Enquist Johan, Sandén Caroline, Skröder Carl, Mathis Sandra A, Leeb-Lundberg L M Fredrik
Department of Experimental Medical Science, Lund University, BMC, A12, 22184, Lund, Sweden.
Neurochem Res. 2014 Jun;39(6):1037-47. doi: 10.1007/s11064-013-1126-9. Epub 2013 Aug 10.
Kinins are potent pro-inflammatory peptides that act through two G protein-coupled receptor subtypes, B1 (B1R) and B2 (B2R). Kinin-stimulated B2R signaling is often transient, whereas B1R signaling is sustained. This was confirmed by monitoring agonist-stimulated intracellular Ca(2+) mobilization in A10 smooth muscle cells expressing human wild-type B2R and B1R. We further studied the role of receptor membrane trafficking in receptor-mediated phosphoinositide (PI) hydrolysis in model HEK293 cell lines stably expressing the receptors. Treatment of cells with brefeldin A, to inhibit maturation of de novo synthesized receptors, or hypertonic sucrose, to inhibit receptor endocytosis, showed that the basal cell surface receptor turnover was considerably faster for B1R than for B2R. Inhibition of endocytosis, which stabilized B1R on the cell surface, inhibited B1R signaling, whereas B2R signaling was not perturbed. Signaling by a B1R construct in which the entire C-terminal domain was deleted remained sensitive to inhibition of receptor endocytosis, whereas signaling by a B1R construct in which this domain was substituted with the corresponding domain in B2R was not sensitive. B2R and B1R co-expression, which also appeared to stabilize B1R on the cell surface, presumably by receptor hetero-dimerization, also inhibited B1R signaling, whereas B2R signaling was slightly enhanced. Furthermore, the B2R-specific agonist bradykinin (BK) directed both receptors through a common endocytic pathway, whereas the B1R-specific agonist Lys-desArg(9)-BK was unable to do so. These results suggest that B1R-mediated PI hydrolysis depends on a step in receptor endocytosis, whereas B2R-mediated PI hydrolysis does not. We propose that B1R uses at least part of the endocytic machinery to sustain agonist-promoted signaling.
激肽是一类强效促炎肽，通过两种G蛋白偶联受体亚型B1（B1R）和B2（B2R）发挥作用。激肽刺激的B2R信号传导通常是短暂的，而B1R信号传导是持续的。这一点通过监测激动剂刺激的表达人野生型B2R和B1R的A10平滑肌细胞内Ca(2+)动员得到了证实。我们进一步研究了受体膜转运在稳定表达这些受体的模型HEK293细胞系中受体介导的磷酸肌醇（PI）水解中的作用。用布雷菲德菌素A处理细胞以抑制新生受体的成熟，或用高渗蔗糖处理以抑制受体内吞作用，结果显示B1R的基础细胞表面受体周转率比B2R快得多。抑制内吞作用可使B1R稳定在细胞表面，但会抑制B1R信号传导，而B2R信号传导不受影响。删除整个C末端结构域的B1R构建体的信号传导仍对受体内吞作用的抑制敏感，而用B2R中相应结构域替代该结构域的B1R构建体的信号传导则不敏感。B2R和B1R共表达似乎也通过受体异二聚化使B1R稳定在细胞表面，这同样抑制了B1R信号传导，而B2R信号传导略有增强。此外，B2R特异性激动剂缓激肽（BK）通过共同的内吞途径引导两种受体，而B1R特异性激动剂Lys-desArg(9)-BK则无法做到这一点。这些结果表明，B1R介导的PI水解依赖于受体内吞作用的一个步骤，而B2R介导的PI水解则不依赖。我们提出B1R利用至少部分内吞机制来维持激动剂促进的信号传导。
