• 제목/요약/키워드: Phosphoinositides

검색결과 14건 처리시간 0.02초

Comparison of Bradykinin- and Platelet-Derived Growth Factor-Induced Phosphoinositide Turnover in NIH 3T3 Cells

  • Lee, Kee-Ho;Ryu, Yong-Wun;Yoo, Young-Do;Bai, Dong-Hoon;Yu, Ju-Hyun;Kim, Chang-Min
    • BMB Reports
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    • 제29권6호
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    • pp.549-554
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    • 1996
  • Phosphoinositide turnover in response to platelet-derived growth factor, epidermal growth factor, and bradykinin was evaluated in NIH 3T3 cells. Platelet-derived growth factor and bradykinin induced a significant increase in incorporation of $^{32}P$ into phosphatidylinositol (PI), phosphatidylinositol 4-monophosphate (PIP), and phosphatidylinositol 4.5-bisphosphate ($PIP_2$) in serum-starved NIH 3T3 cells. However, epidermal growth factor increased incorporation of $^{32}P$ into these phosphoinositides by only a small amount. Stimulation with platelet-derived growth factor, not bradykinin, caused a rapid elevation of PI and PIP kinase activities that were maximally activated within 10 min. The maximal levels of their elevation in cells with plateletderived growth factor stimulation were 3.2-fold for PI kinase, and 2.1-fold for PIP kinase. Short term pretreatment of NIH 3T3 cells with phorbol 12-myristate 13-acetate, activator of protein kinase C. caused an approximately 60% decrease in platelet-derived growth factor-induced PI kinase activities, indicating the feedback regulation of phosphoinositide turnover by protein kinase C. These results suggest that although the enhancement of phosphoinositide turnover is a rapidly occurring response in platelet-derived growth factor- or bradykinin-stimulated NIH 3T3 cells, phosphoinositide kinases may be associated with initial signal transduction pathway relevant to platelet-derived growth factor but not to bradykinin.

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Onion peel extract and its constituent, quercetin inhibits human Slo3 in a pH and calcium dependent manner

  • Wijerathne, Tharaka Darshana;Kim, Ji Hyun;Kim, Min Ji;Kim, Chul Young;Chae, Mee Ree;Lee, Sung Won;Lee, Kyu Pil
    • The Korean Journal of Physiology and Pharmacology
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    • 제23권5호
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    • pp.381-392
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    • 2019
  • Sperm function and male fertility are closely related to pH dependent $K^+$ current (KSper) in human sperm, which is most likely composed of Slo3 and its auxiliary subunit leucine-rich repeat-containing protein 52 (LRRC52). Onion peel extract (OPE) and its major active ingredient quercetin are widely used as fertility enhancers; however, the effect of OPE and quercetin on Slo3 has not been elucidated. The purpose of this study is to investigate the effect of quercetin on human Slo3 channels. Human Slo3 and LRRC52 were co-transfected into HEK293 cells and pharmacological properties were studied with the whole cell patch clamp technique. We successfully expressed and measured pH sensitive and calcium insensitive Slo3 currents in HEK293 cells. We found that OPE and its key ingredient quercetin inhibit Slo3 currents. Inhibition by quercetin is dose dependent and this degree of inhibition decreases with elevating internal alkalization and internal free calcium concentrations. Functional moieties in the quercetin polyphenolic ring govern the degree of inhibition of Slo3 by quercetin, and the composition of such functional moieties are sensitive to the pH of the medium. These results suggest that quercetin inhibits Slo3 in a pH and calcium dependent manner. Therefore, we surmise that quercetin induced depolarization in spermatozoa may enhance the voltage gated proton channel (Hv1), and activate non-selective cation channels of sperm (CatSper) dependent calcium influx to trigger sperm capacitation and acrosome reaction.

Point Mutations in the Split PLC-γ1 PH Domain Modulate Phosphoinositide Binding

  • Kim, Sung-Kuk;Wee, Sung-Mo;Chang, Jong-Soo;Kwon, Taeg-Kyu;Min, Do-Sik;Lee, Young-Han;Suh, Pann-Ghill
    • BMB Reports
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    • 제37권6호
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    • pp.720-725
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    • 2004
  • A number of signaling molecules contain small pleckstrin homology (PH) domains capable of binding phosphoinositides or proteins. Phospholipase C (PLC)-${\gamma}1$ has two putative PH domains, an $NH_2$-terminal (PH1) and a split PH domain ($nPH_2$ and $cPH_2$). We previously reported that the split PH domain of PLC-${\gamma}1$ binds to phosphatidylinositol 4-phosphate (PI(4)P) and phosphatidylinositol 4,5-bisphosphate (PI(4,5)$P_2$) (Chang et al., 2002). To identify the amino acid residues responsible for binding with PI(4)P and PI(4,5)$P_2$, we used site-directed mutagenesis to replace each amino acid in the variable loop-1 (VL-1) region of the PLC-${\gamma}1$ $nPH_2$ domain with alanine (a neutral amino acid). The phosphoinositide-binding affinity of these mutant molecules was analyzed by Dot-blot assay followed by ECL detection. We found that two PLC-${\gamma}1$ nPH2 domain mutants, P500A and H503A, showed reduced affinities for phosphoinositide binding. Furthermore, these mutant PLC-${\gamma}1$ molecules showed reduced PI(4,5)$P_2$ hydrolysis. Using green fluorescent protein (GFP) fusion protein system, we showed that both $PH_1$ and $nPH_2$ domains are responsible for membrane-targeted translocation of PLC-${\gamma}1$ upon serum stimulation. Together, our data reveal that the amino acid residues $Pro^{500}$ and $His^{503}$ are critical for binding of PLC-${\gamma}1$ to one of its substrates, PI(4,5)$P_2$ in the membrane.

$M_1$$M_2$ 무스카린성 수용체에서 아미노산 Triplet Repeat의 Site-Mutagenesis가 수용체기능에 미치는 영향 (Effects of Site-Mutagenesis of an Amino Acid Triplet Repeat at $M_1$ and $M_2$ Muscarinic Receptors on Receptor Function)

  • 이석용;이상복
    • 대한약리학회지
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    • 제32권3호
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    • pp.311-321
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    • 1996
  • $M_1$$M_2$ 무스카린성 수용체의 두 번째 transmembrane domain의 C-말단에는 leucine(L), tyrosine(Y), threonine(T)로 구성된 3중체(triplet)가 있다. 이 3중체는 $M_2$ 무스카린성 수용체에서는 두 번째 transmembrane domain과 첫 번째 세포외 고리사이의 연접부위에서 LYT-LYT의 반복구조로 존재하며 $M_1$ 무스카린성 수용체에서는 흥미롭게도 LYT-TYL의 역상구조로 존재한다. 본 연구에서는 site-directed mutagenesis방법을 사용하여 이와 같은 특이한 구조적차이가 두 subtype의 수용체의 기능상 차이와 관련한 역할을 가지고 있는지를 확인하고자 하였다. $M_1$ 수용체에서는 LYTTYL서열을 $M_2$ 수용체의 서열에 해당하는 LYTLYT로 mutation시켰으며 $M_2$ 수용체에서는 LYTLYT8서열을 $M_1$ 수용체의 서열에 해당하는 LYTTYL로 mutation시켰다. 이와같은 mutation은 $M_1$$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$$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.

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