Lamb Maria E, Zhang Chongwu, Shea Thomas, Kyle Donald J, Leeb-Lundberg L M Fredrik
Department of Biochemistry, The University of Texas Health Science Center, San Antonio, Texas 78229-3900, USA.
Biochemistry. 2002 Dec 3;41(48):14340-7. doi: 10.1021/bi020231d.
To address the targeting of G protein-coupled receptors to caveolae-related lipid rafts (CLR), we studied the human B2 (B2R) and B1 (B1R) bradykinin receptor subtypes in HEK293 cells. CLR were enriched on the basis of their unique buoyant density and composition of cholesterol, caveolin-1, and flotillin-1 but not clathrin. CLR contained B2R and B1R as determined by both receptor immunoblotting and the increase in specific activity of receptor agonist binding to cells at both 4 and 37 degrees C when binding was followed by CLR enrichment. B2R was highly enriched in this fraction, whereas B1R was not enriched. Furthermore, acid washing of cells prior to cell disruption minimally affected the CLR-associated B2R agonist binding, whereas it dissociated a major portion of the CLR-associated B1R agonist binding. In addition, when agonist binding at 4 degrees C was followed by an increase in the temperature to 37 degrees C, B2R agonist binding in CLR transiently increased, and this increase was dependent on the C-terminal domain. On the other hand, B1R agonist binding remained unchanged and was independent of the C-terminal domain. Our results show that B2R is constitutively targeted to CLR in HEK293 cells and appears to shuttle the agonist through these domains, whereas B1R may be there by default.
为了研究G蛋白偶联受体靶向小窝相关脂筏（CLR）的情况，我们在HEK293细胞中研究了人B2（B2R）和B1（B1R）缓激肽受体亚型。基于其独特的浮力密度以及胆固醇、小窝蛋白-1和flotillin-1的组成（而非网格蛋白）对CLR进行了富集。通过受体免疫印迹以及在4℃和37℃下当结合后进行CLR富集时受体激动剂与细胞结合的比活性增加来确定，CLR含有B2R和B1R。B2R在该组分中高度富集，而B1R未富集。此外，在细胞破碎前对细胞进行酸洗对与CLR相关的B2R激动剂结合影响最小，而它使与CLR相关的大部分B1R激动剂结合解离。另外，当在4℃下激动剂结合后将温度升至37℃时，CLR中B2R激动剂结合短暂增加，且这种增加依赖于C末端结构域。另一方面，B1R激动剂结合保持不变且不依赖于C末端结构域。我们的结果表明，在HEK293细胞中B2R组成性地靶向CLR，并且似乎通过这些结构域转运激动剂，而B1R可能是默认存在于那里。
