་ ඹ ᵝ ᘧ 㸯
Ꮫ ㄳ ồ ㄽ ᩥ ࡢ ෆ ᐜ ࡢ せ ᪨
ㄽ ᩥ ᥦ ฟ ⪅ Ặ ྡ ᚠ ⎔ ែ ⛉ Ꮫ 㡿 ᇦ ᚠ ⎔ ែ ෆ ⛉ Ꮫ ᩍ ⫱ ◊ ✲ ศ 㔝 Ặ ྡ ᡂ ⏣ ᠇ ⣖
㸦 ㄽ ᩥ 㢟 ┠ 㸧
Involvement of β-Arrestin in Endothelin Receptor Signaling㸸A Possible Role in the Pathogenesis of Pulmonary Arterial Hypertension
(࢚ ࣥ ࢻ ࢭ ࣜ ࣥ ཷ ᐜ య ࢩ ࢢ ࢼ ࣝ ఏ 㐩 ࠾ ࡅ ࡿ ș ࣞ ࢫ ࢳ ࣥ ࡢ ᙺ :⫵ 㧗 ⾑ ᅽ ࡢ ᪂ ࡓ ࡞
ែ ゎ ᫂ ྥ ࡅ ࡚)
࠙ ⫼ ᬒ ࠚ
࢚ ࣥ ࢻ ࢭ ࣜ ࣥ(ET)ࡣ ᙉ ຊ ࡞ ⾑ ⟶ ⦰ ≀ ㉁ ࡛ ࠶ ࡾ ࠊG ⺮ ⓑ ㉁ ඹ ᙺ ཷ ᐜ య(GPCR)ࢫ ࣮ ࣃ ࣮
ࣇ ࣑ ࣜ ࣮ ᒓ ࡍ ࡿ ࢚ ࣥ ࢻ ࢭ ࣜ ࣥ ཷ ᐜ య(ET-R)ࢆ ࡋ ࡚ ࡑ ࡢ ⏕ ⌮ స ⏝ ࢆ Ⓨ ࡍ ࡿ ࠋET-R
ࡣ A ᆺ ࠾ ࡼ ࡧ B ᆺ ࡢ 2 ࡘ ࡢ ࢧ ࣈ ࢱ ࣉ ࡀ ࠶ ࡿ ࠋA ᆺ ࡣ ⾑ ⟶ ⦰ స ⏝ ࠾ ࡼ ࡧ ᙉ ᚰ స ⏝ ࢆ
᭷ ࡋ ࠊ ୍ ᪉ ࠊBᆺ ࡣ A ᆺ ࡢ స ⏝ ᣕ ᢠ ࡍ ࡿ ࡇ ࡀ ▱ ࡽ ࢀ ࡚ ࠸ ࡿ ࠋ
β-Arrestin (β-Arr)ࡣ GPCR ࡢ ⏝ 㛵 ࡍ ࡿ ศ Ꮚ ࡋ ࡚ Ⓨ ぢ ࡉ ࢀ ࡓ ࡀ ࠊ ㏆ ᖺ ࠊβ-Arr ࡀ G ࢱ ࣥ ࣃ ࢡ ࡣ ู ࡢ ࠊ⊂ ⮬ ࡢ ࢩ ࢢ ࢼ ࣝ ఏ 㐩 ⤒ ㊰ ࠾ ࠸ ࡚ ࡶ 㔜 せ ࡞ ᙺ ࢆ ᢸ ࡗ ࡚ ࠸ ࡿ ࡇ
ࡀ ሗ ࿌ ࡉ ࢀ ࠊ ୡ ⏺ ୰ ࡛ ከ ࡃ ࡢ ◊ ✲ ࡀ ࡞ ࡉ ࢀ ࡚ ࠸ ࡿ ࠋ ࡉ ࡽ ET-R ࡣ ⫵ 㧗 ⾑ ᅽ 㸦PAH㸧 ࡢ ែ ࡶ ῝ ࡃ 㛵 ࡋ ࡚ ࠸ ࡿ ࠋᮏ ◊ ✲ ࡢ ┠ ⓗ ࡣ ࠊET-R ࢩ ࢢ ࢼ ࣝ ఏ 㐩 ࠾ ࡅ ࡿ β-Arrࡢ ᙺ
ࢆ ᫂ ࡽ ࡋ ࠊ ⫵ 㧗 ⾑ ᅽ (PAH)ࡢ ᪂ ࡓ ࡞ ែ ࢆ ゎ ᫂ ࡍ ࡿ ࡇ ࡛ ࠶ ࡿ ࠋ
࠙ ᪉ ἲ ࠚ
1. siRNA ࢆ ⏝ ࠸ ࡚ HEK293 ⣽ ⬊ ࡢ β-Arr1 ࡲ ࡓ ࡣ β-Arr2 ࢆ ࣀ ࢵ ࢡ ࢲ ࢘ ࣥ ࡋ ࠊET ่ ⃭ (10nM) ࡼ ࡿextracellular signal-regulated kinase (ERK)1/2ࡢ ࣜ ࣥ 㓟 ࢆ ࢘ ࢚ ࢫ ࢱ
ࣥ ࣈ ࣟ ࢵ ࢺ ἲ ࡛ ᐃ ࡋ ࡓ(n=10)ࠋ
2. ࣥ ࢪ ࢜ ࢸ ࣥ ࢩ ࣥ II ཷ ᐜ య(ATR)ࡸ β ࢻ ࣞ ࢼ ࣜ ࣥ ཷ ᐜ య(βAR) ࠾ ࠸ ࡚ ࠊ ࣜ ࢞ ࣥ ࢻ
ࡼ ࡿ ཷ ᐜ య ࡢ ά ᛶ ࡣ ࠊG ࢱ ࣥ ࣃ ࢡ ࡢ ࡳ ࡞ ࡽ ࡎ β ࣞ ࢫ ࢳ ࣥ ࢆ ࡋ ࡓ Epidermal growth factor receptor (EGFR)ࡢ ά ᛶ ࡼ ࡗ ࡚ ࡶ ࡑ ࡢ ⏕ ⌮ స ⏝ ࢆ Ⓨ ࡍ ࡿ ࠋᮏ ◊ ✲ ࡛ ࡣ ࠊET-R ࠾ ࡅ ࡿ EGFR ࡢ ά ᛶ (transactivation)ࡢ ᭷ ↓ ࢆ ㄪ ࡿ ࡓ ࡵ ࠊHEK293⣽ ⬊
⥳ Ⰽ ⺯ ග ࢱ ࣥ ࣃ ࢡ ㉁(GFP)ࡀ ຍ ࡉ ࢀ ࡓ EGFR ࡢ ࣉ ࣛ ࢫ ࣑ ࢻ ࢆ ࣜ ࣏ ࣇ ࢙ ࢡ ࢱ ࣑ ࣥ 2000 ࢆ ⏝ ࠸ ࡚ ࢺ ࣛ ࣥ ࢫ ࣇ ࢙ ࢡ ࢩ ࣙ ࣥ ࡋ ࠊ ࡑ ࡢ ⣽ ⬊ ෆ ࡢ ᒁ ᅾ ࢆ ET ࡢ ่ ⃭(10nM)๓ ᚋ
࡛ ඹ ↔ Ⅼ 㢧 ᚤ 㙾 ࢆ ⏝ ࠸ ࡚ ほ ᐹ ࡋ ࡓ ࠋ
3. EGFR ࡢ transactivation ࢆ ࠊEGFR 㜼 ᐖ ⸆ AG1478 ࢆ ⏝ ࠸ ࡚ ࢘ ࢚ ࢫ ࢱ ࣥ ࣈ ࣟ ࢵ ࢺ ἲ ࡛
☜ ㄆ ࡋ ࡓ ࠋAG1478(1μM)ࡲ ࡓ ࡣ G ࢱ ࣥ ࣃ ࢡ ࡢ ୗ ὶ ࢩ ࢢ ࢼ ࣝ ࡛ ࠶ ࡿ Protein kinase C (PKC)ࡢ 㜼 ᐖ ⸆ Ro318425(1μM)ࡢ ๓ ᢞ ୗ HEK293 ⣽ ⬊ ࢆ ET(10nM)࡛ ่ ⃭ ࡋ ࠊ ERK1/2 ࡢ ࣜ ࣥ 㓟 ࢆ ࢘ ࢚ ࢫ ࢱ ࣥ ࣈ ࣟ ࢵ ࢺ ἲ ࡛ ᐃ ࡋ ࡓ(n=8)ࠋ
⤫ ィ ⓗ ᭷ ព ᛶ ࡣ ࠊGraphPad Prism 7 ࢯ ࣇ ࢺ ࢘ ࢙ ࢆ ⏝ ࠸ ࡚ ࠊTukey ࡢ ⿵ ṇ ࢆ ⏝ ࠸ ࡓ ඖ ศ ᩓ ศ ᯒ 㸦ANOVA㸧 ࡼ ࡾ Ỵ ᐃ ࡋ ࡓ ࠋP ್<0.05 ࢆ ⤫ ィ Ꮫ ⓗ ᭷ ព ࡋ ࡓ ࠋ
࠙ ⤖ ᯝ ࠚ
1. β-Arr1ࡲ ࡓ ࡣ β-Arr2ࡢ ࣀ ࢵ ࢡ ࢲ ࢘ ࣥ ࡼ ࡗ ࡚ ET ่ ⃭ ᚋ 5 ศ ᚋ ࠾ ࡼ ࡧ 10ศ ᚋ ࠾ ࡅ
ࡿ ERK 1/2 ࡢ ࣜ ࣥ 㓟 ࡀ ஹ 㐍 ࡋ ࡓ ࠋ
2. EGFR ࡣ ࠊET ่ ⃭ ๓ ࡣ ⣽ ⬊ ⭷ ᒁ ᅾ ࡋ ࡚ ࠸ ࡓ ࡀ ࠊET ่ ⃭ ᚋ ࡣ ⣽ ⬊ ෆ ⛣ ື
(internalization)ࡋ ࠊ 㞟 ⠵ ࡋ ࡚ ࠸ ࡓ(transactivation)ࠋ 㝧 ᛶ ࢥ ࣥ ࢺ ࣟ ࣮ ࣝ ࡋ ࡚ EGF (10ng/ml)ࢆ ⏝ ࠸ ࡓ ࠋ
3. AG1478 ࡲ ࡓ ࡣ Ro318425 ࡢ ๓ ᢞ ࡼ ࡾ ࠊET่ ⃭ ࡼ ࡿ ERK1/2 ࡢ ࣜ ࣥ 㓟 ࡣ ᢚ ไ ࡉ ࢀ ࡓ ࠋ
࠙ ⪃ ᐹ ࠚ
ᮏ ◊ ✲ ࡛ ࡣ HEK293⣽ ⬊ ࡢ ET-R ࠾ ࠸ ࡚ ࠊβ-Arr1ࡲ ࡓ ࡣ β-Arr2ࡢ ࣀ ࢵ ࢡ ࢲ ࢘ ࣥ ࡀ ET
่ ⃭ ࡼ ࡿ ERK1/2 ࡢ ࣜ ࣥ 㓟 ࢆ ஹ 㐍 ࡉ ࡏ ࠊ ࡲ ࡓ ET-R ࡢ ά ᛶ ࡼ ࡗ ࡚ EGFR ࡀ transactivationࡉ ࢀ ࡿ ࡇ ࢆ ♧ ࡋ ࡓ ࠋ
β-Arrࡢ ࣀ ࢵ ࢡ ࢲ ࢘ ࣥ ࡼ ࡗ ࡚ ࠊ ࢦ ࢽ ࢫ ࢺ ࡢ ่ ⃭ ࡼ ࡿ ERK1/2 ࡢ ࣜ ࣥ 㓟 ࡀ ஹ 㐍 ࡍ
ࡿ ᢚ ไ ࡉ ࢀ ࡿ ࡣ ࠊ ཷ ᐜ య ࡼ ࡗ ࡚ ␗ ࡞ ࡿ ࠋβAR ࡛ ࡣ β-Arr1 ࡲ ࡓ ࡣ β-Arr2 ࡢ ࣀ ࢵ ࢡ ࢲ ࢘ ࣥ ࡼ ࡗ ࡚ ERK1/2ࡢ ࣜ ࣥ 㓟 ࡣ ᢚ ไ ࡉ ࢀ ࡿ ࠋ୍ ᪉ ࠊATR ࡛ ࡣ β-Arr2ࡢ ࣀ ࢵ ࢡ ࢲ ࢘
ࣥ ࡼ ࡾ ERK1/2 ࡢ ࣜ ࣥ 㓟 ࡀ ᢚ ไ ࡉ ࢀ ࡿ ࡢ ᑐ ࡋ ࡚ ࠊβ-Arr1 ࡢ ࣀ ࢵ ࢡ ࢲ ࢘ ࣥ ࡛ ࡣ ERK1/2 ࡢ ࣜ ࣥ 㓟 ࡀ ஹ 㐍 ࡍ ࡿ ࠋᮏ ◊ ✲ ࡛ ࡣ β-Arr1ࡲ ࡓ ࡣ β-Arr2 ࡢ ࣀ ࢵ ࢡ ࢲ ࢘ ࣥ ࡼ ࡗ
࡚ ERK1/2 ࡢ ࣜ ࣥ 㓟 ࡀ ஹ 㐍 ࡋ ࡓ ࡀ ࠊࡑ ࡢ ᶵ ᗎ ࡋ ࡚ ET-Rࡢ ⬺ ឤ స ࡀ ᢚ ไ ࡉ ࢀ ࡓ ࡇ ࡀ
⪃ ࠼ ࡽ ࢀ ࡿ ࠋGPCR ࡣ ࢦ ࢽ ࢫ ࢺ ࡢ ⤖ ྜ ࡼ ࡗ ࡚ ά ᛶ ࡉ ࢀ ࡿ ࡍ ࡄ GPCR-related kinase ࡼ ࡗ ࡚ Cᮎ ➃ ࡀ ࣜ ࣥ 㓟 ࡉ ࢀ ࡿ ࡇ ࡼ ࡗ ࡚ ㏿ ࡸ ⬺ ឤ స ࡉ ࢀ ࡿ ࠋ⬺ ឤ స ࡣ β-Arrࡀ ᚲ せ ࡉ ࢀ ࡚ ࠾ ࡾ ࠊET-R ࠾ ࠸ ࡚ ࡣ β-Arrࡀ ࣀ ࢵ ࢡ ࢲ ࢘ ࣥ ࡉ ࢀ ࡿ ࡇ ࡼ ࡗ
࡚ ET-R ࡢ ⬺ ឤ స ࡀ ᢚ ไ ࡉ ࢀ ࡓ ࠋ ࡑ ࡢ ⤖ ᯝ ࡋ ࡚ G ࢱ ࣥ ࣃ ࢡ ࠊPKC ࢆ ࡋ ࡓ ⤒ ㊰ ࡢ ά ᛶ
ࡀ ಁ 㐍 ࡋ ࡚ ࠊERK1/2ࡢ ࣜ ࣥ 㓟 ࡀ ஹ 㐍 ࡋ ࡓ ᥎ ࡉ ࢀ ࡿ ࠋ
EGFRࡣ EGF ࡸ Transforming growth factor-αࠊHeparin-binding EGF-like growth factor
ࡼ ࡗ ࡚ ά ᛶ ࡉ ࢀ ࠊAktࡸ ERK1/2 ࢆ ࡣ ࡌ ࡵ ࡍ ࡿ mitogen-activated protein kinase ࡢ ά ᛶ ࢆ ࡋ ࡚ ᢠ ࣏ ࢺ ࣮ ࢩ ࢫ స ⏝ ࡸ ⥺ ⥔ ࡢ ಁ 㐍 ࢆ ࡶ ࡓ ࡽ ࡍ ࠋ ᮏ ◊ ✲ ࡛ ࡣ ET-R ࡢ ά ᛶ ࡼ ࡗ ࡚ EGFRࡀ transactivationࡉ ࢀ ࡿ ࡇ ࢆ 2 ࡘ ࡢ ᐇ 㦂 ⣔ ࡼ ࡗ ࡚ ♧ ࡋ ࡓ ࠋ
PAH ࡣ ⫵ ື ⬦ ࡢ 㐍 ⾜ ᛶ ࡢ ⊃ ✽ ࠊ ⥺ ⥔ ࢆ ≉ ᚩ ࡍ ࡿ 㞴 ᛶ ࡢ ᝈ ࡛ ࠶ ࡾ ࠊ ⌧ ᅾ ET-R ᣕ ᢠ ⸆ ࢆ ྵ ࡴ ⒪ ⸆ ࡀ ⮫ ᗋ ࡛ ⏝ ࡉ ࢀ ࡚ ࠸ ࡿ ࡀ ࠊࡑ ࡢ ຠ ᯝ ࡣ ᚲ ࡎ ࡋ ࡶ ‶ ㊊ ࡢ ࠸ ࡃ ࡶ ࡢ ࡛ ࡣ ࡞ ࠸ ࠋᮏ ◊ ✲ ࠾ ࠸ ࡚ ࡣ ࠊET-R ࠾ ࠸ ࡚ β-Arr࠾ ࡼ ࡧ EGFR ࡀ ࡑ ࡢ ࢩ ࢢ ࢼ ࣝ ఏ 㐩 ᶵ ᵓ
ྵ ࡲ ࢀ ࠊERK1/2ࡢ ࣜ ࣥ 㓟 㛵 ࡍ ࡿ ࡇ ࡀ ♧ ࡉ ࢀ ࡓ ࠋERK1/2ࡢ ࣜ ࣥ 㓟 ࡣ ⤌ ⧊ ࡢ
⥺ ⥔ ࢆ ಁ 㐍 ࡍ ࡿ ࡇ ࡀ 㐣 ཤ ࡢ ሗ ࿌ ࡽ ♧ ࡉ ࢀ ࠾ ࡾ ࠊβ-Arr ࡀ PAH ࡢ ែ ᙧ ᡂ ࠊ 㐍 ⾜ 㛵 ࡋ ࡚ ࠸ ࡿ ྍ ⬟ ᛶ ࡀ ࠶ ࡿ ࠋࡇ ࢀ ࡽ ࡢ ⤖ ᯝ ࡣ ࠊPAHࡢ ᅉ ᪂ ࡓ ࡞ ▱ ぢ ࢆ ࡶ ࡓ ࡽ ࡋ ࠊ
ࡲ ࡛ ࡣ ␗ ࡞ ࡿ ᪂ ࡓ ࡞ ど Ⅼ ࡽ ⒪ ⸆ ࡢ 㛤 Ⓨ ㈉ ⊩ ࡍ ࡿ ྍ ⬟ ᛶ ࡀ ࠶ ࡿ ࠋET-R ࠾ ࡅ ࡿ β-Arr ࡢ ᙺ ࠊ ࠾ ࡼ ࡧ PAH ࡢ ែ ᙧ ᡂ ࠾ ࡅ ࡿ ࡑ ࡢ 㛵 ࢆ ゎ ᫂ ࡍ ࡿ ࡓ ࡵ ࠊ ࡉ ࡽ ࡞ ࡿ ◊
✲ ࡀ ᚲ せ ࡛ ࠶ ࡿ ࠋ
