• 한국발생생물학회지:발생과생식
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• 제11권1호
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• pp.21-29
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• 2007

본 연구진은 선행 연구에서 progesterone receptor membrane component 2 (pgrmc2) 유전자가 미성숙 난자에서 높게 발현하는 것을 발견하였다. 본 연구는 pgrmc2와 pgrmc1 유전자가 쥐의 생식소와 배아의 발달 단계에 따라 어떻게 발현하는지 알아 보기 위해 수행하였다. 이들 유전자는 난소, 정소, 배아, 그리고 난자의 다양한 발달 단계에서 발현하는 것을 알 수 있었다. 쥐 난자 세포막에 프로게스테론 수용체가 존재하는 것은 progesterone-3-O-carboxymethyl oxime-BSA-fluorescein isothiocyanate (P4-BSA-FITC)의 결합을 이용하여 확인하였다. 본 결과는 프로게스테론 수용체의 세포막 구성 요소들이 생쥐의 난자, 배아, 그리고 생식소에서 발현함을 최초로 확인한 연구 결과다. 스테로이드 수용체의 세포막 구성 요소들의 기능, 특히 프로게스테론 수용체의 세포막 구성 요소들의 기능을 알아 보기 위해 추후 연구가 계속되어야 할 것이다.

• 한국막학회:학술대회논문집
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• 한국막학회 1998년도 춘계 총회 및 학술발표회
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• pp.31-35
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• 1998

1. INTRODUCTION : Recognition and binding of organic substrates by biological molecules are of vital importance in biophysics and biophysical chemistry. Most studies of the application focused on the development of biosensors, which detected reaction products generated by the binding between enzymes and substrates. Other types of biosensors in which membrane proteins (e.g., nicotinic acetylcholine receptor, auxin receptor ATPase, maltose bining protein, and glutmate receptor) were utilized as a receptor function were also developed. In the previous study[1], the shifts in membrane potential, caused by the injection of substrates into a permeation cell, were measured using immobilized glucose oxidase membranes. It was suggested that the reaction product was not the origin of the potential shifts, but the changes in the charge density in the membrane due to the binding between the enzyme and the substrates generated the potential shifts. In this study, $\gamma$-globulin was immobilized (entrapped) in a poly($\gamma$-amino acid) network, and the shifts in the membrane potential caused by the injection of some amino acids were investigated.

• International Journal of Oral Biology
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• 제33권4호
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• pp.217-221
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• 2008

Bicuculline is one of the most commonly used $GABA_A$ receptor antagonists in electrophysiological research. Because of its poor water solubility, bicuculline quaternary ammonium salts such as bicuculline methiodide (BMI) and bicuculline methbromide are preferred. However, a number of studies have shown that BMI has non-$GABA_A$ receptor-mediated effects. The substantia gelatinosa (SG) of the trigeminal subnucleus caudalis (Vc) is implicated in the processing of nociceptive signaling. In this study, we investigated whether BMI has non-GABA receptor-mediated activity in Vc SG neurons using a whole cell patch clamp technique. SG neurons were depolarized by application of BMI ($20{\mu}M$) using a high $Cl^-$ pipette solution. GABA ($30-100{\mu}M$) also induced membrane depolarization of SG neuron. Although BMI is known to be a $GABA_A$ receptor antagonist, GABA-induced membrane depolarization was enhanced by co-application with BMI. However, free base bicuculline (fBIC) and picrotoxin (PTX), a $GABA_A$ and $GABA_C$ receptor antagonist, blocked the GABA-induced response. Furthermore, BMI-induced membrane depolarization persisted in the presence of PTX or an antagonist cocktail consisting of tetrodotoxin ($Na^+$ channel blocker), AP-5 (NMDA receptor antagonist), CNQX (non-NMDA receptor antagonist), and strychnine (glycine receptor antagonist). Thus BMI induces membrane depolarization by directly acting on postsynaptic Vc SG neurons in a manner which is independent of $GABA_A$ receptors. These results suggest that other unknown mechanisms may be involved in BMI-induced membrane depolarization.

• Molecules and Cells
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• 제23권1호
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• pp.1-10
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• 2007

Invading pathogens are recognized by diverse germline-encoded pattern-recognition receptors (PRRs) which are distributed in three different cellular compartments: extracellular, membrane, and cytoplasmic. In mammals, the major extracellular PRRs such as complements may first encounter the invading pathogens and opsonize them for clearance by phagocytosis which is mediated by membrane-associated phagocytic receptors including complement receptors. The major membrane-associated PRRs, Toll-like receptors, recognize diverse pathogens and generate inflammatory signals to coordinate innate immune responses and shape adaptive immune responses. Furthemore, certain membrane-associated PRRs such as Dectin-1 can mediate phagocytosis and also induce inflammatory response. When these more forefront detection systems are avoided by the pathogens, cytoplasmic PRRs may play major roles. Cytoplasmic caspase-recruiting domain (CARD) helicases such as retinoic acid-inducible protein I (RIG-I)/melanoma differentiation-associated gene 5 (MDA5), mediate antiviral immunity by inducing the production of type I interferons. Certain members of nucleotide-binding oligomerization domain (NOD)-like receptors such as NALP3 present in the cytosol form inflammasomes to induce inflammatory responses upon ligand recognition. Thus, diverse families of PRRs coordinately mediate immune responses against diverse types of pathogens.

• The Korean Journal of Physiology
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• 제29권2호
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• pp.203-216
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• 1995

The effects of ${\alpha}_1-adrenergic$ receptor stimulation on membrane potential, intracellular $Na^+$ activity, and twitch force were investigated in ventricular muscles from guinea-pig hearts. Action potentials, intracellular $Na^+$ activity, and twitch force of ventricular papillary muscles were measured simultaneously under various experimental conditions. Stimulation of the ${\alpha}_1-adrenergic$ receptor by phenylephrine produced variable changes in action potential duration, a slight hyperpolarization of the diastolic membrane potential, a decrease in intracellular $Na^+$ activity, and a biphasic inotropic response in which a transient negative inotropic response was followed by a sustained positive inotropic response. These changes were blocked by prazosin, an antagonist of the ${\alpha}_1-adrenergic$ receptor, but not by atenolol, an antagonist of the ${\beta}-adrenergic$ receptor. This indicates that the changes in membrane potential, intracellular $Na^+$ activity, and twitch force are mediated by stimulation of the ${\alpha}_1-adrenergic$ receptor, but not by stimulation of ${\beta}-adrenergic$ receptor. The decrease in intracellular $Na^+$ activity was not observed in quiescent muscles, depending on the rate of the action pontentials in beating muscles. The intracellular $Na^+$ activity decrease was substantially inhibited by tetrodotoxin. However, the decrease in intracellular $Na^+$ activity was not affected by an inhibition of the $Na^+-K^+$ pump. Therefore, the decrease in intracellular $Na^+$ activity mediated by the ${\alpha}_1-adrenergic$ receptor appears to be due to a reduction of $Na^+$ influx during the action potential, perhaps through tetrodotoxin sensitive $Na^+$ channels. Our study also revealed that the decrease in intracellular $Na^+$ activity might be related to the transient negative inotropic response. The intracellular $Na^+$ activity decrease could lower intracellular $Ca^{2+}$ through the $Na^+-Ca^{2+}$ exchanger and thereby produce a decline in twitch force.

• Biomolecules & Therapeutics
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• 제1권2호
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• pp.188-195
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• 1993

In order to understand the machanism of action and regulation of ${\beta}$-adrenergic receptor in terms of molecular level, the purification of receptor protein has a fundamental importance. Moreover, species differences among avian, amphibian and mammalian ${\beta}$-adrenergic receptors make it more important to purify mammalian ${\beta}$-adrenergic receptor. Because ${\beta}$-adrenergic receptor is an integral membrane protein, it must be solubilized from the membrane for the purification. The purpose of the present study was to solubilize and characterize the mammalian $\beta$-adrenergic receptor from guinea pig lung in quantities by more efficient and practical method eventually to purify receptor. Guinea pig lung membrane preparation was solubilized by sequential treatment of buffers containing low and high concentration of digitonin which are 0.2 and 1.2% respectively. About 50% of the total receptor pool was released by this double extraction procedure. The $\beta$-adrenoceptors in the digitonin extract were identified using the ${\beta}$-adrenergic antagonist, (-)-[$^3H$]-dihydroalprenolol ([$^3H$]DHA). The solubilized receptor retained all of the essential characteristics of membrane-bound receptor, namely saturability; stereoselectivity; high affinity to ${\beta}$-adrenergic drugs. For the measurement of soluble receptor activity, Sephadex G-50 chromatography method has been widely used. Inspite of its accuracy and wide acceptance, this technique employed troublesome column work which required long time to assay the activity of receptor. We employed another methods to measure receptor activity. When using 0.5% polyethylenimine pretreated GF/B glass fiber filter, filtration technique could be used to measure soluble receptor activity. This technique enabled us to reduce the total amount of time to assay by a factor of 4 as well as to detect soluble receptor. In the present study, we could establish more efficient and practical solubilization method of mammalian $\beta$-adrenergic receptor. The rapidity and high yield of this solubilization scheme, together with the favorable recovery of the receptor activity, are significant steps toward the ultimate purification of the mammalian $\beta$-adrenergic receptor. The result of this study together with more convenient purification method could provide large amount of purified receptor with ease for various research purposes.

• BMB Reports
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• 제31권5호
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• pp.468-474
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• 1998

Membrane depolarization in PC12 cells induces calcium influx via an L-type voltage-sensitive calcium channel (L-VSCC) and increases intracellular free calcium, which leads to tyrosine phosphorylation of epidermal growth factor (EGF) receptor and the associated adaptor protein, She. This activated EGF receptor complex then can activate mitogen-activated protein (MAP) kinase, as in nerve growth factor (NGF) receptor activation. In the present study, we investigated the role of EGF receptor in the signaling pathway initiated by membrane depolarization of PC12 cells. Prolonged membrane depolarization induced phosphorylation of extracellular signal-regulated kinase (ERK) within 1 min in undifferentiated PC12 cells. Pretreatment of PC12 cells with the calcium chelator EGTA abolished depolarization-stimulated ERK phosphorylation, but NGF-induced phosphorylation of ERK was not affected. The chronic treatment of phorbol ester, which down-regulated the activity of protein kinase C (PKC), did not affect the phosphorylation of ERK upon depolarization. In the presence of an inhibitor of EGF receptor, neither depolarization nor calcium ionophore increased the level of ERK phosphorylation. These data imply that the EGF receptor is functionally necessary to activate ERK and neurite outgrowth in response to the prolonged depolarization in PC12 cells, and also that PKC is apparently not involved in this signaling pathway.

• Archives of Pharmacal Research
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• 제23권6호
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• pp.535-547
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• 2000

Recent findings indicate that mechanical strain (deformation) exerted by the extracellular matrix modulates activation of airway smooth muscle cells. Furthermore, cytoskeletal organization in airway smooth muscle appears to be dynamic, and subject to modulation by receptor activation and mechanical strain. Mechanosensitive modulation of crossbridge activation and cytoskeletal organization may represent intracellular feedback mechanisms that limit the shortening of airway smooth muscle during bronchoconstriction. Recent findings suggest that receptor-mediated signal transduction is the primary target of mechanosensitive modulation. Mechanical strain appears to regulate the number of functional G-proteins and/or phospholipase C enzymes in the cell membrane possibly by membrane trafficking and/or protein translocation. Dense plaques, membrane structures analogous to focal adhesions, appear to be the primary target of cytoskeletal regulation. Mechanical strain and receptor-binding appear to regulate the assembly and phosphorylation of dense plaque proteins in airway smooth muscle cells. Understanding these mechanisms may reveal new pharmacological targets for control1ing airway resistance in airway diseases.

• BMB Reports
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• 제33권6호
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• pp.448-453
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• 2000

In yeast the key gluconeogenic enzyme, fructose-1,6-bisphosphatase (FBPase), is selectively targeted from the cytosol to the lysosome (vacuole) for degradation when glucose starved cells are replenished with glucose. The pathway for glucose induced FBPase degradation is unknown. To identify the receptor-mediated degradation pathway of FBPase, we investigated the presence of the FBPase receptor on the vacuolar membrane by cell fractionation experiments and binding assay using vid mutant (vacuolar import and degradation), which is defective in the glucose-induced degradation of FBPase. FBPase sedimented in the pellets from vid24-1 mutant after centrifugation at $15,000{\times}g$ for 15 min, suggesting that FBPase is associated with subcellular structures. Cell fractionation experiments revealed that FBPase is preferentially associated with the vacuole, but not with other organelles in vid24-1. FBPase enriched fractions that cofractionated with the vacuole were sensitive to proteinase K digestion, indicating that FBPase is peripherally associated with the vacuole. We developed an assay for the binding of FBPase to the vacuole. The assay revealed that FBPase bound to the vacuole with a Kd of $2.3{\times}10^6M$. The binding was saturable and specific. These results suggest that a receptor for FBPase degradation exists on the vacuolar membrane. It implies the existence of the receptor-mediated degradation pathway of FBPase by the lysosome.

• Journal of Plant Biotechnology
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• 제45권4호
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• pp.315-321
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• 2018

Molecular and functional characterization of proteins and their levels is of great interest in understanding the mechanism of diverse cellular processes. In this study, we report on the convenient Escherichia coli-based protein expression system that allows recombinant of soluble proteins expression and cytosolic domain of membrane-localised kinases, followed by the detection of autophosphorylation activity in protein kinases. This approach is applied to regulatory proteins of Arabidopsis thaliana, including 14-3-3, calmodulin, calcium-dependent protein kinase, TERMINAL FLOWER 1(TFL1), FLOWERING LOCUS T (FT), receptor-like cytoplasmic kinase and cytoplasmic domain of leucine-rich repeat-receptor like kinase proteins. Our Western blot analysis which uses phospho-specific antibodies showed that five putative LRR-RLKs and two putative RLCKs have autophosphorylation activity in vitro on threonine and/or tyrosine residue(s), suggesting their potential role in signal transduction pathways. Our findings were also discussed in the broader context of recombinant expression and biochemical analysis of soluble and membrane-localised receptor kinases in microbial systems.