Structure of the porcine thyrotropin receptor: a 200 kilodalton glycoprotein heterocomplex

Summary We have determined that the porcine thyroidal TSH receptor is a glycoprotein heterotetramer composed of two M_r 35,000 () covalently linked subunits which interact noncovalently with two copies of (M_r 66,000) chains.Acknowledgments. This work was supported by grants from the Medical Research Council of Canada.     

Structure of the porcine thyrotropin receptor: a 200 kilodalton glycoprotein heterocomplex

We have determined that the porcine thyroidal TSH receptor is a glycoprotein heterotetramer composed of two Mr approximately 35,000 (epsilon) covalently linked subunits which interact noncovalently with two copies of delta (Mr 66,000) chains.     

Transient and constitutive repression of cytoplasmic translation signaling in cells with mtDNA mutation

Cytoplasmic translation is under sophisticated control but how cells adapt its rate to constitutive loss of mitochondrial oxidative phosphorylation is unknown. Here we show that translation is repressed in cells with the pathogenic A3243G mtDNA mutation or in mtDNA-less ρ⁰ cells by at least two distinct pathways, one transiently targeting elongation factor eEF-2 and the other initiation factor eIF-2α constitutively. Under conditions of exponential cell growth and mammalian target of rapamycin (mTOR) activation, eEF-2 becomes transiently phosphorylated by an AMP-activated protein kinase (AMPK)-dependent pathway, especially high in mutant cells. Independent of AMPK and mTOR, eIF-2α is constitutively phosphorylated in mutant cells, likely a signature of endoplasmic reticulum (ER)-stress response induced by the loss of oxidative phosphorylation. While the AMPK/eEF-2K/eEF-2 pathway appears to function in adaptation to physiological fluctuations in ATP levels in the mutant cells, the ER stress signified by constitutive protein synthesis inhibition through eIF-2α-mediated repression of translation initiation may have pathobiochemical consequences. Received 29 October 2008; received after revision 11 December 2008; accepted 16 December 2008     

LDL receptor relatives at the crossroad of endocytosis and signaling

For many years, the low-density lipoprotein (LDL) receptor and the LDL receptor-related protein (LRP) have been considered to be prototypes of cargo receptors which deliver, via endocytosis, macromolecules into cells. However, the recent identification of additional members of this gene family and examination of their biology has revealed that at least some of these proteins are also signaling receptors. Very low density lipoprotein receptor and ApoER2 transmit the extracellular reelin signal into migrating neurons, and thus are key components of the reelin pathway which governs neuronal layering of the forebrain during embryonic brain development. LRP5 and LRP6 are integral components of the Wnt signaling pathway which is central to many processes of metazoan development, cell proliferation, and tumor formation. Adaptor proteins interacting with the cytosolic domains of these receptors might orchestrate their ability to deliver their cargo or a signal.     

N-acetylglucosaminyltransferase V modifies the signaling pathway of epidermal growth factor receptor

Transfection of sense cDNA of N-acetylglucosamyltransferase V (GnTV-S) into human H7721 hepatocarcinoma cells resulted in an increase in the N-acetylglucosamine1,6mannose1,3- branch (GnT-V product) on the N-glycans of epidermal growth factor (EGF) receptor (EGFR), and promotion of its EGF binding and tyrosine autophosphorylation, but showed little effect on the expression of EGFR protein. The phosphorylation at T308, S473 and tyrosine residue(s) and the activity of protein kinase B (Akt/PKB) as well as the phosphorylation of p42/44 mitogen-activated protein kinase (MAPK) and MAPK kinase (MEK) before and after EGF stimulation were concomitantly increased. Conversely, in the antisense GnT-V (GnTV-AS)-transfected H7721 cells, all the results were the reverse of those with GnTV-S-transfected cells. After the cells were treated with 1-deoxymannojirimycin, an inhibitor of N-glycan processing at high mannose, or antibody against the extracellular glycan domain of EGFR, the differences in PKB activity, p42/44 MAPK and MEK phosphorylation among GnTV-S-, GnTV-AS- and mock-transfected cells were significantly attenuated. These findings indicate that the altered expression of GnT-V will change the glycan structure and function of EGFR, which may modify downstream signal transduction.Received 24 March 2004; received after revision 1 May 2004; accepted 25 May 2004     

比格犬黑皮质素受体3基因291位点突变与体重性状相关性的分析

分析比格犬MC3R基因突变与体重性状的关系。抽取112只比格犬血液,记录体重数据,然后提取DNA;采用PCR-RFLP技术对MC3R基因G291A位点进行分析,并克隆测序。测序结果表明,291位点表现为G、A二个等位基因和GG、GA及AA三种基因型;统计分析结果显示AA型犬与GG型犬体重比较差异显著（P〈0．05）。犬MC3R基因突变可致体重增加,可作为犬体重标记的候选基因。     

The cross-talk between the urokinase receptor and fMLP receptors regulates the activity of the CXCR4 chemokine receptor

The receptor (CXCR4) for the stromal-derived factor-1 (SDF1) and the urokinase-receptor (uPAR) are up-regulated in various tumors. We show that CXCR4-transfected cells migrate toward SDF1 on collagen (CG) and do not on vitronectin (VN). Co-expression of cell-surface uPAR, which is a VN receptor, impairs SDF1-induced migration on CG and allows migration on VN. Blocking fMLP receptors (fMLP-R), alpha-v integrins or the uPAR region capable to interact with fMLP-Rs, impairs migration of uPAR/CXCR4-transfected cells on VN and restores their migration on CG. uPAR co-expression also reduces the adherence of CXCR4-expressing cells to various components of the extracellular matrix (ECM) and influences the partitioning of beta1 and alpha-v integrins to membrane lipid-rafts, affecting ECM-dependent signaling. uPAR interference in CXCR4 activity has been confirmed in cells from prostate carcinoma. Our results demonstrate that uPAR expression regulates the adhesive and migratory ability of CXCR4-expressing cells through a mechanism involving fMLP receptors and alpha-v integrins.     

Comparison of X-prolyl dipeptidyl-aminopeptidase activity in human cerebrospinal fluid with that in serum

Summary X-Prolyl dipeptidyl-aminopeptidase activities in cerebrospinal fluid and serum from the same patients without neurological diseases, undergoing surgery under lumbar anesthesia, were assayed fluorometrically with a newly synthesized fluorogenic substrate, 7-glycylproline-4-methylcoumarinamide; the values were 129.1±19.5 nmoles/min/1 and 54.17±3.11 moles/min/1 (mean±SEM, n=23), respectively, and there was no correlation between both activities (r=0.0894).     

Preliminary evidence that a dopamine receptor antagonist blocks the prolactin-inhibitory effects of melatonin in anosmic male rats

Summary Previous studies have shown that daily afternoon injections of melatonin in anosmic male rats result in depressed accessory sex organ weights and serum prolactin levels. The present data indicate that the prolactin-inhibitory effect of melatonin may be mediated via the dopaminergic system.This work was supported by NIH Biomedical Research Support Grant No. 3150-80.     

Comparison of X-prolyl dipeptidyl-aminopeptidase activity in human cerebrospinal fluid with that in serum.

X-Prolyl dipeptidyl-aminopeptidase activities in cerebrospinal fluid and serum from the same patients without neurological diseases, undergoing surgery under lumbar anesthesia, were assayed fluorometrically with a newly synthesized fluorogenic substrate, 7-glycylproline-4-methylcoumarinamide; the values were 129.1 +/- 19.5 nmoles/min/l and 54.17 +/- 3.11 mumoles/min/l (mean +/- SEM, n = 23), respectively, and there was no correlation between both activities (r = 0.0894).     

Cellular models for the detection and evaluation of drugs that modulate human phagocyte activity

Simple testing models have been developed for the evaluation of chemical or biological compounds that modulate the activity of human phagocytes. Human neutrophils from buffy coats of donor blood are used. They are stimulated with receptor agonists, and the effects of test compounds on exocytosis of different enzymes, the generation of superoxide (respiratory burst), and cytotoxicity are quantified. All assays are performed in microtiter plates and the responses are evaluated by multi-well photometry or fluorimetry. The models are apt to detect compounds acting on phagocytes as agonists or antagonists, signal transduction activators or inhibitors and primers of agonist responses, and to assess cytotoxic effects.     

Dopamine receptor 1 localizes to neuronal cilia in a dynamic process that requires the Bardet-Biedl syndrome proteins

Primary cilia are nearly ubiquitous cellular appendages that provide important sensory and signaling functions. Ciliary dysfunction underlies numerous human diseases, collectively termed ciliopathies. Primary cilia have distinct functions on different cell types and these functions are defined by the signaling proteins that localize to the ciliary membrane. Neurons throughout the mammalian brain possess primary cilia upon which certain G protein-coupled receptors localize. Yet, the precise signaling proteins present on the vast majority of neuronal cilia are unknown. Here, we report that dopamine receptor 1 (D1) localizes to cilia on mouse central neurons, thereby implicating neuronal cilia in dopamine signaling. Interestingly, ciliary localization of D1 is dynamic, and the receptor rapidly translocates to and from cilia in response to environmental cues. Notably, the translocation of D1 from cilia requires proteins mutated in the ciliopathy Bardet-Biedl syndrome (BBS), and we find that one of the BBS proteins, Bbs5, specifically interacts with D1.     

Cellular models for the detection and evaluation of drugs that modulate human phagocyte activity

Summary Simple testing models have been developed for the evaluation of chemical or biological compounds that modulate the activity of human phagocytes. Human neutrophils from buffy coats of donor blood are used. They are stimulated with receptor agonists, and the effects of test compounds on exocytosis of different enzymes, the generation of superoxide (respiratory burst), and cytotoxicity are quantified. All assays are performed in microtiter plates and the responses are evaluated by multi-well photometry or fluorimetry. The models are apt to detect compounds acting on phagocytes as agonists or antagonists, signal transduction activators or inhibitors and primers of agonist responses, and to assess cytotoxic effects.