates the level of extrajunctional acetylcholine receptors by regulating the expression of their mRNAs.     

Orexin-A up-regulates dopamine D2 receptor and mRNA in the nucleus accumbens Shell

Molecular Biology Reports - Orexins-A (OrxA) and -B (OrxB) neuropeptides are synthesized by a group of neurons located in the lateral hypothalamus and adjacent perifornical area, which send their...     

Differential regulation of MyoD and myogenin mRNA levels by nerve induced muscle activity.

The levels of mRNAs coding for the myogenic factors MyoD and myogenin were measured during synapse formation in developing muscle and in adult muscle, after denervation and reinnervation and after muscle paralysis induced by blocking of neuromuscular transmission by neurotoxins known to alter the density and localization of synaptic proteins such as the acetylcholine receptor (AChR). The mRNA levels of both factors depend on usage of the neuromuscular synapses, but they change to different extents. Myogenin mRNA levels decrease drastically with innervation and increase strongly following blocking of transmission whereas the level of MyoD mRNA showed only a small decrease in response to innervation, denervation or muscle paralysis by neurotoxins. Neither mRNA showed a synapse-related cellular distribution. The results suggest that nerve-induced electrical muscle activity determines the cellular ratio of MyoD and myogenin mRNAs in adult muscle.     

Structural model for the mannose receptor family uncovered by electron microscopy of Endo180 and the mannose receptor

The mannose receptor family comprises four members in mammals, Endo180 (CD280), DEC-205 (CD205), phospholipase A(2) receptor (PLA(2)R) and the mannose receptor (MR, CD206), whose extracellular portion contains a similar domain arrangement: an N-terminal cysteine-rich domain (CysR) followed by a single fibronectin type II domain (FNII) and 8-10 C-type lectin-like domains (CTLDs). These proteins mediate diverse functions ranging from extracellular matrix turnover through collagen uptake to homeostasis and immunity based on sugar recognition. Endo180 and the MR are multivalent transmembrane receptors capable of interacting with multiple ligands; in both receptors FNII recognizes collagens, and a single CTLD retains lectin activity (CTLD2 in Endo180 and CTLD4 in MR). It is expected that the overall conformation of these multivalent molecules would deeply influence their function as the availability of their binding sites could be altered under different conditions. However, conflicting reports have been published on the three-dimensional arrangement of these receptors. Here, we have used single particle electron microscopy to elucidate the three-dimensional organization of the MR and Endo180. Strikingly, we have found that both receptors display distinct three-dimensional structures, which are, however, conceptually very similar: a bent and compact conformation built upon interactions of the CysR domain and the lone functional CTLD. Biochemical and electron microscopy experiments indicate that, under a low pH mimicking the endosomal environment, both MR and Endo180 experience large conformational changes. We propose a structural model for the mannose receptor family where at least two conformations exist that may serve to regulate differences in ligand selectivity.