フォトアフィニティーＧＴＰ類似体を用いたＧＴＰ結合タンパク質の解析

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タイトル別名
  • Analysis of GTP-binding potein function with a photoaffinity GTP analog.
  • フォト アフィニティー GTP ルイジタイ オ モチイタ GTP ケツゴウ タ

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抄録

The mechanism whereby hormones or neurotransmitters activate G proteins and their intracellular effectors can be studied in reconstituted systems using purified components. However, the regulation of receptor-G protein signaling appears to be substantially more complex in the cell and several additional components participate in this event. To study the relationship among G proteins receptors, and effectors in complex systems, such as membranes of permeable cells, it is necessary to employ methods that selectively allow the examination of G protein activation. One such method is photoaffinity labeling using a hydrolysis-resistant, photoaffinity GTP analog, P3 (4-azidoanilido)-P1-guanosine 5'-triphosphate (AAGTP). Here we describe the synthesis and purification of [32P] AAGTP as well as a procedure suitable studying the G protein function in membrane preparations. Photoaffinity labeling in rat cerebral cortex membranes showed that at least four G proteins (GsH, GsL, Gi, and Go) were labeled by [32P] AAGTP. [32P] AAGTP labeling on Gs and Gi was altered in concert with the activation states of those G proteins. An agonistspecific increase in [32P] AAGTP labeling of the G protein α-subunit in a membrane preparation has also been demonstrated. Thus, the photoaffinity labeling method with [32P] AAGTP makes it possible to investigate the behavior of individual G proteins in complex systems such as membrane preparations.

収録刊行物

  • 日本薬理学雑誌

    日本薬理学雑誌 105 (6), 431-436, 1995

    公益社団法人 日本薬理学会

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