Gage Robert M, Matveeva Elena A, Whiteheart Sidney W, von Zastrow Mark
Department of Pharmaceutical Chemistry, University of California at San Francisco, San Francisco California 94143, USA.
J Biol Chem. 2005 Feb 4;280(5):3305-13. doi: 10.1074/jbc.M406934200. Epub 2004 Nov 17.
Molecular sorting of G protein-coupled receptors (GPCRs) between divergent recycling and lysosomal pathways determines the functional consequences of agonist-induced endocytosis. The carboxyl-terminal cytoplasmic domain of the beta2 adrenergic receptor (beta2AR) mediates both PDZ binding to Na+/H+ exchanger regulatory factor/ezrin/radixin/moesin-binding phosphoprotein of 50 kDa (NHERF/EBP50) family proteins and non-PDZ binding to the N-ethylmaleimide-sensitive factor (NSF). We have investigated whether PDZ interaction(s) are actually sufficient to promote rapid recycling of endocytosed receptors and, if so, whether PDZ-mediated sorting is restricted to the beta2AR tail or to sequences that bind NHERF/EBP50. The trafficking effects of short (10 residue) sequences differing in PDZ and NSF binding properties were examined using chimeric mutant receptors. The recycling activity of the beta2AR-derived tail sequence was not blocked by a point mutation that selectively disrupts binding to NSF, and naturally occurring PDZ ligand sequences were identified that do not bind detectably to NSF yet function as strong recycling signals. The carboxyl-terminal cytoplasmic domain of the beta1-adrenergic receptor, which does not bind either to NSF or NHERF/EBP50 and interacts selectively with a distinct group of PDZ proteins, promoted rapid recycling of chimeric mutant receptors with efficiency similarly high as that of the beta2AR tail. These results indicate that PDZ domain-mediated protein interactions are sufficient to promote rapid recycling of GPCRs, independent of binding to NSF. They also suggest that PDZ-directed recycling is a rather general mechanism of GPCR regulation, which is not restricted to a single GPCR, and may involve additional PDZ domain-containing protein(s) besides NHERF/EBP50.
G蛋白偶联受体（GPCRs）在不同的再循环和溶酶体途径之间的分子分选决定了激动剂诱导的内吞作用的功能后果。β2肾上腺素能受体（β2AR）的羧基末端胞质结构域介导与Na+/H+交换调节因子/埃兹蛋白/根蛋白/膜突蛋白结合磷蛋白50 kDa（NHERF/EBP50）家族蛋白的PDZ结合以及与N-乙基马来酰亚胺敏感因子（NSF）的非PDZ结合。我们研究了PDZ相互作用是否实际上足以促进内吞受体的快速再循环，如果是这样，PDZ介导的分选是否仅限于β2AR尾部或与NHERF/EBP50结合的序列。使用嵌合突变受体检查了在PDZ和NSF结合特性上不同的短（10个残基）序列的运输效应。β2AR衍生的尾部序列的再循环活性没有被选择性破坏与NSF结合的点突变所阻断，并且鉴定出天然存在的PDZ配体序列，其与NSF没有可检测的结合，但作为强再循环信号起作用。β1肾上腺素能受体的羧基末端胞质结构域既不与NSF也不与NHERF/EBP50结合，而是与一组不同的PDZ蛋白选择性相互作用，它促进嵌合突变受体的快速再循环，效率与β2AR尾部相似。这些结果表明，PDZ结构域介导的蛋白质相互作用足以促进GPCR的快速再循环，而与与NSF的结合无关。它们还表明，PDZ指导的再循环是GPCR调节的一种相当普遍的机制，不限于单一的GPCR，并且除了NHERF/EBP50之外可能还涉及其他含PDZ结构域的蛋白质。
