Expression of the calcitonin receptor, calcitonin receptor-like receptor, and receptor activity modifying proteins during osteoclast differentiation

The expressions of the calcitonin receptor (CTR), the calcitonin receptor-like receptor (CLR), the receptor activity-modifying proteins (RAMP) 1-3, and of the receptor component protein (RCP) have been studied in mouse bone marrow macrophages (BMM) during osteoclast differentiation, induced by treatment with M-CSF and RANKL. Analyses of mRNA showed that CLR and RAMP1-3, but not CTR, were expressed in M-CSF stimulated BMM. RANKL gradually increased CTR mRNA, transiently enhanced CLR and transiently decreased RAMP1 mRNA, but did not affect RAMP2, RAMP3, or RCP mRNA. However, RANKL did not affect protein levels of CLR or RAMP1-3 as assessed by Western blots or FACS analyses, whereas immunocytochemistry showed enhanced CTR protein. Analyses of cAMP production showed that BMM cells expressed functional receptors for calcitonin gene-related peptide (CGRP), amylin, adrenomedullin, and intermedin, but not for calcitonin and calcitonin receptor stimulating peptide (CRSP), but that RANKL induced the expression of receptors for calcitonin and CRSP as well. Calcitonin, CGRP, amylin, adrenomedullin, intermedin, and CRSP all down regulated the CTR mRNA, but none of the peptides caused any effects on the expression of CLR or any of the RAMPs. Our data show that BMM cells express receptors for CGRP, amylin, adrenomedullin, and intermedin and that RANKL induces the formation of receptors for calcitonin and CRSP in these cells. We also show, for the first time, that the CTR is not only down regulated by signaling through the CTR but also by the peptides signaling through CLR/RAMPs.     

The ecdysteroid receptor

Summary

The steroid molting hormone of insects and other arthropods regulates gene activity in target tissues through its association with a specific, high affinity receptor protein. In this review we summarize recent advances in several areas of ecdysteroid receptor research, including efforts to characterize and purify the receptor protein, cytochemical studies of its tissue distribution and subcellular localization during development, and current molecular genetic studies ecdysteroid action.     

Molecular defects of the androgen receptor

Defects of the androgen receptor cause a wide spectrum of abnormalities of phenotypic male development, ranging from individuals with mild defects of virilization to those with complete female phenotypes. In parallel with this phenotypic spectrum, a large number of different mutations have been identified that alter the synthesis or functional activity of the receptor protein. In many instances, the genetic mutations identified lead to an absence of the intact, full-length receptor protein. Such defects (splicing defects, termination codons, partial or complete gene deletions) invariably result in the phenotype of complete androgen insensitivity (complete testicular feminization). By contrast, single amino acid substitutions in the androgen receptor protein can result in the entire phenotypic spectrum of androgen resistant phenotypes and provide far more information on the functional organization of the receptor protein. Amino acid substitutions in different segments of the AR open-reading frame disturb AR function by distinct mechanisms. Substitutions in the DNA binding domain of the receptor appear to comprise a relatively homogeneous group. These substitutions impair the capacity of the receptor to bind to specific DNA sequence elements and to modulate the function of responsive genes. Amino acid substitutions in the hormone-binding domain of the receptor have a more varied effect on receptor function. In some instances, the resulting defect is obvious and causes an inability of the receptor to bind hormone. In other instances, the effect is subtler, and may result in the production of a receptor protein that displays qualitative abnormalities of hormone binding or from which hormone dissociates more rapidly. Often it is not possible to correlate the type of binding defect with the phenotype that is observed. Instead, it is necessary to measure the capacity of the receptor that is synthesized in functional assays in order to discern any type of correlation with phenotype. Finally, two types of androgen receptor mutation do not fit such a categorization. The first of these—the glutamine repeat expansion that is observed in spinal and bulbar muscular atrophy—leads to a reduction of receptor function that can be measured in heterologous cells or in fibroblasts established from such patients. The expression of ARs containing such expanded repeats in men is associated with a degeneration of motor neurons in the spinal cords of affected patients. Likewise, the alterations of androgen receptor structure that have been detected in advanced forms of prostate cancer also behave as gain-of-function mutations. In this latter type of mutation, the exquisite specificity of the normal androgen receptor is relaxed and the mutant receptors can be activated by a variety of steroidal and non-steroidal ligands.     

Rapsyn‐mediated clustering of acetylcholine receptor subunits requires the major cytoplasmic loop of the receptor subunits

During synaptogenesis at the neuromuscular junction, nicotinic acetylcholine receptors (AChRs) are organized into high‐density postsynaptic clusters that are critical for efficient synaptic transmission. Rapsyn, an AChR associated cytoplasmic protein, is essential for the aggregation and immobilization of AChRs at the neuromuscular junction. Previous studies have shown that when expressed in nonmuscle cells, both assembled and unassembled AChR subunits are clustered by rapsyn, and the clustering of the α subunit is dependent on its major cytoplasmic loop. In the present study, we investigated the mechanism of rapsyn‐induced clustering of the AChR β, γ, and δ subunits by testing mutant subunits for the ability to cocluster with rapsyn in transfected QT6 cells. For each subunit, deletion of the major cytoplasmic loop, between the third and fourth transmembrane domains, dramatically reduced coclustering with rapsyn. Furthermore, each major cytoplasmic loop was sufficient to mediate clustering of an unrelated transmembrane protein. The AChR subunit mutants lacking the major cytoplasmic loops could assemble into αδ dimers, but these were poorly clustered by rapsyn unless at least one mutant was replaced with its wild‐type counterpart. These results demonstrate that the major cytoplasmic loop of each AChR subunit is both necessary and sufficient for mediating efficient clustering by rapsyn, and that only one such domain is required for rapsyn‐mediated clustering of an assembly intermediate, the αδ dimer. © 2003 Wiley Periodicals, Inc. J Neurobiol 54: 486–501, 2003     

褪黑素受体

褪黑素是松果体分泌的主要激素,其功能活动通过特异的Ｇ蛋白耦联受体介导,本文综述褪黑素受体 分布、药理学特性,受体的克隆及受体基因结构特点。     

Desensitization of the nicotinic acetylcholine receptor by diisopropylfluorophosphate

The interaction of diisopropylfluorophosphate (DFP) with the nicotinic acetylcholine (ACh) receptor of Torpedo electric organ was studied, using [³H]-phencyclidine ([³H]-PCP) as a reporter probe. Phencyclidine binds with different kinetics to resting, activated, and desensitized receptor conformations. Although DFP did not inhibit binding of [³H]-ACh or ¹²⁵I-α-bungarotoxin (BGT) to the receptor recognition sites and potentiated in a time-dependent manner [³H]-PCP binding to the receptor's high-affinity allosteric site, it inhibited the ACh or carbamylcholine-stimulated [³H]-PCP binding. This suggested that DFP bound to a third kind of site on the receptor and affected receptor conformation. Preincubation of the membranes with DFP increased the receptor's affinity for carbamylcholine by eightfold and raised the pseudo-first-order rate of [³H]-PCP binding to that of an agonist-desensitized receptor. Accordingly, it is suggested that DFP induces receptor desensitization by binding to a site that is distinct from the recognition or high-affinity noncompetitive sites.     

On the expanding terminology in the GPCR field: The meaning of receptor mosaics and receptor heteromers

The oligomerization of G protein-coupled receptors (GPCRs) is a fact that deserves further attention as increases both the complexity and diversity of the receptor-mediated signal transduction, thus enriching the cell signaling. Consequently, in the present review we tackle among others the problems concerning the terminology used to describe aspects surrounding the GPCRs oligomerization phenomenon. Therefore, the theoretical implications of the GPCR oligomerization will be briefly discussed together with possible implications of this phenomenon especially for new strategies in drug development.     

Antibody to the phosphomannosyl receptor inhibits recycling of receptor in fibroblasts

The 215-kd phosphomannosyl receptor is involved in the transport of newly synthesized acid hydrolases to lysosomes and also mediates the pinocytosis of lysosomal enzymes by fibroblasts in culture. Recycling of receptors to the sorting sites is an integral part of both these processes. In this report, we describe the inhibition in human fibroblasts of both functions of the phosphomannosyl receptor by a rabbit antiserum to the bovine liver receptor. This inhibition cannot be completely accounted for by inhibition of ligand-receptor interaction. Rather the antibody appears to cross-link receptors and cause a removal of receptors from the sorting sites (plasma membrane and Golgi apparatus) and their accumulation in a compartment from which they do not recycle. Removal of receptors from the recycling pool by antibody is irreversible, and return of receptors requires synthesis of new protein. Degradation of "trapped receptors" is enhanced (t1/2 = 7.5 hr), but much more gradual than their removal from the functional receptor pool (t1/2 = 30 min).     

雌激素受体亚型及其配体调节基因转录机制的研究

本文综述雌激素受体亚型（ERα和ERβ）的结构,功能,组织分布,生理作用及雌激素受体配体调节基因转录的机制,目的是深入系统地了解植物雌激素和选择性雌激素受体调节剂的作用路径及其组织特异性的发生机制,最终为提高雌激素类药物的选择性,优化以临床为基础的药物设计提供一条较为系统的思路。结果表明,ERα和ERβ对不同雌激素类化合物产生不同应答,配体的结构不同,调节基因转录的路径不同和募集的辅调节蛋白的不同是雌激素受体两种亚型组织特异性激活或抑制的主要原因。     

Triterpenes from Alisma orientalis act as androgen receptor agonists,progesterone receptor antagonists,and glucocorticoid receptor antagonists

Alisma orientalis, a well-known traditional medicine, exerts numerous pharmacological effects including anti-diabetes, anti-hepatitis, and anti-diuretics but its bioactivity is not fully clear. Androgen receptor (AR), progesterone receptor (PR), and glucocorticoid receptor (GR) are three members of nuclear receptor superfamily that has been widely targeted for developing treatments for essential diseases including prostate cancer and breast cancer. In this study, two triterpenes, alisol M 23-acetate and alisol A 23-acetate from Alisma orientalis were determined whether they may act as androgen receptor (AR), progesterone receptor (PR), or glucocorticoid receptor (GR) modulators. Indeed, in the transient transfection reporter assays, alisol M 23-acetate and alisol A 23-acetate transactivated AR in dose-dependent manner, while they transrepressed the transactivation effects exerted by agonist-activated PR and GR. Through molecular modeling docking studies, they were shown to respectively interact with AR, PR, or GR ligand binding pocket fairly well. All these results indicate that alisol M 23-acetate and alisol A 23-acetate from Alisma orientalis might possess therapeutic effects through their modulation of AR, PR, and GR pathways.     

阿片受体的研究进展

阿片及其衍生物在神经系统中具有很强的镇痛作用,对阿片受体的研究已有20多年的历史。20世纪70年代发现了阿片受体的存在并先后发现了脑啡肽、β-内啡肽和强啡肽等阿片肽,随后发现了孤啡肽。如年代3种阿片受体的基因均已克隆成功,氨基酸序列表明它们均属G蛋白偶联受体,为7螺旋跨膜受体家族的成员,具有很高的同源性,功能包括介导腺苷酸环化酶的抑制作用以及一些离子通道的激活和抑制作用等。阿片受体基因的克隆将有利于新型临床药物的开发以及耐受和药物成瘾性分子基础的研究。目前阿片受体基因敲除、计算机结构模拟分析以及寻找新型阿片受体基因的研究均在深入进行。     

Expression of estrogen receptors,androgen receptor and steroid receptor coactivator-3 is negatively correlated to the differentiation of astrocytic tumors

Astrocytic tumors are the most common primary brain tumors. It has been reported that androgen receptor (AR), estrogen receptors alpha (ERα) and beta (ERβ) and their coactivator SRC-1 and SRC-3 are involved in the regulation of the growth and development of many tumors, but their expression profiles and significances in the astrocytic tumors remain largely unknown. In this study, the expression of AR, ERs, and SRCs, and the possible roles of them in astrocytic neoplasm were evaluated and compared to normal brain tissues by nickel-intensified immunohistochemistry with tissue microarrays. The results showed that there were no age- or gender-differences regarding to the levels of these receptors or coactivators in astrocytic or normal brain tissues. In the high-grade astrocytic tissue, the levels of AR, ERs and SRC-3 were significantly decreased when compared to the low-grade astrocytic tissues, but the levels of SRC-1 remain unchanged. Correlation analysis revealed that the levels of AR, ERs and SRC-3 were negatively correlated to tumor differentiation, and the levels of SRC-3 were positively correlated to that of ERα. Furthermore, the decreased levels of SRC-3 were associated with an increase of ERβ in astrocytic tumors when compared to that of normal brain tissues. These above results indicate a combination of decreased expression of ERs, AR and SRC-3 but not SRC-1 may be involved in the tumorigenesis of gliomas, ERα/SRC-3 axis may play central role in the regulation these tumors.     

EP24.15 interacts with the angiotensin II type I receptor and bradykinin B2 receptor

The carboxyl-terminal cytoplasmic domain of the angiotensin II type 1 receptor (AT1) is known to interact with several classes of intracellular proteins that may modulate receptor function. Employing yeast two-hybrid screening of a human embryonic kidney cDNA library with the carboxyl-terminal cytoplasmic domain of the AT1 receptor as a bait, we have isolated EP24.15 (EC 3.4.24.15, thimet oligopeptidase) as a potentially interacting protein. EP24.15 is widely distributed and is known to degrade bioactive peptides such as angiotensin I and II and bradykinin. In addition, EP24.15 was previously identified as a putative soluble angiotensin II binding protein. Two-hybrid screening also determined that EP24.15 can interact with the B2 bradykinin receptor. Transient expression of EP24.15 in a porcine kidney epithelial cell line stably expressing full length AT1 and full length B2 followed by affinity chromatography and co-immunoprecipitation confirmed EP24.15 association with both AT1 and B2 receptors. EP24.15 was also co-immunoprecipitated with AT1 and B2 in rat kidney brush border membranes (BBM) and basolateral membranes (BLM). Both AT1 and B2 undergo ligand-induced endocytosis. Analysis of endosomal fractions following immunoprecipitation with AT1 or B2 antibodies detected strong association of EP24.15 with the receptors in both light and heavy endosomal populations. Therefore, the present study indicates that EP24.15 associates with AT1 and B2 receptors both at the plasma membrane and after receptor internalization and suggests a possible mechanism for endosomal disposition of ligand that may facilitate receptor recycling.     

Formation of the 67-kDa laminin receptor by acylation of the precursor

Even though the involvement of the 67-kDa laminin receptor (67LR) in tumor invasiveness has been clearly demonstrated, its molecular structure remains an open problem, since only a full-length gene encoding a 37-kDa precursor protein (37LRP) has been isolated so far. A pool of recently obtained monoclonal antibodies directed against the recombinant 37LRP molecule was used to investigate the processing that leads to the formation of the 67-kDa molecule. In soluble extracts of A431 human carcinoma cells, these reagents recognize the precursor molecule as well as the mature 67LR and a 120-kDa molecule. The recovery of these proteins was found to be strikingly dependent upon the cell solubilization conditions: the 67LR is soluble in NP-40-lysis buffer whereas the 37LRP is NP-40-insoluble. Inhibition of 67LR formation by cerulenin indicates that acylation is involved in the processing of the receptor. It is likely a palmitoylation process, as indicated by sensitivity of NP-40-soluble extracts to hydroxylamine treatment. Immunoblotting assays performed with a polyclonal serum directed against galectin3 showed that both the 67- and the 120-kDa proteins carry galectin3 epitopes whereas the 37LRP does not. These data suggest that the 67LR is a heterodimer stabilized by strong intramolecular hydrophobic interactions, carried by fatty acids bound to the 37LRP and to a galectin3 cross-reacting molecule. J. Cell. Biochem. 69:244–251, 1998. © 1998 Wiley-Liss, Inc.     

Fate of the sea urchin egg receptor for sperm following fertilization

The sea urchin egg receptor for sperm is a 350 kDa glycoprotein containing a large extracellular domain that contains the sperm binding site, a transmembrane domain and a short COOH- terminal intracellular domain. During oogenesis, the receptor protein is first detected in Golgi-associated vesicles and cortical granules. Not until the egg is mature does the receptor appear on the cell surface; at this stage the intact receptor is found in approximately equal quantities on the egg cell surface and in cortical granules. As a potentially unique type of receptor, we were interested in its fate following fertilization. Several techniques have revealed that, following sperm binding, the amount of receptor markedly decreases. Using western blot analysis as well as direct measurement of the receptor protein, it was found that the membrane-bound form of the receptor rapidly disappeared following sperm binding to the egg, with only 3% of the receptor remaining after 30 s. Analysis by immupoelectron microscopy revealed that 30 s after sperm binding, 30% of the initial level of receptor was present. This remaining 30% was found mostly within the perivitelline space formed by the raised fertilization envelope. The disparity between these two sets of results (i.e. 3 vs 30%) is most likely accounted for by the exocytosis of receptor molecules from cortical granules; this fraction of the receptor would have been lost during isolation of the membrane-bound form of the receptor. Thus, unlike other cell surface receptors, the sea urchin egg receptor for sperm is not endocytosed and recycled following ligand binding. Rather, it disappears, presumably as a result of proteolysis. Transiently, the cortical granule form of the receptor is found released into the perivitelline space where it may bind to sperm and thereby prevent polyspermy. Despite the apparent secretion of this form of the receptor, experiments with antibodies to the extracellular and intracellular domains indicate that the receptors in cortical granules and in the plasmic membrane are similar, if not identical.     

Keratinocyte growth factor receptor ligands target the receptor to different intracellular pathways

The keratinocyte growth factor receptor (KGFR)/fibroblast growth factor receptor 2b is activated by high-affinity-specific interaction with two different ligands, keratinocyte growth factor (KGF)/fibroblast growth factor (FGF)7 and FGF10/KGF2, which are characterized by an opposite requirement of heparan sulfate proteoglycans and heparin for binding to the receptor. We investigated here the possible different endocytic trafficking of KGFR, induced by the two ligands. Immunofluorescence and immunoelectron microscopy analysis showed that KGFR internalization triggered by either KGF or FGF10 occurs through clathrin-coated pits. Immunofluorescence confocal microscopy using endocytic markers as well as tumor susceptibility gene 101 (TSG101) silencing demonstrated that KGF drives KGFR to the degradative pathway, while FGF10 targets the receptor to the recycling endosomes. Biochemical analysis showed that KGFR is ubiquitinated and degraded after KGF treatment but not after FGF10 treatment, and that the alternative fate of KGFR might depend on the different ability of the receptor to phosphorylate the fibroblast growth factor receptor substrate 2 (FRS2) substrate and to recruit the ubiquitin ligase c-Cbl. The recycling endocytic pathway followed by KGFR upon FGF10 stimulation correlates with the higher mitogenic activity exerted by this ligand on epithelial cells compared with KGF, suggesting that the two ligands may play different functional roles through the regulation of the receptor endocytic transport.