Böhm S K, Khitin L M, Smeekens S P, Grady E F, Payan D G, Bunnett N W
Department of Surgery, University of California, San Francisco, California 94143, USA.
J Biol Chem. 1997 Jan 24;272(4):2363-72. doi: 10.1074/jbc.272.4.2363.
Although agonist-induced endocytosis of G-protein-coupled receptors is critical for receptor desensitization and resensitization, receptor motifs that interact with the endocytic apparatus have not been adequately characterized. We examined the effects of mutating the rat neurokinin-1 receptor on endocytosis using 125I-substance P, fluorescent substance P, and receptor antibodies. Substance P induced rapid internalization of wild-type receptors that were targeted to perinuclear endosomes. Truncation of the C-tail at residues 324, 342, and 354 reduced internalization up to 60% and caused retention of receptors at the cell surface and in superficial endosomes. Mutation of Tyr-341 and Tyr-349 in potential tyrosine-containing endocytic motifs of the C-tail also impaired internalization. A Y305A mutant within the putative NPX2-3Y endocytic motif of the seventh transmembrane domain showed impaired signaling and was minimally expressed at the plasma membrane but was found in cytoplasmic vesicles. In contrast, a Y305F mutant signaled normally and was normally expressed at the plasma membrane but showed impaired internalization. Thus, endocytosis of the neurokinin 1 receptor relies on several tyrosine-containing sequences in the C-tail and seventh transmembrane domain.
尽管激动剂诱导的G蛋白偶联受体内吞作用对于受体脱敏和再敏化至关重要，但与内吞装置相互作用的受体基序尚未得到充分表征。我们使用125I-物质P、荧光物质P和受体抗体研究了大鼠神经激肽-1受体突变对内吞作用的影响。物质P诱导野生型受体快速内化，这些受体靶向核周内体。在第324、342和354位残基处截断C末端可使内化作用降低多达60%，并导致受体滞留在细胞表面和浅表内体中。C末端潜在含酪氨酸内吞基序中的Tyr-341和Tyr-349突变也损害内化作用。第七跨膜结构域假定的NPX2-3Y内吞基序内的Y305A突变体显示信号传导受损，在质膜上的表达极少，但存在于细胞质囊泡中。相比之下，Y305F突变体信号传导正常，在质膜上正常表达，但内化作用受损。因此，神经激肽1受体的内吞作用依赖于C末端和第七跨膜结构域中的几个含酪氨酸序列。
