Binding Profiles of Oxomemazine to the Muscarinic Receptor Subtypes

이신웅,김정구,Lee, Shin-Woong,Kim, Jeung-Gu The Korean Society of Pharmacology 1994 대한약리학잡지 Vol.30 No.1

Oxomemazine이 muscarinic receptor subtypes에 대하여 선택성을 가지는지에 관한 지견을 얻고자, 대뇌, 심실 및 회장 muscarinic receptor에 대한 oxomemazine의 결합성질을 조사, 비교하였다. $[^3H]QNB$ 포화결합실험 결과 세 조직의 muscarinic receptor는 $[^3H]QNB$에 대해서는 affinity가 약 60pM인 단일 receptor인 것으로 추정되었다. 대뇌에서 pirenzepine과 oxomemazine의 $[^3H]QNB$ 결합억제에 대한 Hill coefficient는 각각 0.67 및 0.8로서 대뇌에는 이들 약물에 대하여 affinity가 서로 다른 두 종류의 muscarinic receptor subtypes가 존재하는 것으로 나타났으며, pirenzepine에 대한 high $affinity(M_1)$와 $low affinity(M_2)$ receptor 및 oxomemazine에 대한 high $affinity(O_H)$와 $low\;affinity (O_L)$ receptor의 분포비는 약 60:40 및 40:60이었고, $M_1$과 $M_2$ receptor에 대한 pirenzepine의 $K_i$치는 16nM 및 431 nM, $O_H$와 $O_L$, receptor에 대한 oxomemazine 의 $K_i$치는 80nM 및 1350nM이었다. 그러나 심실과 회장에서 이들 약물의 $[^3H]QNB$ 결합억제에 대한 Hill coefficient는 1에 가까웠다. 심실과 회장 muscarinic receptor에 대한 pirenzepine의 $K_i$치는 850nM 및 250nM, oxomemazine의 $K_i$치는 1460nM 및 670nM로서 대피에서 이들 약물의 low affinity receptor에 대한 $K_i$치에 가까웠다. 즉, muscarinic receptor에 대한 affinity면에서 oxomemazine은 pirenzepine과 같이 대뇌에서 가장 높았으며, 회장에 대해서는 중등도였고, 심실에서 가장 낮았다. 이로 보아 oxomemazine은 $M_1\;receptor$에 선택성이 있는 것으로 추정된다. The binding properties of oxomemazine to muscarinic receptors using the ability of oxomemazine to inhibit $[^3H]QNB$ binding in membrane fractions of rat cerebrum and guinea pig ventricle and ileum were investigated. $[^3H]QNB$ bound to a single class of muscarinic receptors with a dissociation constant of approximately 60 pM in three tissue preparations. Pirenzepine and oxomemazine inhibited $[^3H]QNB$ binding in cerebrum with a Hill coefficient lower than unity, and the inhibition data were best described by a two-site model. The relative densities of the high $(M_1)\;and\;low\;(M_2)$ affinity sites for pirenzepine were 60 and 40%, with corresponding Ki values of 16 and 431 nM, and those $(O_H\;and\;O_L)$ for oxomemazine 40 and 60%, with corresponding Ki values of 80 and 1350 nM. However, the inhibition data of both drugs vs $[^3H]QNB$ in ventricle and ileum appeared to obey the law of mass-action (Hill coefficient close to 1). The apparent Ki values of pirenzepine were 850 and 250 nM, and those of oxomemazine 1460 and 570 nM in ventricle and ileum, respectively. Thus, oxomemazine like pirenzepine has high affinity for cerebrum, moderate affinity for ileum and low affinity for ventricle. These results suggest that oxomemazine could recognize the muscarinic receptor subtypes with a high affinity for the $M_1$ sites.

Selectivity of Oxomemazine for the M₁ Muscarinic Receptors

Lee, Shin Woong,Woo, Chang Woo,Kim, Jeung Gu 영남대학교 약품개발연구소 1995 영남대학교 약품개발연구소 연구업적집 Vol.5 No.-

The binding characteristics of pirenzepine and oxomemazine to muscarinic receptor were studied to evaluate the selectivity of oxomemazine for the muscarinic receptor subtypes in rat cerebral microsomes. Equilibrium dissociation constant (K_(D)) of (-)-[³H]quinuclidnyl benzilate([³H]QNB) determined from saturation isotherms was 64 pM. Analysis of the pirenzepine inhibition curve of [³H]QNB binding to cerebral microsome indicated the presence of two receptor subtypes with high (K_(i)=16 nM, M₁ receptor) and low (K_(i)=400 nM, M₃receptor) affinity for pirenzepine. Oxomemazine also identified two receptor subtypes with about 20-fold difference in the affinity for high (K_(i)=84nM, O_(H) receptor) and low (K_(i)=1.65 μM, O_(L) receptor) affinity sites. The percentage populations of M₁ and M₃ receptors to the total receptors were 61:39, and those of O_(H) and O_(L) receptors 39:61, respectively. Both pirenzepine wd oxomemazine increased the K_(D) value for [³H]QNB without affecting the binding site concentrations and Hill coefficient for the [³H]QNB binding. Oxomemazine had a 10-fold higher affinity at M₁ receptors than at M₃ receptors, and pirenzepine a 8-fold higher affinity at O_(H) receptors than at O_(L) receptors. Analysis of the shallow competition binding curves of oxomemazine for M₁ receptors and pirenzepine for O_(L) receptors yielded that 69% of M₁ receptors were of O_(H) receptors and remaining 31% of O_(L) receptors, and that 29% of O_(L) receptors were of M₁ receptors and 71% of M₃ receptors. However, M₃ for oxomemazine and O_(H) for pirenzepine were composed of a uniform population. These results suggest that oxomemazine could be classified as a selective drug for M₁ receptors and also demonstrate that rat cerebral microsomes contain three different subtypes of M₁, M₃ and the other site which is different from M₁, M₂ and M₃ receptors.

Functions of Eel Luteinizing Hormone Receptor Mutants (Activating and Inactivating Receptors)

Munkhzaya Byambaragchaa,Jeong-Soo Kim,Hun-Ki Seong,Kwan-Sik Min 한국동물생명공학회(구 한국동물번식학회) 2017 Reproductive & Developmental Biology(Supplement) Vol.41 No.2

Gonadotropin receptors are members of the seven transmembrane (TM) receptor families. Several point mutations in TM II, III, V and VI have been identified in the luteinizing hormone receptor (LHR) gene, leading to constitutive activation and inactivation of the receptor. In eelLHR, we generated 3 types of constitutive activating mutations (M410T, L469R and D590Y) and 2 types of constitutive inactivating mutations (D383N and Y546F) to investigate how they work on hormone-receptor interaction and receptor activation system on eel. To assess the functional effects of 5 receptor mutations directly, wt and mutant eel- LHRs were transiently expressed in CHO-K1 cells, and basal and recombinant eel LH-stimulated cAMP and IP-1 accumulations were measured. Rec-eelLH (0.076~1,200 ng/mL) produced a concentration-dependent increase in cAMP production in wt eelLHR expressing cells with an EC50 of 160 ng/mL and basal cAMP level of 2.6 nM. In contrast, the L469R activation mutant had most elevated (16.88 fold higher than wt) basal cAMP production (basal cAMP level=43.9 nM). Compared with the wt eelLHR, all the activation mutant receptors produced higher basal levels of cAMP (18.4 nM for D590Y and 7.9 nM for M410T). However, eelLH-stimulated (0.076~1,200 ng/mL) basal cAMP levels in the constitutive inactivation mutants D383N and Y546F did not obviously altered from that in wt eelLHR. D383N mutation increased the EC50 value to 185 ng/mL (inhibited receptor activity to 86%), while Y546F mutation increased that to 170 ng/mL which implies that receptor activity was inhibited to 94% only. As seen in IC50 values in IP-1 accumulation, activity for M410T mutant was 19% higher than that for wt receptor, but other activation mutants did not show any difference in IP-1 production. In case of inactivation mutants, there were no significant differences in IP-1 production and only 7% decreased activity was identified in D383N mutant. In summary, we have demonstrated 3 mutations that are responsible for constitutive activation of eelLHR. Although predicted 2 inactivation mutations led to slightly diminished activation of receptor, those could not impair signal transduction of eelLHR.

Differential Coupling of G$\alpha$q Family of G-protein to Muscarinic $M_1$ Receptor and Neurokinin-2-Receptor

Lee, Chang-Ho,Shin, In-Chul,Kang, Ju-Seop,Koh, Hyun-Chul,Ha, Ji-Hee,Min, Chul-Ki The Pharmaceutical Society of Korea 1998 Archives of Pharmacal Research Vol.21 No.4

The ligand binding signals to a wide variety of seven transmembrane cell surface receptors are transduced into intracellular signals through heterotrimeric G-proteins. Recently, there have been reports which show diverse coupling patterns of ligand-activated receptors to the members of Gq family $\alpha$ subunits. In order to shed some light on these complex signal processing networks, interactions between G$\alpha$q family of G protein and neurokinin-2 receptor as well as muscarinic M$_{1}$ receptor, which are considered to be new thearpeutic targets in asthma, were studied. Using washed membranes from Cos-7 cells co-transfected with different G.alpha.q and receptor cDNAs, the receptors were stimulated with various concentrations of carbachol and neurokinin A and the agonist-dependent release of [$^3H$]inositol phosphates through phospholipase C beta-1 activation was measured. Differential coupling of Gaq family of G-protein to muscarinic M$_{1}$ receptor and neurokinin-2 receptor was observed. The neurokinin-2 receptor shows a ligand-mediated response in membranes co-transfected with G$\alpha$q, G$\alpha$11 and G$\alpha$14 but not G$\alpha$16 and the ability of the muscarinic $M_1$ receptor to activate phospholipase C through G$\alpha$/11 but not G$\alpha$14 and G$\alpha$16 was demonstrated. Clearly G$\alpha$/11 can couple $\M_1$ and neurokinin-2 receptor to activate phospholipase C. But, there are differences in the relative coupling of the G$\alpha$14 and G$\alpha$16 subunits to these receptors.

Binding Profiles of Oxomemazine to the Muscarinic Receptor Subtypes

Lee, Shin Woong,Kim, Jeung Gu 영남대학교 약품개발연구소 1994 영남대학교 약품개발연구소 연구업적집 Vol.4 No.-

Oxomemazine이 muscarinic receptor subtypes에 대하여 선택성을 가지는지에 관한 지견을 얻고자, 대뇌, 심실 및 회장 muscarinic receptor에 대한 oxomemazine의 결합성질을 조사, 비교하였다. [³H]QNB 포화결합실험 결과 세 조직의 muscarinic receptor는 [³H]QNB에 대해서는 affinity가 약 60pM인 단일 receptor인 것으로 추정되었다. 대뇌에서 pirenzepine과 oxomemazine의 [³H]QNB 결합억제에 대한 Hill coefficient는 각각 0.67 및 0.8로서 대뇌에는 이들 약물에 대하여 affinity가 서로 다른 두 종류의 muscarinic receptor subtypes가 존재하는 것으로 나타났으며, pirenzepine에 대한 high affinity(M₁)와 low affinity(M₂) receptor 및 oxomemazine에 대한 high affinity(O_(H))와 low affinity (O_(L)) receptor의 분포비는 약 60:40 및 40:60이었고, M₁과 M₂ receptor에 대한 pirenzepine의 K_(i)치는 16nM 및 431 nM, O_(H)와 O_(L) receptor에 대한 oxomemazine 의 K_(i)치는 80nM 및 1350nM이었다. 그러나 심실과 회장에서 이들 약물의 [³H]QNB 결합억제에 대한 Hill coefficient는 1에 가까웠다. 심실과 회장 muscarinic receptor에 대한 pirenzepine의 K_(i)치는 850nM 및 250nM, oxomemazine의 K_(i)치는 1460nM 및 670nM로서 대뇌에서 이들 약물의 low affinity receptor에 대한 K_(i)치에 가까웠다. 즉, muscarinic receptor에 대한 affinity면에서 oxomemazine은 pirenzepine과 같이 대뇌에서 가장 높았으며, 회장에 대해서는 중등도였고, 심실에서 가장 낮았다. 이로 보아 oxomemazine은 M₁ receptor에 선택성이 있는 것으로 추정된다. The binding properties of oxomemazine to muscarinic receptors using the ability of oxomemazine to inhibit [³H]QNB binding in membrane fractions of rat cerebrum and guinea pig ventricle and ileum were investigated. [³H]QNB bound to a single class of muscarinic receptors with a dissociation constant of approximately 60 pM in three tissue preparations. Pirenzepine and oxomemazine inhibited [³H]QNB binding in cerebrum with a Hill coefficient lower than unity, and the inhibition data were best described by a two-site model. The relative densities of the high (M₁) and low (M₂) affinity sites for pirenzepine were 60 and 40%, with corresponding Ki values of 16 and 431 nM, and those (O_(H) and O_(L)) for oxomemazine 40 and 60%, with corresponding Ki values of 80 and 1350 nM. However, the inhibition data of both drugs vs [³H]QNB in ventricle and ileum appeared to obey the law of mass-action (Hill coefficient close to 1). The apparent Ki values of pirenzepine were 850 and 250 nM, and those of oxomemazine 1460 and 570 nM in ventricle and ileum, respectively. Thus, oxomemazine like pirenzepine has high affinity for cerebrum, moderate affinity for ileum and low affinity for ventricle. These results suggest that oxomemazine could recognize the muscarinic receptor subtypes with a high affinity for the M₁ sites.

GABA와 Benzodiazepine 수용체 및 그 기능

정영조,한기석 大韓神經精神醫學會 1991 신경정신의학 Vol.30 No.3

GABA is ubiquitously distributed throughout the CNS. It is probably the major central inhibitory aminoacid neurotransmitter which hyperpolarizes the postsynapic neurons and inhibits the release of neurotransmitters. Generally inhibition of GABA activity causes excitation leading to anxiety and convulsions, whereas activation of its activity causes antiexpressive. anticonvulsive activity and sedation. The GABA receptors have been divided into two distinct groups named GABA(A) and GABA(B) receptors by their pharmacological and physiological properties. GABA(A) receptors are coupled with Bz binding site and that in conjunction with a CI­ channel they form a supramolecular receptor complex which mediates rapid increasing of CI­ influx into postsynaptic neurons. thus contributing to the prompt inhibition of cellular excitability. On the contrary, the GABA(B) receptor does not contain an integral ion channel and is responsible for slow responses through receptor-G protein-effector complexes. GABA(B) receptors appear to be localized on presynaptic nerve terminals and stimulation of presynaptic GABA(B) receptors reduced Ca²+ influx, resulting in decreased release of neurotransmitters. Stimulation of presynaptic GABA(B) receptors increases K efflux, resulting in inhibition of cell firing. A division of function among the two types of GABA receptors appears to exist : GABA(A) receptor complex mediates anxiety, anticonvulsive activity and feeding, GABA(B) receptors, on the other hand, are involved in depression and analgesia. In those cases where GABA(A) and GABA(B) receptors mediate similar functions (e.g., cardiovascular regulation), they do so by affecting different transmitter systems and cellular mechanisms. The putative involvement of GABA(A) and GABA(B) receptors in various behavioral and physiological effects is summarized in Table 3. There are two types of Bz receptors in brain which are central and peripheral type Bz receptors. Central Bz receptors are in many regions of the brain, coupled with the receptors for GABA and they mediate the acute actions of Bz in CNS. Although there is general acceptance that Bz evert their major actions via the GABA(A) receptor, more recent studies suggest that other systems may be involved. In addition, endogenous anxiogenic ligands that interact with Bz receptors in several ways have been discovered, which of these,β-carbolines and DBI may produce anxiety and convulsion in human, The recent investigation of endogenous ligands and specific receptor agonist or antagonist and inverse agonist provide important new conceptual tools for the studies of anxiety and depression mechanisms. It is expected that a better understanding of GABA and Bz receptors will eventually help to demonstrate the biology of anxiety and depression.

Brain Receptor Mosaics and Their Intramembrane Receptor-Receptor Interactions: Molecular Integration in Transmission and Novel Targets for Drug Development

Kjell Fuxe,Daniel Marcellino,Diego Guidolin,Amina S. Woods,Luigi Agnati 사단법인약침학회 2009 Journal of Acupuncture & Meridian Studies Vol.2 No.1

The concept of intramembrane receptor-receptor interactions and evidence for their existence was introduced by Agnati and Fuxe in 1980/81 suggesting the existence of heteromerization of receptors. In 1982, they proposed the existence of aggregates of multiple receptors in the plasma membrane and coined the term receptor mosaics (RM). In this way, cell signaling becomes a branched process beginning at the level of receptor recognition at the plasma membrane where receptors can directly modify the ligand recognition and signaling capacity of the receptors within a RM. Receptorreceptor interactions in RM are classified as operating either with classical cooperativity, when consisting of homomers or heteromers of similar receptor subtypes having the same transmitter, or non-classical cooperativity, when consisting of heteromers. It has been shown that information processing within a RM depends not only on its receptor composition, but also on the topology and the order of receptor activation determined by the concentrations of the ligands and the receptor properties. The general function of RM has also been demonstrated to depend on allosteric regulators (e.g., homocysteine) of the receptor subtypes present. RM as integrative nodes for receptor-receptor interactions in conjunction with membrane associated proteins may form horizontal molecular networks in the plasma membrane coordinating the activity of multiple effector systems modulating the excitability and gene expression of the cells. The key role of electrostatic epitope-epitope interactions will be discussed for the formation of the RM. These interactions probably represent a general molecular mechanism for receptor-receptor interactions and, without a doubt, indicate a role for phosphorylation-dephosphorylation events in these interactions. The novel therapeutic aspects given by the RMs will be discussed in the frame of molecular neurology and psychiatry and combined drug therapy appears as the future way to go. The concept of intramembrane receptor-receptor interactions and evidence for their existence was introduced by Agnati and Fuxe in 1980/81 suggesting the existence of heteromerization of receptors. In 1982, they proposed the existence of aggregates of multiple receptors in the plasma membrane and coined the term receptor mosaics (RM). In this way, cell signaling becomes a branched process beginning at the level of receptor recognition at the plasma membrane where receptors can directly modify the ligand recognition and signaling capacity of the receptors within a RM. Receptorreceptor interactions in RM are classified as operating either with classical cooperativity, when consisting of homomers or heteromers of similar receptor subtypes having the same transmitter, or non-classical cooperativity, when consisting of heteromers. It has been shown that information processing within a RM depends not only on its receptor composition, but also on the topology and the order of receptor activation determined by the concentrations of the ligands and the receptor properties. The general function of RM has also been demonstrated to depend on allosteric regulators (e.g., homocysteine) of the receptor subtypes present. RM as integrative nodes for receptor-receptor interactions in conjunction with membrane associated proteins may form horizontal molecular networks in the plasma membrane coordinating the activity of multiple effector systems modulating the excitability and gene expression of the cells. The key role of electrostatic epitope-epitope interactions will be discussed for the formation of the RM. These interactions probably represent a general molecular mechanism for receptor-receptor interactions and, without a doubt, indicate a role for phosphorylation-dephosphorylation events in these interactions. The novel therapeutic aspects given by the RMs will be discussed in the frame of molecular neurology and psychiatry and combined drug therapy appears as the future way to go.

IM-9 Lymphocyte에서 포도당과 인슐린이 인슐린 수용체 유전자의 발현에 미치는 영향

정수경 ( Jeong Su Gyeong ),김성운 ( Kim Seong Un ),양인명 ( Yang In Myeong ),김진우 ( Kim Jin U ),김영설 ( Kim Yeong Seol ),김광원 ( Kim Gwang Won ),최영길 ( Choe Yeong Gil ),정해원 ( Jeong Hae Won ) 대한내과학회 1992 대한내과학회지 Vol.42 No.5

연구배경 : 인슐린 수용체의 조절은 인슐린과 수용제의 복합체가 세포내로 내재화한 후 분해되는 비율과 합성된 수용체가 세포막으로 출현하는 비율의 조화에 의해 이루어진다. 세포막 수준의 인슐린 수용체수의 저하는 인슐린 수용체의 합성의 저하즉 인슐린 수용체 mRNA의 발현이 저하된 것으로 고려해 볼 수 있다. 인슐린 구용체의 발현에 영향을 미치는 요인을 분석하여 그중 인슐린과 포도당이 인슐린 수용체 유전자의 발현에 미치는 영향을 보고한다. 방법 : 인슐린 수용체의 조절기전을 연구하기 위하여 인슐린 수용체가 다량으로 세포막에 표현되는 세포인 IM-9 lymphocyte를 이용하여 인슐린과 포도당 농도의 변화가 인슐린 수용체의 변동과 그 유전자의 발현에 미치는 영향을 조사하였다. 포도당은 생체의 저혈당 농도인 2.BmM/L에서 고혈당 농도인 22mM/L까지, 그리고 인슐린은 인슐린이 없는 대조군에서부터 최고 10^(-6)M까지 노출하여 16시간까지 배양한 후 방사수용체 분석법으로 인슐린 수용체를 분석하였다. RNA를 추출하여 Northern blot을 시행하여 분자 수준의 변화인 mRNA의 전사양상을 관찰하였다. 결과 : 인슐린의 농도가 증가됨에 따라 인슐린 수용체의 최대 결합율은 대조군에서 13.6%였으나, 점차 감소하여 10^(-6)M에서 2%로 유의하게 감소하였다(p<0.01). 고친화성 인슐린 수용체의 수는 대조군이 5.14×10^(3)sites/cell 이었으며, 최고 인슐린 농도인 10^(-6)M에서 0.26×10^(3) sites/cell로 감소하였으나(p<0.01), 저친화성 인슐린 수용체는 변화하지 않았다. 포도당의 농도가 증가됨에 따라 인슐린 수용체의 최대 결합율은 2.8~5.5 mM/L까지는 23% 정도로 변화가 없었으나, 11-22mM/L에서는 약 15%로 유의하게 감소하었다 (p<0.05). 고친화성 인슐린 수용체의 수는 생체내 저혈당 농도인 2.8mM/L에서 5.08×10^(3) sites/cell 이었으나, 고혈당 농도인 22mM/L에서는 2.53×10^(-3) sites/cell로 감소하였으며(p<0.05), 저친화성 수용체 역시 감소하는 경향이었다(p<0.01). Northern blot을 시행한 결과 인슐린 수용체 mRNA는 11과 8.5 kb의 두 종류로 발현되었다. 인슐린 수용체 mRNA 발현양상은 인슐린 농도의 증가에도 유의한 변화가 없었다. 포도당의 농도가 증가됨에 따라 lIkb의 mRNA는 전사량이 증가하였으나, 8.5kb의 전사량은 감소하었다. 이 두 종류 mRNA의 비(8.5kb/11kn)는 인슐린 수용체 수와 정상관 관계에 있었다. 결론 : 이러한 연구결과를 토대로 인슐린 수용체의 조절은 전사과정에서부터 두 종류의 mRNA로 전사됨을 확인할 수 있었다. 수용체 조절의 첫단계인 전사와 마지막 단계인 수용체 결합을 측정한 결과 인슐린은 수용제의 전사단계에는 영향을 미치지 않고 세포막에서 수용체 단백의 표현에 영향을 주며. 포도당은 인슐린 수용체의 전사단계에 영향을 미치며 이를 통하여 세포막 수용체의 조절에 영향을 미치는 것으로 생각된다. 포도당의 농도가 증가됨에 따라 두 종류 mRNA의 전사비(8.5kb/11kb)가 감소하는 것으로 미루어 인슐린 수용체 mRNA는 전사량 뿐만 아니라 전사비의 변화도 세포막 인슐린 수용체의 증가나 감소에 관여하리라고 사료되었다. Backgroundhw level of cell surface insulin receptor seems to result either from a low level of insulin receptor gene expression or from structural changes in the receptor which interfere with proper processing of the primary gene product. The balance of insulin receptor degradation and de novo synthesis determines the final number of receptor on the plasma membrane. We have studied the factors influencing mRNA levels of the insulin receptor, and report on the effects of insulin and glucose on insulin receptor mRNA levels in IM-9 lymphoblastic cells. Methode:IM-9 cells were cultured in RPMI 1640 (glucose free) media containing various concentrations of glucose and insulin for 16 hours. mRNA levels was quantified by Northern blot analysis using a labled cDNA (phINSR-13.1) probe for the insulin receptor. And the number of cell surface insulin receptor was estimated by Scatchard plot simultaneously. Results:The number of insulin receptor was decreased with increasing glucose and insulin concentration in the culture media. Northern blot analysis of insulin receptor gene mRNA revealed two major size of 11 kb (band Ⅰ) and 8.5 kb (band Ⅱ). The concentration of insulin up to 1 pM had no effect on hybridizable insulin receptor mRNA levels. The level of transcripted mRNA band Ⅰ (11 kb) was increased, while band Ⅱ (8.5 kb) was decreased with increasing glucose concentration. The changes of band ratio (band Ⅱ/band Ⅰ) correlated well with the decreased number of insulin receptor. Conclusion: These results suggested that glucose had suppressive effect on the expression of insulin receptor mRNA, but insulin had no direct effect on the expression of insulin receptor mRNA. And it was suggested that the changes of band ratio as well as the amount of transcripted mRNA might play a role in regulaiton of the cell surface insulin receptor.

Effects of Site-Mutagenesis of an Amino Acid Triplet Repeat at $M_1$ and $M_2$ Muscarinic Receptors on Receptor Function

이석용,이상복,Lee, Seok-Yong,Lee, Sang-Bok The Korean Society of Pharmacology 1996 대한약리학잡지 Vol.32 No.3

$M_1$과 $M_2$ 무스카린성 수용체의 두 번째 transmembrane domain의 C-말단에는 leucine(L), tyrosine(Y), threonine(T)로 구성된 3중체(triplet)가 있다. 이 3중체는 $M_2$ 무스카린성 수용체에서는 두 번째 transmembrane domain과 첫 번째 세포외 고리사이의 연접부위에서 LYT-LYT의 반복구조로 존재하며 $M_1$ 무스카린성 수용체에서는 흥미롭게도 LYT-TYL의 역상구조로 존재한다. 본 연구에서는 site-directed mutagenesis방법을 사용하여 이와 같은 특이한 구조적차이가 두 subtype의 수용체의 기능상 차이와 관련한 역할을 가지고 있는지를 확인하고자 하였다. $M_1$ 수용체에서는 LYTTYL서열을 $M_2$ 수용체의 서열에 해당하는 LYTLYT로 mutation시켰으며 $M_2$ 수용체에서는 LYTLYT8서열을 $M_1$ 수용체의 서열에 해당하는 LYTTYL로 mutation시켰다. 이와같은 mutation은 $M_1$과 $M_2$ 수용체에서 효능제 carbachol의 수용체 결합친화력에 유의한 변화를 주지 않았다. 또한 $M_1$ 수용체에서의 mutation은 cyclic AMP 증가작용에 대한 coupling은 변화시키지 않고 phosphoinositides (PI) hydrolysis 촉진작용과 세포내 $Ca^{2+}$ 농도 상승을 현저히 증가시켰다. 또한 $M_2$ 수용체에서의 mutation은 adenylate cyclase 억제에 대한 coupling은 변화시키지 않고 PI hydrolysis 촉진을 약간 증가 시켰다. 이상의 결과는 $M_1$과 $M_2$ 수용체에서 LYTTYL/LYTLYT 아미노산 서열의 차이는 두 수용체의 PI hydrolysis에 대한 coupling을 조절하는 역할을 하지만, 두 수용체 사이에서 ligand 결합과 신호전달계의 차이를 구분하는데 중요한 역할을 하지는 않는다. Both $M_1$ and $M_2$ muscarinic receptors contain a triplet of amino acid residues consisting of leucine (L), tyrosine (Y) and threonine (T) at C-terminus ends of the second putative transmembrane domains. This triplet is repeated as LYT-LYT in $M_2$ receptors at the interface between the second transmembrane domain and the first extracellular loop. Interestingly, however, it is repeated in a transposed fashion (LYT-TYL) in the sequence of $M_1$ receptors. In this work, we employed site-directed mutagenesis to investigate the possible significance of this unique sequence diversity for determining the distinct differential cellular function at the two receptor subtypes. Mutation of the LYTTYL sequence of $M_1$ receptors to the corresponding $M_2$ receptor LYTLYT sequence did not result in a significant change in the binding affinity of the agonist carbachol. The reverse mutation at the $M_2$ receptor also did not modify agonist affinity. Surprisingly, the LYTLYT $M_1$ receptor mutant demonstrated markedly enhanced coupling to activation of phospholipase C without a change in its coupling to increased cyclic AMP formation. There was also an enhanced receptor sensitivity in transducing elevation of intracellular $Ca^{2+}$. On the other hand, the reverse $LYTLYT{\rightarrow}LYTTYL$ mutation in the $M_2$ receptor did not alter its coupling to inhibition of adenylate cyclase, but slightly enhanced its coupling to stimulation of phosphoinositide (PI) hydrolysis. Our data suggest that the LYTTYL/LYTLYT sequence differences between $M_1$ and $M_2$ muscarinic receptors are not important for specifying ligand binding and coupling of various subtypes of muscarinic receptors to different cellular signaling pathways although they might play a role in the modulation of muscarinic reseptor coupling to PI hydrolysis.

Heterogeneity in the Molecular Composition of Excitatory Postsynaptic Sites during Development of Hippocampal Neurons in Culture

Rao, Anuradha,Kim, Eunjoon,Sheng, Morgan,Craig, Ann Marie 부산대학교 유전공학연구소 1998 분자생물학 연구보 Vol.14 No.-

To dertermine their roles in the assemly of glutamatergic postsynaptic sites, we studied the distributions of NMDA- and AMPA-type glutamate receptors; the NMDA receptorinteracting proteins α-actinin-2, PSD-95, and chapsyn;and the PSD-95-associated protein GKAP during the development of hippocampal nerons in culture. NMDA receptors first formed nonsynaptic proximal dendrite shaft clusters within 2-5 d. AMPA receptors were diffuse at this stage anf began to cluster on spines at 9-10 d. NMDA receptor clusters remained partially nonsynaptic and mainly distinct from AMPA receptor clusters until after 3 weeks in culture, when the two began to colocalize at spiny synatic sites. thus, the localization of NMDA and AMPA receptors must be regulated by different mechanisms. α-Actinin-2 colocalize with the NMDA receptor only at spiny synaptic clusters, but not at shrft nonsynaptic or synaptic clusters, suggesting a modulatory role in the anchoring of NMDA receptor at spines. PSD-95, chapsyn, and GKAP were present at some, but not all, nonsynaptic NMDA receptor clusters during the first 2 weeks, indicating that none is essential for NMDA receptor cluster formation. When NMDA receptor clusters becames synaptic, PSD-95 and GKAP were always present, consistent with an essential function in synaptic localization of NMDA receptors. Furthermore, PSD-95 and GKAP clustered opposite presynaptic terminals serveral days before either NMDA or AMPA receptors clustered at these presumptive postsynaptic sites. These results suggest that synapse development proceeds by formation of a postsynaptic scaffold containing PSD-95 and GKAP in concert with presynaptic vesicle clustering, followed by regulated attachment of glutamate receptor subtypes to this scaffold.