Nooh Mohammed M, Mancarella Salvatore, Bahouth Suleiman W
Department of Pharmacology, The University of Tennessee Health Sciences Center, 71 S. Manassas, Memphis, TN 38103, USA; Department of Biochemistry, Faculty of Pharmacy Cairo University, Kasr El-Aini St., Cairo 11562, Egypt.
Department of Physiology, The University of Tennessee Health Sciences Center, 71 S. Manassas, Memphis, TN 38103, USA.
Biochem Pharmacol. 2016 Nov 15;120:22-32. doi: 10.1016/j.bcp.2016.09.011. Epub 2016 Sep 16.
β-Adrenergic receptor (β-AR) agonists and antagonists are widely used in the treatment of major cardiovascular diseases such as heart failure and hypertension. The β-AR like other G protein-coupled receptors (GPCRs) are endocytosed in response to intense agonist activation. Recycling of the agonist-internalized β-AR is dependent on its carboxy-terminal type-1 PSD-95/DLG/ZO1 (PDZ) and on phospho-serine in the third intracellular loop of the β-AR. Progressive elongation of the β-AR at its C-tail inactivated the PDZ-biding domain and inhibited the recycling of the β-AR. However, fusing a twenty amino acid peptide derived from the multiple cloning region of the mammalian expression vector pCDNA3 to the C-tail of the β-AR (β-AR[+20]) produced a chimeric β-AR that recycled rapidly and efficiently. The β-AR[+20] recycled in a type-1 PDZ and phospho-Ser-independent manner, indicating that this peptide provided a general GPCR recycling signal. Fusing the enhanced yellow fluorescent protein (EYFP) down-stream of β-AR[+20] generated a β-AR-EYFP chimera that was expressed on the membrane and recycled efficiently after agonist-induced internalization. This construct trafficked in a PDZ-SNX27/retromer-independent manner. We also fused EYFP to the N-terminus of the β-AR to created EYFP-WT β-AR. This construct recycled in PDZ and SNX27/retromer dependent manner. These β-AR-EYFP constructs would be useful for high throughput screening (HTS) programs to identify new entities that would interfere with the recycling of agonist internalized GPCR that traffic in PDZ-dependent vs. PDZ-independent roadmaps.
β-肾上腺素能受体（β-AR）激动剂和拮抗剂广泛应用于治疗心力衰竭和高血压等主要心血管疾病。与其他G蛋白偶联受体（GPCR）一样，β-AR在激动剂强烈激活时会发生内吞作用。激动剂内化的β-AR的再循环依赖于其羧基末端的1型PSD-95/DLG/ZO1（PDZ）以及β-AR第三细胞内环中的磷酸丝氨酸。β-AR的C末端逐渐延长会使PDZ结合结构域失活，并抑制β-AR的再循环。然而，将源自哺乳动物表达载体pCDNA3多克隆区域的20个氨基酸肽融合到β-AR的C末端（β-AR[+20]），产生了一种嵌合β-AR，其能快速且有效地再循环。β-AR[+20]以一种不依赖1型PDZ和磷酸丝氨酸的方式再循环，表明该肽提供了一个通用的GPCR再循环信号。在β-AR[+20]的下游融合增强型黄色荧光蛋白（EYFP），产生了一种β-AR-EYFP嵌合体，其在膜上表达，并在激动剂诱导的内化后有效再循环。该构建体以一种不依赖PDZ-SNX27/retromer的方式运输。我们还将EYFP融合到β-AR的N末端，以创建EYFP-WT β-AR。该构建体以依赖PDZ和SNX27/retromer的方式再循环。这些β-AR-EYFP构建体将有助于高通量筛选（HTS）项目，以识别那些会干扰以依赖PDZ与不依赖PDZ路线运输的激动剂内化GPCR再循环的新物质。
