Kohout T A, Lin F S, Perry S J, Conner D A, Lefkowitz R J
Howard Hughes Medical Institute, Department of Medicine, Duke University Medical Center, Durham, NC 27710, USA.
Proc Natl Acad Sci U S A. 2001 Feb 13;98(4):1601-6. doi: 10.1073/pnas.98.4.1601. Epub 2001 Feb 6.
The two widely coexpressed isoforms of beta-arrestin (termed beta arrestin 1 and 2) are highly similar in amino acid sequence. The beta-arrestins bind phosphorylated heptahelical receptors to desensitize and target them to clathrin-coated pits for endocytosis. To better define differences in the roles of beta-arrestin 1 and 2, we prepared mouse embryonic fibroblasts from knockout mice that lack one of the beta-arrestins (beta arr1-KO and beta arr2-KO) or both (beta arr1/2-KO), as well as their wild-type (WT) littermate controls. These cells were analyzed for their ability to support desensitization and sequestration of the beta(2)-adrenergic receptor (beta(2)-AR) and the angiotensin II type 1A receptor (AT(1A)-R). Both beta arr1-KO and beta arr2-KO cells showed similar impairment in agonist-stimulated beta(2)-AR and AT(1A)-R desensitization, when compared with their WT control cells, and the beta arr1/2-KO cells were even further impaired. Sequestration of the beta(2)-AR in the beta arr2-KO cells was compromised significantly (87% reduction), whereas in the beta arr1-KO cells it was not. Agonist-stimulated internalization of the AT(1A)-R was only slightly reduced in the beta arr1-KO but was unaffected in the beta arr2-KO cells. In the beta arr1/2-KO cells, the sequestration of both receptors was dramatically reduced. Comparison of the ability of the two beta-arrestins to sequester the beta(2)-AR revealed beta-arrestin 2 to be 100-fold more potent than beta-arrestin 1. Down-regulation of the beta(2)-AR was also prevented in the beta arr1/2-KO cells, whereas no change was observed in the single knockout cells. These findings suggest that sequestration of various heptahelical receptors is regulated differently by the two beta-arrestins, whereas both isoforms are capable of supporting receptor desensitization and down-regulation.
β-抑制蛋白的两种广泛共表达的亚型（称为β抑制蛋白1和2）在氨基酸序列上高度相似。β-抑制蛋白与磷酸化的七螺旋受体结合，使其脱敏并将其靶向网格蛋白包被的小窝进行内吞作用。为了更好地界定β抑制蛋白1和2作用的差异，我们从缺乏其中一种β-抑制蛋白（βarr1基因敲除小鼠和βarr2基因敲除小鼠）或两种都缺乏（βarr1/2基因敲除小鼠）的基因敲除小鼠以及它们的野生型（WT）同窝对照小鼠中制备了小鼠胚胎成纤维细胞。分析了这些细胞支持β2-肾上腺素能受体（β2-AR）和血管紧张素II 1A型受体（AT1A-R）脱敏和隔离的能力。与野生型对照细胞相比，βarr1基因敲除细胞和βarr2基因敲除细胞在激动剂刺激的β2-AR和AT1A-R脱敏方面均表现出相似的损伤，而βarr1/2基因敲除细胞的损伤更严重。β2-AR在βarr2基因敲除细胞中的隔离显著受损（减少87%），而在βarr1基因敲除细胞中则没有。激动剂刺激的AT1A-R内化在βarr1基因敲除细胞中仅略有减少，但在βarr2基因敲除细胞中未受影响。在βarr1/2基因敲除细胞中，两种受体的隔离均显著减少。比较两种β-抑制蛋白隔离β2-AR的能力发现，β-抑制蛋白2的效力比β-抑制蛋白1高100倍。βarr1/2基因敲除细胞中β2-AR的下调也受到抑制，而在单基因敲除细胞中未观察到变化。这些发现表明，两种β-抑制蛋白对各种七螺旋受体隔离的调节方式不同，而两种亚型都能够支持受体脱敏和下调。
