• Title/Summary/Keyword: $G{\alpha}_{16}$

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Characterization of Segments of $G{\alpha}_{16}$ Subunit Required for Efficient Coupling with Chemoattractant C5a, IL-8, and fMLP Receptors

  • Eia, Ji-Hee;Lee, Chul-Hoon;Lee, Chang-Ho
    • Journal of Microbiology and Biotechnology
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    • v.14 no.5
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    • pp.1031-1037
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    • 2004
  • The interaction of chemoattractant receptors and $G{\alpha}_{16}$ was studied to provide the molecular basis to elucidate the interaction of chemoattractant receptors with $G{\alpha}_{16}$ subunit, thereby possibly contributing to finding novel targets for designing new type of G protein antagonists with anti-inflammatory effects. Experiments were performed to characterize the $G{\alpha}_{16}$ subunit domains responsible for efficient coupling to chemoattractant receptors. Thus, a series of chimeric $G{\alpha}_{11}G{\alpha}_{16}$ and $G{\alpha}_{16}G{\alpha}_{11}$ cDNA constructs were expressed, and the ability of chimeric proteins to mediate C5a, IL-8, and fMLP-induced release of inositol phosphate in transfected Cos-7 cells was tested. The results showed that short stretches of residues 154 to residue 167 and from residue 174 to residue 195 of $G{\alpha}_{16}$ contribute to efficient coupling to the C5a receptor. On the other hand, a stretch of amino acid residues 220-240 of $G{\alpha}_{16}$ that is necessary for interacting with C5a receptor did not play any role in the interaction with IL-8 receptor. However, a stretch from residue 155 to residue 195 of $G{\alpha}_{16}$ was found to be crucial for efficient coupling to IL-8 receptor in concert with C-terminal 30 amino acid residues of this ${\alpha}$ subunit. Coupling profiles of a variety of chimeras, composed of $G{\alpha}_{11}G{\alpha}_{16}$ to fMLP receptor indicate that the C-terminal 30 amino acids are most critical for the coupling of $G{\alpha}_{16}$ to fMLP receptor. Taken together, $G{\alpha}_{16}$ subunit recruits multiple and distinctive coupling regions, depending on the type of receptors, to interact.

Molecular Interaction Between Interleukin-8 Receptor and G$_\alpha$16 subunit G protein (Interleukin-8 수용체와 G$_\alpha$ 16 subunit G protein 간의 분자상호 작용에 관한 연구)

  • 하지희;강주섭;고현철;신인철;이창호
    • Biomolecules & Therapeutics
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    • v.8 no.3
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    • pp.276-280
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    • 2000
  • In order to identify the domains of the G$_{\alpha}$16 subunit G protein that are responsible for its activation by the Interleukin-8 receptor, a serious of chimeras between G$_{\alpha}$16 and G$_{\alpha}$11 were assessed for their abilities to be activated by these receptors. Co-expression of IL-8 receptor and chimeras in which the carboxyl-terminal regions of G$_{\alpha}$11 were replaced from 30 up to 156 amino acid residues with the corresponding regions of G$_{\alpha}$16 demonstrated that C-terminal 156 amino acid residues of the G$_{\alpha}$16 were not sufficient to confer IL-8 receptor interaction specificity. Testing of a reciprocal serious of chimeras composed of G$_{\alpha}$16 sequences at the amino terminus and G$_{\alpha}$11 sequences at the carboxyl terminals revealed that sequences extending from the amino tar- minus to amino acid 209 of G$_{\alpha}$16 were sufficient to 7ndow the chimera with 75-80% of interaction specificity for 7-8-induced activation. These results suggest th,.7t combined interactions of the C-terminal 30 amino acid residues and certain domains extending from the arts.ino terminus to amino acid 209 of Gal 6 protein may be involved in its couplings to IL-8 receptor.tain domains extending from the arts.ino terminus to amino acid 209 of Gal 6 protein may be involved in its couplings to IL-8 receptor.

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Possible target for G protein antagonist: Identification of specific amino acid residue responsible for the molecular interaction of G$\alpha$ 16 with chemoattractant C5a receptor.

  • 이창호
    • Proceedings of the Korean Society of Applied Pharmacology
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    • 2000.04a
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    • pp.17-19
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    • 2000
  • Heterotrimeric G Proteins transduce ligand binding to a wide variety of seven transmembrane cell surface receptors into intracellular signals. The currently accepted model for the activation of G protein suggests that ligand-activated receptor accelerates GDP-GTP exchange reactions on the ${\alpha}$ subunit of the heterotrimeric G protein. At least seventeen distinct isoforms of the G${\alpha}$ subunit protein have been identified in mammalian organisms. Among them, the G${\alpha}$q family consists of five members whose ${\alpha}$ subunits show different expression patterns. G${\alpha}$q and G${\alpha}$11 seem to be almost ubiquitously expressed, whereas G${\alpha}$14 is predominantly expressed in spleen, lung, kidney and testis. G${\alpha}$16 and its murine counterpart G${\alpha}$15 are expressed in hematopoietic cells and has been shown to couple a wide variety of receptors to phosphoinositide-specific phospholipase C activity. Beta-isoforms of phospholipase C were shown to be activated by all members of G${\alpha}$q family, i.e., G${\alpha}$q, G${\alpha}$11, G${\alpha}$l4 and G${\alpha}$16 subunits either in reconstitution system. or in experiments using cDNA transfection with intact Cos-7 cells.

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Comparative studies of various transfection processes for the optimal luminescence signal analysis (최적의 luminescence 신호 분석을 위한 유전자 전달 방법의 비교연구)

  • Park, Seohyun;Lee, Sunghou
    • Journal of the Korea Academia-Industrial cooperation Society
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    • v.17 no.11
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    • pp.640-647
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    • 2016
  • By minimizing fluorescence interference phenomena, aequorin-based luminescence technology can provide a relatively sensitive detection platform with integration of $G{\alpha}16$ protein in order to track internal calcium mobilization by G protein-coupled receptors (GPCR). In this type of cell-based functional assay format, it is essential to optimize the transfection process of a receptor and $G{\alpha}16$ protein. For this study, corticotropin releasing factor receptor subtype 2(CRF2) was set as a model system to generate three stable cells with CRF2 and $G{\alpha}16$ in addition to transiently transfected cells under three different conditions. Agonist (sauvagine) and antagonist (K41498) responses in those cells were analyzed to develop the optimum transfection process. As a result, the effective signal ratio in the dose response experiments of sauvagine and K41498 were at least 10-fold higher (z'=0.77) in CRF2-$G{\alpha}16$ stable cells. For the transient transfection cells, stable expression of $G{\alpha}16$ prior to the CRF2 represented a two-fold higher signal (z'=0.84) than the other cases of transient transfection. In conclusion, for the utilization of transient transfection processes to develop a cell-based GPCR functional assay system, it is suggested to introduce various target receptors after stable expression of $G{\alpha}16$ protein.

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
    • Archives of Pharmacal Research
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    • v.21 no.4
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    • pp.423-428
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    • 1998
  • 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.

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Anti-melanogenesis in B16F0 Melanoma Cells by Extract of Fermented Cordyceps militaris Containing High Cordycepin (Cordycepin-고함유 동충하초(Cordyceps militaris) 발효 추출물의 미백효과)

  • Cha, Jae-Young;Kim, Sung-Young
    • Journal of Life Science
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    • v.23 no.12
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    • pp.1516-1524
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    • 2013
  • To find a novel skin whitening agent, the effect of cordycepin-enriched Cordyceps militaris (CM${\alpha}$) extract fermented by fungi on anti-melanogenesis in B16F0 mouse melanoma cells was investigated. Fermented CM${\alpha}$ was prepared with fungi, including Monascus purpureus (Mp), Aspergillus oryzae (Ao), Aspergillus kawachii (Ak), and Rhizopus oryzae (Ro), respectively. When the content of the phenolics and the flavonoids and the activities of the antioxidant and the mushroom tyrosinase inhibition were measured in the CM fermented by Ak (AkF-CM), the highest content of the phenolics was 46 mg/g dry weight and the highest content of the flavonoids was 0.93 mg/g; the highest activity of the DPPH radical scavenging was 62.74% and the highest activity of the mushroom tyrosinase inhibition was 79.97% CM${\alpha}$CM${\alpha}$. From this result, AkF-CM${\alpha}$ exhibited the highest mushroom tyrosinase inhibitory activity and so it was used in subsequent anti-melanogenesis. B16F0 melanoma cells were treated with 1-10 mg/ml concentrations of AkF-CM${\alpha}$ and 200 ${\mu}M$ arbutin as the positive control. The melanin content and cell viability of the melanoma cells by arbutin treatment decreased to 43% and 92% of the control, respectively. AkF-CM${\alpha}$ treatment at 1, 3, and 5 mg/ml concentrations decreased the extracellular melanin release induced by IBMX treatment by 35%, 45%, and 53%, respectively. AkF-CM${\alpha}$ showed inhibitory activity against both intracellular tyrosinase in melanoma cells and mushroom tyrosinase. AkF-CM${\alpha}$ reduced the protein level of tyrosinase in the IBMX-stimulated cells. These results indicate that AkF-CM${\alpha}$ suppressed the activity and protein content of cellular tyrosinase and decreased the total melanin content in cultured B16F0 melanoma cells.

Enterohepatic Recycling of Estrogen and its Relevance with Female Fertility

  • Sher, Alam;Rahman, M.Ataur
    • Archives of Pharmacal Research
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    • v.23 no.5
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    • pp.513-517
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    • 2000
  • Enterohepatic recycling of estrogen after oral administration of 1 mg non-radioactive estriol was studied in fourteen women selected as the control subjects and ten infertile women in whom the infertility was appearing to be of endocrine origin. The extent of enterohepatic recycling of estriol ($E_3$) during the early follicular phase of menstrual cycle was assessed by monitoring during 48 h the urinary excretion of its two major metabolites i.e; estriol 16 $\alpha$-glucuronide ($E_3-16$$\alpha-G$) and estriol-3 glucuronide ($E_3$-3-G). The change in urinary level of $E_3$-3-G with respect to ($E_3-16$$\alpha-G$G was considered to reflect the extent of enterohepatic recycling of estriol. Lower values of urinary output of both metabolites in the infertile women as compared with the control subjects and the urinary excretion profile of both metabolites during 48 h after estriol ingestion reveal that the reduced extent of enterohepatic recycling could possibly be one of the factors which contribute towards the incidence of infertility in women.

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Inhibitory Effects of Polyopes affinis Ethanol Extract on Melanogenesis in B16F10 Melanoma Cells (참까막살 에탄올 추출물이 B16F10 흑색종 세포에서의 멜라닌합성에 미치는 영향연구)

  • Kim, Hyang Suk;Choi, Yung Hyun;Hwang, Hye Jin
    • Journal of Life Science
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    • v.29 no.9
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    • pp.972-976
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    • 2019
  • Polyopes affinis is a kind of red algae found in the South coast and near Jeju Island of Korea. The purpose of this study was to investigate the effects of Polyopes affinis ethanol extract (PAEE) on melanogenesis in ${\alpha}-MSH$ stimulated B16F10 melanoma cells. Melanoma cells were cultured for 72 hr treated with PAEE. Total melanin content and the activity of tyrosinase, a key enzyme in melanogenesis, were measured. When the melanin content in B16F10 melanoma cells was tested, PAEE was decreased in a dose-dependent manner: treatment with 100, 300, and $500{\mu}g/ml$ caused 25%, 30%, and 35% reduction, respectively. Treatment of 100, 300, and $500{\mu}g/ml$ of PAEE caused 6%, 12%, and 21% reduction of tyrosinase activities in B16F10 melanoma cells. Also, PAEE suppressed the expression of tyrosinase, tyrosinase-related protein-1, tyrosinase-related protein-2, and melanocyte-inducing transcription factor in B16F10 melanoma cells. A concentration of $500{\mu}g/ml$ of PAEE showed a greater decrease in tyrosinase activity, melanin content, and melanogenic enzyme protein expression. These results indicate that PAEE inhibits melanin synthesis and tyrosinase activity, and Polyopes affinis ethanol extract could be used as a functional whitening agent.

Structure and $Ca^{2+}$-ion effects on the function of $\alpha$-cyclodextrin Glucanotransferase from B. macerans : An X-ray study (Bacillus macerans에서 정제한 $\alpha$-cyclooextrin glucanotransferase의 구조와 칼슘이온이 기능에 미치는 영향 : X-ray 연구)

  • 최희욱;홍순강
    • KSBB Journal
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    • v.19 no.2
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    • pp.159-163
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    • 2004
  • The X-ray structure of the cydodextrin-glucanotransferase of Bacillus macerans was solved by molecular replacement at 2.0 ${\AA}$ resolution. The refined structure has a crystallographic R-factor of 16.6%, (R$\sub$free/ = 20.5%). A new metal binding site occupied by two Ca$\^$2+/-ions was found at an accession channel of the active site. There is a large accumulation of negative charges that bind these Ca$\^$2+/-ions, thereby connecting segment ${\beta}$13-${\alpha}$G (residue 254-276) to the main body of domain A (at ${\alpha}$H, residue 283-297). The segment 313-${\alpha}$G contains the catalytic residue Glu258 between subsite 1 and -1 and Tyr260 (subsite 2) which is located at the entrance of the active site. The Ca$\^$2+/-site 3a,b may have a major role for the activity and specificity of this CGTase, although it is not even conserved for the a-subclass of CGTases.

Inhibitory Effect of Muscat Bailey A Seed Extract on Melanin Production in $\alpha$-Melanin Stimulating Hormone-stimulated B16 Cell (머루포도 씨 추출물의 $\alpha$-Melanin Stimulating Hormone으로 자극한 B16세포에서 melanin 생성억제 효과)

  • Lee, Pyeong-Jae
    • Korean Journal of Plant Resources
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    • v.22 no.5
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    • pp.477-482
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    • 2009
  • Inhibitory effect of skin and seed of three species grape cultivated in Korea on melanogenesis was investigated. Melanin generation was examined in $\alpha$-Melanin Stimulating Hormone-stimulated B16 cell, mouse melanoma, in the presence of samples. All skin sample did not show the inhibitory effect. Seed extract of Campbell early and Neo Muscat had negative effect on cell viability. When $50{\mu}g/ml$ seed extract of Muscat Bailey A was treated, amount of generated melanin and cell viability were $51.6{\pm}20.5%$ and $90.4{\pm}11.3%$ compared to control, respectively. Seed extract of Muscat Bailey A reduced the tyrosinase protein induced by $\alpha$-Melanin Stimulating Hormone, which suggests that inhibitory effect of seed extract of Muscat Bailey A on melanin is partly due to suppression of tyrosinase that is responsible for melanin production.