Bradykinin sequesters B2 bradykinin receptors and the receptor-coupled G alpha subunits G alpha(q) and G alpha(i) in caveolae in DDT1 MF-2 smooth muscle cells
Bradykinin sequesters B2 bradykinin receptors and the receptor-coupled G alpha subunits G alpha(q) and G alpha(i) in caveolae in DDT1 MF-2 smooth muscle cells
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DOI：
10.1074/jbc.272.28.17858
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发表时间：
1997-07-11
影响因子：
4.8
通讯作者：
LeebLundberg, LMF
中科院分区：
文献类型：
--
作者：
deWeerd, WFC;LeebLundberg, LMF
在本报告中，我们表明，当与DDT1 MF-2平滑肌上的B2 BK受体结合时，血管活性肽激动剂（BK）。 cells promotes the recruitment and sequestration of the occupied receptors and the receptor-coupled G-protein alpha subunits G alpha(q) and G alpha(i) in caveolae, Association of ligand receptor complexes and G alpha subunits with caveolae was indicated by their co-enrichment on density gradients with caveolin, a marker protein for caveolae.可以通过免疫印迹监测可爱素和Gα亚基，而受体则被监测为通过具有激动剂BK的标记受体或拮抗剂NPC17731形成的配体受体复合物，或者在细胞扰动之前的拮抗剂NPC17731形成，并通过radioligandies检测到caveolae富集，并通过brotunobodies检测。 Galpha亚基与小窝蛋白的直接相互作用也通过其共免疫沉淀表示，免疫电子显微镜表明，富集的小窝蛋白，GA亚基和BK受体复合物存在于0.1-0.2 mu m的结构中。在Caveolae中检测到4度C，BH和NPC17731受体复合物，并且两个复合物在细胞干扰和小窝富集之前对酸洗涤敏感。温度升高到37度C的升高增加了小窝中的BK受体复合物的量，在10分钟时最大反应（连续标记或20分钟）（单发标记），并且这些络合物变得耐酸，这些条件也增加了GALPHA（Q）和G alpha（i）的量，在5-10分钟内增加了GALPHA（Q）（Q）的量。受体复合物在此温度下保持酸敏感并解离，并且拮抗剂并没有增加小窝中的获得亚基的量，这些结果表明，一些通过G蛋白偶联受体起作用的激动剂会促进其受体和受体偶联的关联，并与Caveolae相关。
In this report, we show that the vasoactive peptide agonist bradykinin (BK) when bound to B2 BK receptors on DDT1 MF-2 smooth muscle. cells promotes the recruitment and sequestration of the occupied receptors and the receptor-coupled G-protein alpha subunits G alpha(q) and G alpha(i) in caveolae, Association of ligand receptor complexes and G alpha subunits with caveolae was indicated by their co-enrichment on density gradients with caveolin, a marker protein for caveolae. Caveolin and G alpha subunits were monitored by immunoblotting, whereas receptors were monitored as ligand receptor complexes formed by labeling receptors with the agonist BK or the antagonist NPC17731 prior to cell disruption and caveolae enrichment, These complexes were detected with radioligand and by immunoblotting with BR antibodies. A direct interaction of G alpha subunits with caveolin was also indicated by their co-immunoprecipitation, Immunoelectron microscopy revealed that the enriched caveolin, Ga subunits, and BK receptor complexes were present in structures of 0.1-0.2 mu m. At 4 degrees C, BH and NPC17731 receptor complexes were detected in caveolae, and both complexes were sensitive to acid washing prior to cell disruption and caveolae enrichment. Elevation of the temperature to 37 degrees C increased the amount of BK receptor complexes in caveolae with a maximal response at 10 min (continuous labeling or 20 min (single-round labelling), and the complexes became acid-resistant, These conditions also increased the amount of G alpha(q), and G alpha(i) in caveolae with a maximal response at 5-10 min, In contrast, the NPC17731 receptor complexes remained acid-sensitive and dissociated at this temperature, and antagonists did not increase the amount of Get subunits in caveolae, These results show that some agonists that act through G-protein-coupled receptors promote the association of their receptors and receptor-coupled Get subunits with caveolae.