我々は、いくつかの財団や慈善ドナーの寛大な支援を（SakmarLab.orgを参照）に感謝します。

1。 GPCRの中に非天然アミノ酸の部位特異的な遺伝子の組み込み<ol><li> 5％CO 2雰囲気中、37℃で10％ウシ胎児血清（FBS）を補充した（グルコース、2mMのグルタミンを4.5g / L）DMEMにHEK293T細胞を維持する。 </li><li>リポフェクトアミンプラス試薬を用いて、10cmのプレートで60〜80％コンフルエンスまで増殖させた細胞をトランスフェクトする。 <ol><li> 750μlのDMEM中に、所望の位置にアンバー終止コドンを含む10μlのプラス試薬、GPCR cDNAの3.5μgの（pMT4. RhoのかをpcDNA 3.1。CCR5）、3.5サプレッサーtRNAのcDNA（pSVB.Yam）μgの0.35μgのを追加p-アジド-L-フェニルアラニン（pcDNA.RS）する変異アミノアシルtRNA合成酵素cDNAの。 15分間室温でインキュベートする。また、コントロールとして機能するように重量のGPCRのcDNA（アンバー終止コドンを含まない）を使用して同様のトランスフェクションを行う。 17μlのリポフェクタミンを含むDMEM750μlのこの混合物を追加します。室温で15分間平衡化した後、持参4ミリリットルの総量。 </li><li> 10-10cmプレート上の吸引メディアは、細胞へのトランスフェクション混合物を適用し、5％CO 2雰囲気下で37℃に戻る。 4-6時間後、20％FBSおよび1mM AZFを含有する4ミリリットルDMEMで細胞を補充する。 </li></ol></li><li>次の日、DMEM中10％FBSおよび0.5 mMのAZFを含有する増殖培地を交換してください。 </li><li>細胞を回収し、48時間トランスフェクション後、発現を解析するか、次のセクションで説明する光架橋または標識手続きに進みます。 <ol><li>増殖培地中のUAAの存在下でのGPCRアンバー変異体の発現を確認。我々は、ウェスタン免疫ブロット検出によってこれを行う。溶解液を4℃で1時間、20mMのトリス-HCl、pH7.5中の1％で細胞ペレット（w / v）のドデシル-β-D-マルトシド（DDM）、100mMのNaCl 16,000×gで溶解液を遠心分離し、SDS-PAGEにより、還元条件下で、上清を解決します。 PVDF膜に移し、目を用いて、完全長受容体の発現を検出するためのイムノブロット分析を行っ受容体のC末端に設計されるエピトープを認識するモノクローナル抗体E 1D4（国立細胞培養センター）。31 </li></ol></li></ol> 2。ターゲット光架橋を使用して、リガンド結合部位のマッピング<ol><li> 48時間のトランスフェクション後、細胞から吸引メディアと4ミリリットル1Xリン酸緩衝生理食塩水（PBS）と交換してください。 15分間37℃に戻ります。 </li><li> 1X PBSで細胞を再懸濁し、ファルコンチューブに移す。細胞をペレットと上清を吸引する2分間1,500 rpmで遠心分離する。 </li><li> 20mMのHEPES、pH7.5で、0.2％BSAおよび受容体 - リガンド複合体形成を確実にするために時間および温度の必要な長さの結合飽和濃度でトリチウム化したリガンドを含有する1×ハンクス緩衝塩類溶液中に細胞ペレットを再懸濁する。 </li><li> 4℃で15分間（ 例えば、UVランプを使用して）365nmでAZFの光活性化のために、24ウェルまたは96ウェルプレートに細胞懸濁液を転送するMaintainは氷上でプレートを試料加熱及び細胞溶解を回避する。 </li><li>エッペンドルフチュー​​ブに細胞を移し、遠心分離機、細胞をペレット上清を除去し、分析に進みます。ペレットは、将来の使用のために-20℃で保存することができる。 </li><li> 1％（w / v）のDDM、20mMのトリス-HCl、pH7.5およびプロテアーゼ阻害剤を含む溶解緩衝液中で細胞ペレットを再懸濁します。 4℃で1時間のインキュベーション後、16,000×gで10分間、細胞溶解物を遠心分離し、上清を新しいチューブに移す。 </li><li> 1D4モノクローナル抗体 - セファロース2B樹脂は上清に追加して設計され、C末端1D4のmAbエピトープを使用してGPCRを免疫精製するために4℃で一晩インキュベートする。翌日溶解緩衝液でビーズを数回洗浄する。振とうしながら1時間、37℃で1％SDSで溶出サンプル。 </li><li>シンチレーション液を含有する15ミリリットルバイアルに溶出液の一部を移し、目にトリチウム化したリガンドの特異的結合の量を定量し、βシンチレーションカウンターで数える電子受容体である。 </li><li>標準的なゲル電気泳動により、残りの1D4モノクローナル抗体精製された溶離液を解決します。我々は、4〜12％SDS-PAGEゲルおよびイムノ用PVDF膜にして、半乾燥転送を使用しています。また、分子量を視覚的に近似を導くために、標準的なタンパク質ラダーとレーンが含まれています。 <ol><li> 0.05％のTween-20を含むTBS緩衝液中の5％ミルクでPVDF膜をブロックする。 HRP結合抗マウスIgG二次抗体1D4 mAbと完全長受容体の発現を確認するために、膜をプローブ。 </li><li>増強化学発光（ECL）試薬で処理し、バンドを可視化し、オートラジオグラフィーフィルムに膜を公開します。 </li></ol></li><li>特定の分子量マーカーで切断し、その後、各サンプルレーンを分離するためにカミソリの刃を有する膜をカットします。シンチレーション液を含むバイアルに膜セグメントを転送します。ベータシンチレーションカウンターでカウントすることにより、各膜セグメント内のトリチウムの量を定量化する。 </li><li>正を識別GPCRとリガンドとの和に等しい見かけの分子量におけるトリチウムの検出を光架橋する。 </li></ol> 3。エピトープタギングおよび細胞表面酵素結合免疫吸着アッセイ（ELISA）を標的化<ol><li> 24時間のトランスフェクション後、1ミリリットル×PBSで細胞を洗浄し、3分間0.25％トリプシン1mlでトリプシン処理。 10％FBSおよび0.5 mMのAZFを含む9ミリリットルDMEMで補う。細胞を再懸濁し、細胞密度をカウントします。私たちは、標準的な血球計数器を使用しています。 </li><li>ウェル当たりのポリ-D-リジン溶液は0.01mg / 100mlで高結合性、透明底96ウェルプレートの事前処置する。室温で30分間インキュベートし、1×PBSおよび空気乾燥で繰り返し洗浄する。 </li><li>プレートトランスフェクトされた細胞を200μlの4×10 6の密度で、 - 8×10 4細胞/ウェルで96ウェルプレートに、5％CO 2雰囲気中で37℃に戻る。 </li><li>翌日標識用細胞を調製。静かに100μl/ウェル1X PBS Tで3回洗浄OどんなAZF含む増殖培地を除去します。細胞は、例えば、のCa 2 +およびMg 2 +を含有するPBSを用いて、付着したままであることを確認してください。 </li><li> PBS中で5月20日mMのに維持原液から1X HBSS / PBSに50〜200μmのFLAG-トリアリール標識試薬を準備します。 （各アンバー変異体のための三連で）を各ウェルに60ミリリットルを適用し、4時間まで30分、37℃に戻ります。 1×HBSS / PBS中のラベル処理をせずに井戸のセットを維持する。またAZF-GPCR変異型と比較する重量GPCRコントロールが含まれています。 </li><li>よくブロッキングバッファーを100μl/で細胞を3回洗浄、BB（1×PBS、0.5％BSA）は、完全に未反応のラベルを削除します。 </li><li>細胞に100μl/ウェルの（1×PBS中16％ストックから調製した）を4％パラホルムアルデヒドを適用します。室温で20分間インキュベートする。 BBで3回洗浄する。 </li><li>標識受容体および受容体の発現を検出するために、標準的な細胞表面ELISAプロトコルを実行する。二AN続く氷上1.5時間一次抗体とインキュベートします1時間室温でtibodyインキュベーション。 <ol><li> FLAG-トリアリール標識試薬のFLAGペプチドエピトープを検出するために、抗FLAG M2抗体（BBで行われた抗体の例 1:2,000希釈）を100μlを追加します。また、ネガティブコントロールとして非ラベル処理したウェルを探る。抗CCR5 2D7は、（我々は1:500希釈を使用）抗体重量やAZF-GPCR細胞表面発現を決定するために、井戸の別々のセットに追加します。 </li><li> BBで細胞を3回洗浄し、HRP結合抗マウスIgG二次抗体（1:2,000希釈）100μlでインキュベートする。 </li><li>慎重に、BBで細胞を5回洗浄する。 50μlの検出緩衝液を追加しアンプレックスレッド、20mMのH 2 O 2、1×PBS（1:10:90比）、室温で15分間インキュベートする。 EMは 590 =λEX = 530で蛍光マルチウェルプレートリーダー上のスペクトルデータを収集し、λ。 </li></ol></li></ol> 4。 GPCRのバイオ直交蛍光標識<ol><li>溶解するwtまたはAZF-ロドプシンが1％を含有する1ミリリットルの可溶化緩衝液中で変異（w / v）のDM、50mMのHEPESまたはTris-塩酸、pH 6.8、100mMのNaCl、1mMのCaCl 2およびプロテアーゼ阻害剤の1を発現する10 7採取した細胞4℃での時間10分間15,000×gで細胞溶解物を遠心分離し、上清画分を収集します。 </li><li>設計C末端1D4エピトープを使用して、受容体を捕捉するために、100μlの1D4-モノクローナル抗体セファロース2B樹脂を追加します。 4℃で12時間インキュベートする0.1 Mリン酸ナトリウム緩衝液、pH 7.3中の0.1％（w / v）のDDMで樹脂を3回洗浄する。 </li><li>免疫アフィニティーマトリックス（1D4-モノクローナル抗体セファロース2B）上に固定化された受容体を標識する0.3〜0.5ミリリットルの全容量0.1 mMのフルオレセイン - ホスフィンを追加します。室温で12時間インキュベートする。上清を遠心分離して除去します。未反応の標識を除去し、樹脂を洗浄します。 </li><li> 0.1％を含む100mlの溶出緩衝液（w / v）のDDMおよび0.33 mg / mlのノナペプチド（1D4エピトープに対するC9ペプチド）で標識された受容体を溶出させる2 mMリン酸緩衝液中で、1時間氷上でインキュベートすることによりpHを6.0。 </li><li>還元条件下でSDS-PAGEによって標識されたサンプルを解決する。 PBSで簡単にゲルを洗った後、ゲル内蛍光によってAZF-GPCRのラベル付けを視覚化。例えば、フルオレセインで修飾体を検出するために、488 nmの波長のレーザー共焦点蛍光スキャナを使用。 </li></ol>

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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incorporation+of+azides+into+recombinant+proteins+for+chemoselective+modification+by+the+Staudinger+ligation.">Incorporation of azides into recombinant proteins for chemoselective modification by the Staudinger ligation.</a> Proceedings of the National Academy of Sciences of the United States of America. 99, 19-24 (2002).</li><li>Agard, N. J., Prescher, J. A., Bertozzi, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+strain-promoted+[3+++2]+azide-alkyne+cycloaddition+for+covalent+modification+of+biomolecules+in+living+systems.">A strain-promoted [3 + 2] azide-alkyne cycloaddition for covalent modification of biomolecules in living systems.</a> Journal of the American Chemical Society. 126, 15046-15047 (2004).</li><li>Kolb, H. C., Sharpless, K. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+growing+impact+of+click+chemistry+on+drug+discovery.">The growing impact of click chemistry on drug discovery.</a> Drug discovery today. 8, 1128-1137 (2003).</li><li>Huber, T., Botelho, A. V., Beyer, K., Brown, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+Model+for+the+G-Protein-Coupled+Receptor+Rhodopsin:+Hydrophobic+Interface+and+Dynamical+Structure.">Membrane Model for the G-Protein-Coupled Receptor Rhodopsin: Hydrophobic Interface and Dynamical Structure.</a> Biophys. J. 84, 2078-2100 (2004).</li><li>Okada, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+retinal+conformation+and+its+environment+in+rhodopsin+in+light+of+a+new+2.2+&#197;+crystal+structure.">The retinal conformation and its environment in rhodopsin in light of a new 2.2 &#197; crystal structure.</a> J. Mol. Biol. 342, 571-583 (2004).</li><li>Huber, T., Tian, H., Ye, S., Naganathan, S., Kazmi, M. A., Duarte, T., Sakmar, T. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioorthogonal+labeling+of+functional+G+protein-coupled+receptors+at+genetically+encoded+azido+groups.">Bioorthogonal labeling of functional G protein-coupled receptors at genetically encoded azido groups.</a> , (2013).</li><li>Naganathan, S., Ye, S., Sakmar, T. P., Huber, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Site-specific+epitope+tagging+of+GPCRs+by+bioorthogonal+modification+of+a+genetically-encoded+unnatural+amino+acid.">Site-specific epitope tagging of GPCRs by bioorthogonal modification of a genetically-encoded unnatural amino acid.</a> Biochemistry. , (2013).</li><li>Molday, R. S., MacKenzie, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monoclonal+antibodies+to+rhodopsin:+characterization,+cross-reactivity,+and+application+as+structural+probes.">Monoclonal antibodies to rhodopsin: characterization, cross-reactivity, and application as structural probes.</a> Biochemistry. 22, 653-660 (1983).</li><li>Gether, U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agonists+induce+conformational+changes+in+transmembrane+domains+III+and+VI+of+the+beta2+adrenoceptor.">Agonists induce conformational changes in transmembrane domains III and VI of the beta2 adrenoceptor.</a> The EMBO journal. 16, 6737-6747 (1997).</li><li>Hubbell, W. L., Altenbach, C., Hubbell, C. M., Khorana, H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rhodopsin+structure,+dynamics,+and+activation:+A+perspective+from+crystallography,+site-directed+spin+labeling,+sulfhydryl+reactivity,+and+disulfide+cross-linking.">Rhodopsin structure, dynamics, and activation: A perspective from crystallography, site-directed spin labeling, sulfhydryl reactivity, and disulfide cross-linking.</a> Advances in Protein Chemistry; Membrane Proteins. 63, 243-290 (2003).</li><li>Cai, K., Itoh, Y., Khorana, H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+of+contact+sites+in+complex+formation+between+transducin+and+light-activated+rhodopsin+by+covalent+crosslinking:+use+of+a+photoactivatable+reagent.">Mapping of contact sites in complex formation between transducin and light-activated rhodopsin by covalent crosslinking: use of a photoactivatable reagent.</a> Proceedings of the National Academy of Sciences of the United States of America. 98, 4877-4882 (2001).</li><li>Dorman, G., Prestwich, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Benzophenone+photophores+in+biochemistry.">Benzophenone photophores in biochemistry.</a> Biochemistry. 33, 5661-5673 (1994).</li><li>Zhang, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+strategy+for+the+site-specific+modification+of+proteins+in+vivo.">A new strategy for the site-specific modification of proteins in vivo.</a> Biochemistry. 42, 6735-6746 (2003).</li><li>Dirksen, A., Hackeng, T. M., Dawson, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleophilic+catalysis+of+oxime+ligation.">Nucleophilic catalysis of oxime ligation.</a> Angew. Chem. Int. Ed. 45, 7581-7584 (2006).</li><li>Yanagisawa, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multistep+engineering+of+pyrrolysyl-tRNA+synthetase+to+genetically+encode+N(epsilon)-(o-azidobenzyloxycarbonyl)+lysine+for+site-specific+protein+modification.">Multistep engineering of pyrrolysyl-tRNA synthetase to genetically encode N(epsilon)-(o-azidobenzyloxycarbonyl) lysine for site-specific protein modification.</a> Chemistry &amp; biology. 15, 1187-1197 (2008).</li></ol>

著者らは、開示することは何もありません。

我々のGPCRに、ここでは反応性プローブ​​、AZFの部位特異的組み込みのための強固な方法論を記述し、GPCRの構造やダイナミクスを研究するために、このツールの便利な3つのアプリケーションを示しています。部位特異的に本手法UAAsを組み込むには、化学的に32、33をラベルまたは単一アクセス可能なシステイン変異体に光架橋34を取り付けるに基づく代替戦略の基本的な問?...

ヘプタヘリカルGタンパク質共役受容体（GPCR）は、重要かつ多様な細胞外シグナルを媒介する高度に動的な膜タンパク質のスーパーファミリーを含む。古典パラダイムでは、受容体の活性化は、リガンド誘導性立体構造変化に連結されている。1は、GPCRの構造生物学における2近年の進歩は膜貫通シグナル伝達の分子機構に重要な洞察を提供してきました。3-5が、より高い化学的精度で理解する作用機序およびGPCRシグナル伝達の構造力学は、アプローチのツールキットは、活性なシグナル伝達複合体を調べるために有益な分子や化学プローブを組み込むことが必要である。 この目的のために、我々は、具体的には、部位以前シュルツとCによって開拓アンバーコドン抑制技術を使用して非または非天然アミノ酸に基づいて発現する受容体への侵襲摂動プローブ（UAA）突然変異誘発を導入する方法を適応さoworkers。6私たちは、このような哺乳動物の細胞以外のほとんどの異種系の中で表現するのが難しいのGPCRなどのタンパク質のための高収量発現および変異誘発システムを実現するために、UAA突然変異誘発法を最適化した。直交設計サプレッサーtRNAを使用して、特定のUAAのためのアミノアシルtRNA合成酵素のペアを進化させ、我々は部位特異的に発現される標的GPCRをでUAAsを導入しました。 UAAsの成功した組み込みは、p-アセチル-L-フェニルアラニン（ACF）に、p-ベンゾイル-L-フェニルアラニン（BZF）、およびp-アジド-L-フェニルアラニン（AZF）は、我々のモデルのGPCRにおいて実証されている-ロドプシンおよびヒトCCケモカイン受容体、CCR5 図7、図8 原理的には、UAAは、遺伝的にタンパク質配列内の任意の位置で符号化することができ、それは、標的GPCRの単一コドン走査を可能にするように、このプロパティは、貴重な生化学的ツールである。私たちは、反応性庵治を持つUAA、AZFの汎用性に特に焦点を絞り部分を行う。ユニークな赤外線（IR）プローブ、8として機能することに加えて、9 AZFはまた、隣接する第一級アミンまたは脂肪族の水素と反応して光架橋基として機能することができる。さらに、生物学的に不活性なアジド基は、バイオ直交標識反応における選択的化学ハンドルとして参加することができます。ここでは、そのようなトラップに目標と光架橋受容体 - リガンド複合体、およびバイオ直交エピトープ標識、蛍光標識ストラテジーによるGPCRの変更などのGPCRの中にAZFの部位特異的組み込みの有用な応用を説明するための例を紹介します。 光活性化試薬は、1960年代から生物学的システムを研究するために使用されてきた。10この間、受容体-リガンド架橋実験の存在量は、光親和性リガンドの使用を含むほとんどがGPCR複合体を研究することが報告されている。11、12しかし、これらの彼らはrequirなどのアプリケーションでは、技術的に限られている架橋性基を有する配位子の電子合成。13-15また、リガンド中の架橋部分では、GPCR上の架橋の位置を特定するために挑戦している。部位特異的に抑制技術アンバーコドンを使用して蛋白質にUAAsとして光架橋基を導入することは、貴重な進歩です。16、17我々は内受容体-リガンド複合体の形成に関与している受容体に結合界面を識別するために、光架橋技術を開発GPCRの中に光解離性基を導入することによって、細胞を生き。18,19ここでは、小分子リガンドの結合部位、CCR5にトリチウム化マラビロックを識別するために、この標的化光架橋技術を適用するための実験プロトコルおよびデータ分析の方法を記載している。この方法は、天然のリガンドの化学構造を保持することに加えて、放射性リガンド上のハンドルの正確な定量化を利用する。 蛍光ベースの技術は、直接、受容体の立体配座状態をプローブすることによって受容体活性化の構造的基盤を正確に理解してサポートしています。20は、21しかし 、GPCRの中に蛍光標識を導入する柔軟性を有する技術は部位特異的に限られている。我々は、GPCRのシグナル伝達複合体の単一分子検出（SMD）を容易にするために、バイオ直交化学修飾戦略を採用することに興味を持っている。22アジド基は、シュタウディンガー·ベルトッツィライゲーション、23、24銅を含まない株をプロモートアジドなどのバイオ直交化学反応に参加することができますアルキン環（SpAAC）25、銅触媒によるアジド-アルキン環（CuAAC）。26我々は、アジドとホスフィンとの間の特定の反応を含むシュタウディンガー·ベルトッツィライゲーション反応にここに焦点を当てています。我々は、ペプチドエピトープ（FLAGペプチド）または蛍光標識に結合つの異なるホスフィン誘導体の使用を示すGPCRのサイト固有の変更を達成するために（フルオレセイン）。 ロドプシンAZFの部位特異的標識が露出し、溶剤である標的部位を選択するためにX線結晶構造と動的シミュレーションを用いて変異体についての我々は以前の条件を最適化した。27、28我々はまた、シュタウディンガー·バックグラウンドのないラベルを達成するための実現可能性を示すベルトッツィライゲーション。29ここでは、免疫親和性マトリックス上に固定し、その後、ゲル内の蛍光により可視化された界面活性剤で可溶化受容体の蛍光標識を達成するために使用の一般的な手順を示しています。さらに、私たちは未知の構造、CCR5の受容体上の標識に適した位置を特定するため、このラベル戦略に関する有用な拡張を示しています。これはAZF-GPCRは、細胞ベースの半ハイスループット形式で変異体のバイオ直交修飾に依存している標的化ペプチド-エピトープタギング戦略を用いて行われる。30本この方法は、標識事象を監視するために細胞表面ELISAの多段階検出特性を利用するものである。 我々はここで議論する方法論は一般的なものであり、原則的に抑制技術アンバーコドンを使用してAZFを組み込んだ任意のGPCRに適用することができます。我々の詳細を非天然アミノ酸突然変異誘発法を用いて、例えばAZFとしてUAAsとGPCRの構造的および動的な研究を促進するそれらの後続のアプリケーションに組み込まれた受容体の哺乳動物細胞発現に関与する工程、ここで提示されたプロトコルである。

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Plasmid pSVB. Yam</td>
			<td></td>
			<td></td>
			<td>(Ye et al., 2008)</td>
		</tr>
		<tr>
			<td>Plasmid pcDNA.RS for azF</td>
			<td></td>
			<td></td>
			<td>(Ye et al., 2009)</td>
		</tr>
		<tr>
			<td>Plasmids pMT4.Rho and pcDNA 3.1.CCR5</td>
			<td></td>
			<td></td>
			<td>Optionally contain the amber stop codon (TAG) at a desired position</td>
		</tr>
		<tr>
			<td>HEK 293T cells</td>
			<td></td>
			<td></td>
			<td>Adherent cells</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM)</td>
			<td>Gibco</td>
			<td>10566</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline</td>
			<td>Gibco</td>
			<td>14200</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Gemini Bio-products</td>
			<td>100-106</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine Plus</td>
			<td>Invitrogen</td>
			<td>Lipofectamine: 18324-012 
			Plus: 11514-015</td>
			<td></td>
		</tr>
		<tr>
			<td>p-azido-L-phenylalanine</td>
			<td>Chem-Impex International</td>
			<td>6162</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Table 1. Site-specific genetic incorporation of unnatural amino acids into GPCRs materials</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>[header]</td>
		</tr>
		<tr>
			<td>Maxima ML-3500S UV-A lamp</td>
			<td>Spectronics Corporation</td>
			<td></td>
			<td>azF is activated by 365-nm light</td>
		</tr>
		<tr>
			<td>Hank&#39;s Buffered Salt Solution (HBSS)</td>
			<td>Gibco</td>
			<td>14065</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Irvine Scientific</td>
			<td>9319</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Roche</td>
			<td>3117405001</td>
			<td></td>
		</tr>
		<tr>
			<td>Tritium-labeled ligand</td>
			<td></td>
			<td></td>
			<td>From collaborator (Grunbeck et al., 2012)</td>
		</tr>
		<tr>
			<td>1% (w/v) n-dodecyl-&#946;-D-maltoside</td>
			<td>Anatrace</td>
			<td>D310LA</td>
			<td></td>
		</tr>
		<tr>
			<td>1D4-sepharose resin</td>
			<td></td>
			<td></td>
			<td>1D4 mAb immobilized on CNBr-activated sepharose 2B resin</td>
		</tr>
		<tr>
			<td>1% (w/v) sodium dodecyl sulfate (SDS)</td>
			<td>Fisher Scientific</td>
			<td>BP166</td>
			<td></td>
		</tr>
		<tr>
			<td>NuPAGE Novex 4 - 12% SDS gels</td>
			<td>Invitrogen</td>
			<td>NP0322BOX</td>
			<td>SDS-PAGE performed on NuPAGE apparatus</td>
		</tr>
		<tr>
			<td>Trans-Blot SD apparatus</td>
			<td>Biorad</td>
			<td></td>
			<td>Apparatus for semi-dry transfer</td>
		</tr>
		<tr>
			<td>Immobilon polyvinylidene difluoride (PVDF) membrane</td>
			<td>Millipore</td>
			<td>IPVH00010</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-fat powered milk</td>
			<td>Fisher Scientific</td>
			<td>NC9934262</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Aldrich</td>
			<td>274348</td>
			<td></td>
		</tr>
		<tr>
			<td>Ecoscint A</td>
			<td>National Diagnostics</td>
			<td>LS-273</td>
			<td>Scintillation fluid</td>
		</tr>
		<tr>
			<td>Scintillation vials</td>
			<td>Fisher Scientific</td>
			<td>333726</td>
			<td></td>
		</tr>
		<tr>
			<td>LKB Wallac 1209 Rackbeta Liquid Scintillation Counter</td>
			<td>Perkin Elmer</td>
			<td></td>
			<td>Beta-Scintillation counter</td>
		</tr>
		<tr>
			<td>Anti-rhodopsin 1D4 mAb</td>
			<td>National Cell Culture Center</td>
			<td>custom</td>
			<td></td>
		</tr>
		<tr>
			<td>Horseradish peroxidase (HRP)-conjugated anti-mouse IgG</td>
			<td>KPL, Inc.</td>
			<td>474-1806</td>
			<td></td>
		</tr>
		<tr>
			<td>SuperSignal West Pico Chemiluminescent Substrate</td>
			<td>Thermo Scientific</td>
			<td>34080</td>
			<td></td>
		</tr>
		<tr>
			<td>Hyblot CL AR film</td>
			<td>Denville</td>
			<td>E3018</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Table 2. Targeted photocrosslinking materials</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>[header]</td>
		</tr>
		<tr>
			<td>0.25% Trypsin</td>
			<td>Invitrogen</td>
			<td>15050065</td>
			<td></td>
		</tr>
		<tr>
			<td>FLAG-triarylphosphine</td>
			<td>Sigma</td>
			<td>GPHOS1</td>
			<td></td>
		</tr>
		<tr>
			<td>M2 FLAG mAb</td>
			<td>Sigma</td>
			<td>F1804</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-CCR5 2D7 mAb</td>
			<td>BD Biosciences</td>
			<td>555990</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-D-lysine</td>
			<td>Sigma</td>
			<td>P6407</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plate</td>
			<td>Costar</td>
			<td>3601</td>
			<td>Clear bottom, high binding EIA/RIA</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (Calcium, Magnesium)</td>
			<td>Gibco</td>
			<td>14040</td>
			<td></td>
		</tr>
		<tr>
			<td>16% Paraformaldehyde</td>
			<td>EMS</td>
			<td>28908</td>
			<td></td>
		</tr>
		<tr>
			<td>HRP-conjugated</td>
			<td>KPL, Inc.</td>
			<td>474-1516</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-rabbit IgG</td>
			<td>KPL, Inc.</td>
			<td>474-1516</td>
			<td></td>
		</tr>
		<tr>
			<td>Amplex Red</td>
			<td>Invitrogen</td>
			<td>A12222</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrogen peroxide</td>
			<td>DE Healthcare Products</td>
			<td>97-93399</td>
			<td></td>
		</tr>
		<tr>
			<td>CytoFluor II fluorescence multi-well plate reader</td>
			<td>Perseptive Biosystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Table 3. Targeted peptide-epitope tagging and cell surface ELISA materials</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>[header]</td>
		</tr>
		<tr>
			<td>Fluorescein-phosphine</td>
			<td></td>
			<td></td>
			<td>(Huber et al., submitted 2012)</td>
		</tr>
		<tr>
			<td>Nonapeptide (C9 peptide)</td>
			<td>AnaSpec</td>
			<td>62190</td>
			<td>Peptide mimicking the 1D4-epitope NH2-TETSQVAPA-COOH</td>
		</tr>
		<tr>
			<td>1% (w/v) n-dodecyl-&#946;-D-maltoside</td>
			<td>Anatrace</td>
			<td>D310LA</td>
			<td></td>
		</tr>
		<tr>
			<td>1D4-sepharose resin</td>
			<td></td>
			<td></td>
			<td>1D4 mAb immobilized on CNBr-activated sepharose 2B resin</td>
		</tr>
		<tr>
			<td>NuPAGE Novex 4 - 12% SDS gels</td>
			<td>Invitrogen</td>
			<td>NP0322BOX</td>
			<td>SDS-PAGE performed on NuPAGE apparatus</td>
		</tr>
		<tr>
			<td>Confocal Typhoon 9400 fluorescence scanner</td>
			<td>GE Healthcare</td>
			<td>discontinued</td>
			<td>Scanner with 488-nm wavelength laser</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Table 4. Fluorescent labeling materials</td>
		</tr>
	</tbody>
</table>

genetically-encoded molecular probes to study g protein-coupled receptors

複合体シグナル伝達Gタンパク質共役型受容体（GPCR）の構造的および動的研究を容易にするために、新しいアプローチは、受容体機能を撹乱しないで発現される受容体に有益なプローブ又は標識を導入するために必要とされる。我々は、遺伝的にエンコードにアンバーコドン抑圧技術を使用した非天然アミノ酸、異種哺乳動物細胞において発現されたGPCRの様々な標的位置でのp-アジド-L-フェニルアラニン（AZF）。アジド基の汎用性をネイティブな細胞環境や洗剤可溶化条件の下でのGPCRを研究するさまざまなアプリケーションで、ここに示されている。まず、トリチウム標識した小分子リガンドは、遺伝的にコードされたアジドアミノ酸に架橋されるGPCRのリガンド結合ポケット内の残基を同定するための細胞ベースの標的化光架橋技術を実証する。次に、バイオ直交シュタウディンガー·ベルトッツィライゲーションREACによるGPCRのサイト固有の変更を実証ホスフィン誘導体を用いたアジド基を標的たTiON。私たちは、全細胞ベースのELISA法を用いて標的ペプチドエピトープでの培養発現する膜タンパク質のタグ付けおよびその検出のための一般的な戦略を議論する。最後に、我々はAZF-GPCRが選択的蛍光プローブでタグ付けすることができることを示している。それらは原理的に活性なシグナル伝達複合体を調べるためにGPCRを発現したいずれかにおける任意のアミノ酸位置に適用することができるという点で論じ方法は、一般的である。

私たちは、部位特異的抑制技術コドン琥珀を使用して、GPCRに分子プローブを導入する非天然アミノ酸の変異導入方法論を採用。 図1の方式では、方法論の主要なステップとのGPCRに、汎用性のUAA、p-アジド-L-フェニルアラニン（AZF）を組み込む様々なアプリケーションの概要を説明します。哺乳動物細胞におけるAZF-GPCRの発現は、リガンドをターゲットに光架橋を可能にし、...

Watch this Scientific Journal Video about Genetically-encoded Molecular Probes to Study G Protein-coupled Receptors at JoVE.com

Genetically-encoded Molecular Probes to Study G Protein-coupled Receptors

我々のGPCRの様​​々な標的位置に非天然アミノ酸p-アジド-L-フェニルアラニンを遺伝的に符号化し、異なる用途におけるアジド基の汎用性を示している。これらは、GPCRのリガンド結合ポケットに残基を同定するために目標と光架橋技術、およびペプチドエピトープタグまたは蛍光プローブでのGPCRの部位特異的バイオ直交修飾を含む。

遺伝的にコードされた分子プローブは、Gタンパク質共役受容体を研究する

To facilitate structural and dynamic studies of G protein-coupled receptor (GPCR) signaling complexes, new approaches are required to introduce ...

genetically-encoded-molecular-probes-to-study-g-protein-coupled

Research

JoVE Journal

Biology

15.1K ビュー.  The Rockefeller University. 我々のGPCRの様​​々な標的位置に非天然アミノ酸p-アジド-L-フェニルアラニンを遺伝的に符号化し、異なる用途におけるアジド基の汎用性を示している。これらは、GPCRのリガンド結合ポケットに残基を同定するために目標と光架橋技術、およびペプチドエピトープタグまたは蛍光プローブでのGPCRの部位特異的バイオ直交修飾を含む。 

ビデオ: Genetically-encoded Molecular Probes to Study G Protein-coupled Receptors

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

トカゲの運動の速度と方向の視覚感度のテスト

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

生物学

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite morphogenesis at the functional and molecular levels. To aid in these analyses, we have developed a strategy for the isolation of a subclass of PNS neurons called dendritic arborization (da) neurons that have been widely used for studying dendrite morphogenesis1,2. These neurons are very difficult to isolate as a pure population, due in part to their extremely low occurrence and their difficult-to-reach location below the tough chitinous larval cuticle. Our newly developed method overcomes these challenges, and is based on a fast and specific cell enrichment using antibody-coated magnetic beads. For our magnetic bead sorting studies, we have used age-matched third instar larvae expressing a mouse CD8 tagged GFP fusion protein (UAS-mCD8-GFP)3 under the control of either the class IV dendritic arborization (da) neuron-specific pickpocket (ppk)-GAL4 driver4 or the control of the pan-da neuron-specific GAL421-7 driver5. Although this protocol has been optimized for isolating PNS cells which are attached to the inner wall of the larval cuticle, by varying a few parameters, the same protocol could be used to isolate many different cell types attached to the cuticle at larval or pupal stages of development (e.g. epithelia, muscle, oenocytes etc.), or other cell types from larval organs depending upon the GAL4-specific driver expression pattern. The RNA isolated by this method is of high quality and can be readily used for downstream genomic analyses such as microarray gene expression profiling studies. This approach offers a powerful new tool to perform studies on isolated Drosophila dendritic arborization (da) neurons thereby providing novel insights into the molecular mechanisms underlying dendrite morphogenesis.

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

In this video-article we present a method for the isolation and purification of Drosophila peripheral neurons using a fast magnetic bead assisted cell sorting strategy. RNA obtained from the isolated cells can be readily used for downstream applications including microarray analyses.

の単離と精製ショウジョウバエ</em磁気ビーズソート別>末梢ニューロン

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite ...

In vivo Quantification of G Protein Coupled Receptor Interactions using Spectrally Resolved Two-photon Microscopy

The study of protein interactions in living cells is an important area of research because the information accumulated both benefits industrial applications as well as increases basic fundamental biological knowledge. F&ouml;rster (Fluorescence) Resonance Energy Transfer (FRET) between a donor molecule in an electronically excited state and a nearby acceptor molecule has been frequently utilized for studies of protein-protein interactions in living cells. The proteins of interest are tagged with two different types of fluorescent probes and expressed in biological cells. The fluorescent probes are then excited, typically using laser light, and the spectral properties of the fluorescence emission emanating from the fluorescent probes is collected and analyzed. Information regarding the degree of the protein interactions is embedded in the spectral emission data. Typically, the cell must be scanned a number of times in order to accumulate enough spectral information to accurately quantify the extent of the protein interactions for each region of interest within the cell. However, the molecular composition of these regions may change during the course of the acquisition process, limiting the spatial determination of the quantitative values of the apparent FRET efficiencies to an average over entire cells. By means of a spectrally resolved two-photon microscope, we are able to obtain a full set of spectrally resolved images after only one complete excitation scan of the sample of interest. From this pixel-level spectral data, a map of FRET efficiencies throughout the cell is calculated. By applying a simple theory of FRET in oligomeric complexes to the experimentally obtained distribution of FRET efficiencies throughout the cell, a single spectrally resolved scan reveals stoichiometric and structural information about the oligomer complex under study. Here we describe the procedure of preparing biological cells (the yeast Saccharomyces cerevisiae) expressing membrane receptors (sterile 2 &alpha;-factor receptors) tagged with two different types of fluorescent probes. Furthermore, we illustrate critical factors involved in collecting fluorescence data using the spectrally resolved two-photon microscopy imaging system. The use of this protocol may be extended to study any type of protein which can be expressed in a living cell with a fluorescent marker attached to it.

In vivo Quantification of G Protein Coupled Receptor Interactions using Spectrally Resolved Two-photon Microscopy

By employing a spectrally resolved two-photon microscopy imaging system, pixel-level maps of F&ouml;rster Resonance Energy Transfer (FRET) efficiencies are obtained for cells expressing membrane receptors hypothesized to form homo-oligomeric complexes. From the FRET efficiency maps, we are able to estimate stoichiometric information about the oligomer complex under study.

スペクトル的に解決された二光子顕微鏡を用いたGタンパク質共役受容体相互作用の in vivo定量で

The study of protein interactions in living cells is an important area of research because the information accumulated both benefits industrial ...

Monitoring Dynamic Changes In Mitochondrial Calcium Levels During Apoptosis Using A Genetically Encoded Calcium Sensor

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, differentiation, and apoptosis1. During apoptosis, calcium accumulation in mitochondria promotes the release of pro-apoptotic factors from the mitochondria into the cytosol2. It is therefore of interest to directly measure mitochondrial calcium in living cells in situ during apoptosis. High-resolution fluorescent imaging of cells loaded with dual-excitation ratiometric and non-ratiometric synthetic calcium indicator dyes has been proven to be a reliable and versatile tool to study various aspects of intracellular calcium signaling. Measuring cytosolic calcium fluxes using these techniques is relatively straightforward. However, measuring intramitochondrial calcium levels in intact cells using synthetic calcium indicators such as rhod-2 and rhod-FF is more challenging. Synthetic indicators targeted to mitochondria have blunted responses to repetitive increases in mitochondrial calcium, and disrupt mitochondrial morphology3. Additionally, synthetic indicators tend to leak out of mitochondria over several hours which makes them unsuitable for long-term experiments. Thus, genetically encoded calcium indicators based upon green fluorescent protein (GFP)4 or aequorin5 targeted to mitochondria have greatly facilitated measurement of mitochondrial calcium dynamics. Here, we describe a simple method for real-time measurement of mitochondrial calcium fluxes in response to different stimuli. The method is based on fluorescence microscopy of 'ratiometric-pericam' which is selectively targeted to mitochondria. Ratiometric pericam is a calcium indicator based on a fusion of circularly permuted yellow fluorescent protein and calmodulin4. Binding of calcium to ratiometric pericam causes a shift of its excitation peak from 415 nm to 494 nm, while the emission spectrum, which peaks around 515 nm, remains unchanged. Ratiometric pericam binds a single calcium ion with a dissociation constant in vitro of ~1.7 &mu;M4. These properties of ratiometric pericam allow the quantification of rapid and long-term changes in mitochondrial calcium concentration. Furthermore, we describe adaptation of this methodology to a standard wide-field calcium imaging microscope with commonly available filter sets. Using two distinct agonists, the purinergic agonist ATP and apoptosis-inducing drug staurosporine, we demonstrate that this method is appropriate for monitoring changes in mitochondrial calcium concentration with a temporal resolution of seconds to hours. Furthermore, we also demonstrate that ratiometric pericam is also useful for measuring mitochondrial fission/fragmentation during apoptosis. Thus, ratiometric pericam is particularly well suited for continuous long-term measurement of mitochondrial calcium dynamics during apoptosis.

This protocol describes a method for real-time measurement of mitochondrial calcium fluxes by fluorescent imaging. The method takes advantage of a circularly permutated YFP-based dual-excitation ratiometric calcium sensor (ratiometric pericam-mt) selectively expressed in mitochondria.

遺伝的にコードされたカルシウムのセンサを用いたアポトーシス時のミトコンドリア内カルシウム濃度の動的変更の監視

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, ...

Imaging G-protein Coupled Receptor (GPCR)-mediated Signaling Events that Control Chemotaxis of Dictyostelium Discoideum

Many eukaryotic cells can detect gradients of chemical signals in their environments and migrate accordingly 1. This guided cell migration is referred as chemotaxis, which is essential for various cells to carry out their functions such as trafficking of immune cells and patterning of neuronal cells 2, 3. A large family of G-protein coupled receptors (GPCRs) detects variable small peptides, known as chemokines, to direct cell migration in vivo 4. The final goal of chemotaxis research is to understand how a GPCR machinery senses chemokine gradients and controls signaling events leading to chemotaxis. To this end, we use imaging techniques to monitor, in real time, spatiotemporal concentrations of chemoattractants, cell movement in a gradient of chemoattractant, GPCR mediated activation of heterotrimeric G-protein, and intracellular signaling events involved in chemotaxis of eukaryotic cells 5-8. The simple eukaryotic organism, Dictyostelium discoideum, displays chemotaxic behaviors that are similar to those of leukocytes, and D. discoideum is a key model system for studying eukaryotic chemotaxis. As free-living amoebae, D. discoideum cells divide in rich medium. Upon starvation, cells enter a developmental program in which they aggregate through cAMP-mediated chemotaxis to form multicullular structures. Many components involved in chemotaxis to cAMP have been identified in D. discoideum. The binding of cAMP to a GPCR (cAR1) induces dissociation of heterotrimeric G-proteins into G&gamma; and G&beta;&gamma; subunits 7, 9, 10. G&beta;&gamma; subunits activate Ras, which in turn activates PI3K, converting PIP2 into PIP3 on the cell membrane 11-13. PIP3 serve as binding sites for proteins with pleckstrin Homology (PH) domains, thus recruiting these proteins to the membrane 14, 15. Activation of cAR1 receptors also controls the membrane associations of PTEN, which dephosphorylates PIP3 to PIP2 16, 17. The molecular mechanisms are evolutionarily conserved in chemokine GPCR-mediated chemotaxis of human cells such as neutrophils 18. We present following methods for studying chemotaxis of D. discoideum cells. 1. Preparation of chemotactic component cells. 2. Imaging chemotaxis of cells in a cAMP gradient. 3. Monitoring a GPCR induced activation of heterotrimeric G-protein in single live cells. 4. Imaging chemoattractant-triggered dynamic PIP3 responses in single live cells in real time. Our developed imaging methods can be applied to study chemotaxis of human leukocytes.

Imaging G-protein Coupled Receptor GPCR-mediated Signaling Events that Control Chemotaxis of Dictyostelium Discoideum

Here, we describe detailed live cell imaging methods for investigating chemotaxis. We present fluorescence microscopic methods to monitor spatiotemporal dynamics of signaling events in migrating cells. Measurement of signaling events permits us to further understand how a GPCR-signaling network achieves gradient sensing of chemoattractants and controls directional migration of eukaryotic cells.

イメージングG -タンパク質共役受容体（GPCR）を介するシグナル伝達事象その制御走キイロタマホコリカビ</em

Many eukaryotic cells can detect gradients of chemical signals in their environments and migrate accordingly 1.  This guided cell migration is referred as ...

Quantifying Agonist Activity at G Protein-coupled Receptors

When an agonist activates a population of G protein-coupled receptors (GPCRs), it elicits a signaling pathway that culminates in the response of the cell or tissue. This process can be analyzed at the level of a single receptor, a population of receptors, or a downstream response. Here we describe how to analyze the downstream response to obtain an estimate of the agonist affinity constant for the active state of single receptors.
Receptors behave as quantal switches that alternate between active and inactive states (Figure 1). The active state interacts with specific G proteins or other signaling partners. In the absence of ligands, the inactive state predominates. The binding of agonist increases the probability that the receptor will switch into the active state because its affinity constant for the active state (Kb) is much greater than that for the inactive state (Ka). The summation of the random outputs of all of the receptors in the population yields a constant level of receptor activation in time. The reciprocal of the concentration of agonist eliciting half-maximal receptor activation is equivalent to the observed affinity constant (Kobs), and the fraction of agonist-receptor complexes in the active state is defined as efficacy (&#949;) (Figure 2).
Methods for analyzing the downstream responses of GPCRs have been developed that enable the estimation of the Kobs and relative efficacy of an agonist 1,2. In this report, we show how to modify this analysis to estimate the agonist Kb value relative to that of another agonist. For assays that exhibit constitutive activity, we show how to estimate Kb in absolute units of M-1.
Our method of analyzing agonist concentration-response curves 3,4 consists of global nonlinear regression using the operational model 5. We describe a procedure using the software application, Prism (GraphPad Software, Inc., San Diego, CA). The analysis yields an estimate of the product of Kobs and a parameter proportional to efficacy (&tau;). The estimate of &tau;Kobs of one agonist, divided by that of another, is a relative measure of Kb (RAi) 6. For any receptor exhibiting constitutive activity, it is possible to estimate a parameter proportional to the efficacy of the free receptor complex (&tau;sys). In this case, the Kb value of an agonist is equivalent to &tau;Kobs/&tau;sys 3.
Our method is useful for determining the selectivity of an agonist for receptor subtypes and for quantifying agonist-receptor signaling through different G proteins.

A method for estimating the affinity constant of an agonist for the active state (Kb) of a G protein-coupled receptor is described. The analysis provides absolute or relative measures of Kb depending on whether constitutive receptor activation is measurable. Our method applies to various responses downstream from receptor activation.

Gタンパク質共役受容体でアゴニスト活性を定量化

When an agonist activates a population of G protein-coupled receptors (GPCRs), it elicits a signaling pathway that culminates in the response of the cell ...

Characterization of G Protein-coupled Receptors by a Fluorescence-based Calcium Mobilization Assay

For more than 20 years, reverse pharmacology has been the preeminent strategy to discover the activating ligands of orphan G protein-coupled receptors (GPCRs). The onset of a reverse pharmacology assay is the cloning and subsequent transfection of a GPCR of interest in a cellular expression system. The heterologous expressed receptor is then challenged with a compound library of candidate ligands to identify the receptor-activating ligand(s). Receptor activation can be assessed by measuring changes in concentration of second messenger reporter molecules, like calcium or cAMP. The fluorescence-based calcium mobilization assay described here is a frequently used medium-throughput reverse pharmacology assay. The orphan GPCR is transiently expressed in human embryonic kidney 293T (HEK293T) cells and a promiscuous G&#945;16 construct is co-transfected. Following ligand binding, activation of the G&#945;16 subunit induces the release of calcium from the endoplasmic reticulum. Prior to ligand screening, the receptor-expressing cells are loaded with a fluorescent calcium indicator, Fluo-4 acetoxymethyl. The fluorescent signal of Fluo-4 is negligible in cells under resting conditions, but can be amplified more than a 100-fold upon the interaction with calcium ions that are released after receptor activation. The described technique does not require the time-consuming establishment of stably transfected cell lines in which the transfected genetic material is integrated into the host cell genome. Instead, a transient transfection, generating temporary expression of the target gene, is sufficient to perform the screening assay. The setup allows medium-throughput screening of hundreds of compounds. Co-transfection of the promiscuous G&#945;16, which couples to most GPCRs, allows the intracellular signaling pathway to be redirected towards the release of calcium, regardless of the native signaling pathway in endogenous settings. The HEK293T cells are easy to handle and have proven their efficacy throughout the years in receptor deorphanization assays. However, optimization of the assay for specific receptors may remain necessary.

The here described fluorescence-based calcium mobilization assay is a medium-throughput reverse pharmacology screening system for the identification of functionally activating ligand(s) of orphan G protein-coupled receptors (GPCRs).

蛍光ベースのカルシウム動員アッセイによるGタンパク質共役受容体のキャラクタリゼーション

For more than 20 years, reverse pharmacology has been the preeminent strategy to discover the activating ligands of orphan G protein-coupled receptors ...

Visualizing Clathrin-mediated Endocytosis of G Protein-coupled Receptors at Single-event Resolution via TIRF Microscopy

Many important signaling receptors are internalized through the well-studied process of clathrin-mediated endocytosis (CME). Traditional cell biological assays, measuring global changes in endocytosis, have identified over 30 known components participating in CME, and biochemical studies have generated an interaction map of many of these components. It is becoming increasingly clear, however, that CME is a highly dynamic process whose regulation is complex and delicate. In this manuscript, we describe the use of Total Internal Reflection Fluorescence (TIRF) microscopy to directly visualize the dynamics of components of the clathrin-mediated endocytic machinery, in real time in living cells, at the level of individual events that mediate this process. This approach is essential to elucidate the subtle changes that can alter endocytosis without globally blocking it, as is seen with physiological regulation. We will focus on using this technique to analyze an area of emerging interest, the role of cargo composition in modulating the dynamics of distinct clathrin-coated pits (CCPs). This protocol is compatible with a variety of widely available fluorescence probes, and may be applied to visualizing the dynamics of many cargo molecules that are internalized from the cell surface.

Clathrin-mediated endocytosis, a rapid and highly dynamic process internalizes many proteins, including signaling receptors. The protocol described here directly visualizes the kinetics of individual endocytic events. This is essential for understanding how core members of the endocytic machinery coordinate with each other, and how protein cargo influence this process.

TIRF顕微鏡を経由してシングルイベント解像度のGタンパク質共役型受容体のクラスリン依存性エンドサイトーシスの可視化

Many important signaling receptors are internalized through the well-studied process of clathrin-mediated endocytosis (CME). Traditional cell biological ...

Hydroponics: A Versatile System to Study Nutrient Allocation and Plant Responses to Nutrient Availability and Exposure to Toxic Elements

Hydroponic systems have been utilized as one of the standard methods for plant biology research and are also used in commercial production for several crops, including lettuce and tomato. Within the plant research community, numerous hydroponic systems have been designed to study plant responses to biotic and abiotic stresses. Here we present a hydroponic protocol that can be easily implemented in laboratories interested in pursuing studies on plant mineral nutrition.
This protocol describes the hydroponic system set up in detail and the preparation of plant material for successful experiments. Most of the materials described in this protocol can be found outside scientific supply companies, making the set up for hydroponic experiments less expensive and convenient.
The use of a hydroponic growth system is most advantageous in situations where the nutrient media need to be well controlled and when intact roots need to be harvested for downstream applications. We also demonstrate how nutrient concentrations can be modified to induce plant responses to both essential nutrients and toxic non-essential elements.

Here, we present an easy-to-follow protocol to establish a successful hydroponic system for plant nutrition studies. This protocol has been extensively tested in Arabidopsis and can easily be adapted to other plant species to study specific nutritional requirements or the effect of non-essential elements on plant growth and development.

水耕：栄養アベイラビリティと毒性元素への曝露に栄養の割り当てと植物の応答を研究するための汎用性の高いシステム

Hydroponic systems have been utilized as one of the standard methods for plant biology research and are also used in commercial production for several ...

Imaging G Protein-coupled Receptor-mediated Chemotaxis and its Signaling Events in Neutrophil-like HL60 Cells

Eukaryotic cells sense and move towards a chemoattractant gradient, a cellular process referred as chemotaxis. Chemotaxis plays critical roles in many physiological processes, such as embryogenesis, neuron patterning, metastasis of cancer cells, recruitment of neutrophils to sites of inflammation, and the development of the model organism Dictyostelium discoideum. Eukaryotic cells sense chemo-attractants using G protein-coupled receptors. Visual chemotaxis assays are essential for a better understanding of how eukaryotic cells control chemoattractant-mediated directional cell migration. Here, we describe detailed methods for: 1) real-time, high-resolution monitoring of multiple chemotaxis assays, and 2) simultaneously visualizing the chemoattractant gradient and the spatiotemporal dynamics of signaling events in neutrophil-like HL60 cells.

Visual chemotaxis assays are essential for a better understanding of how eukaryotic cells control chemoattractant-mediated directional cell migration. Here, we describe detailed methods for: 1) real-time, high-resolution monitoring of multiple chemotaxis assays, and 2) simultaneously visualizing the chemoattractant gradient and the spatiotemporal dynamics of signaling events in neutrophil-like HL60 cells.

イメージングGタンパク質共役型受容体媒介走化性及び好中球様HL60細胞におけるそのシグナル伝達のイベント

Eukaryotic cells sense and move towards a chemoattractant gradient, a cellular process referred as chemotaxis. Chemotaxis plays critical roles in many ...