Botto J M, Chabry J, Nouel D, Paquet M, Séguéla P, Vincent J P, Beaudet A, Mazella J
Institut de Pharmacologie Moléculaire et Cellulaire, CNRS UPR 411, Valbonne, France.
Brain Res Mol Brain Res. 1997 Jun;46(1-2):311-7. doi: 10.1016/s0169-328x(97)00006-5.
In order to identify charged amino-acid residues of the cloned rat brain neurotensin (NT) receptor (NTR) that are critical for NT binding, we performed site-directed mutagenesis on the cDNA encoding this protein, followed by transient expression into mammalian COS-7 cells and in Xenopus laevis oocytes. Point substitutions of charged residues in the N-terminal part and in the 2nd and 3rd extracellular loop of the receptor either did not affect (125)I-Tyr3-NT binding or resulted in a decrease in binding affinity by a factor of 2-3. Mutations of amino acids Asp113 in the second transmembrane domain (TM) and of Arg149 or Asp150 in TM III yielded receptors that bound NT as efficiently as the native receptor. By contrast, replacement of the Asp139 residue in the 1st extracellular loop, or of Arg143 or Arg327-Arg328 residues at the top of TM III and in TM VI, respectively, completely abolished ligand binding. Confocal and EM immunocytochemical studies of the expression of these affected receptors, tagged with the C-terminal sequence of the vesicular stomatitis virus glycoprotein (VSV-G), indicated that this loss of binding was not due to altered receptor expression or to their improper insertion into the plasma membrane. When these mutated forms of neurotensin receptor were expressed into Xenopus oocytes, Asp139-Gly- and Arg143-Gly-modified receptors remained functional in spite of a lowered response to NT whereas the Arg327-Arg328 mutant form was totally insensitive to NT at concentrations up to 10 microM. In the case of the Arg327-Arg328 mutation, the observed insensibility to NT could be the result of a drastic conformational alteration of this mutant protein. By contrast, it would appear that Asp139 and Arg143 residues located in the first extracellular loop of the receptor may be directly involved in the interaction of the receptor with neurotensin.
为了确定克隆的大鼠脑内神经降压素（NT）受体（NTR）中对NT结合至关重要的带电荷氨基酸残基，我们对编码该蛋白的cDNA进行了定点诱变，随后将其瞬时表达于哺乳动物COS-7细胞和非洲爪蟾卵母细胞中。受体N端部分以及第二和第三细胞外环中带电荷残基的点突变，要么不影响（125）I-Tyr3-NT结合，要么导致结合亲和力下降2至3倍。第二跨膜结构域（TM）中的天冬氨酸113以及TM III中的精氨酸149或天冬氨酸150发生突变后产生的受体，与天然受体一样有效地结合NT。相比之下，第一细胞外环中天冬氨酸139残基的替换，或分别在TM III顶部和TM VI中的精氨酸143或精氨酸327 - 精氨酸328残基的替换，完全消除了配体结合。对这些受影响的受体（用水泡性口炎病毒糖蛋白（VSV-G）的C端序列标记）的表达进行共聚焦和电子显微镜免疫细胞化学研究表明，这种结合丧失并非由于受体表达改变或其插入质膜不当所致。当这些神经降压素受体的突变形式在非洲爪蟾卵母细胞中表达时，尽管对NT的反应降低，但天冬氨酸139 - 甘氨酸和精氨酸143 - 甘氨酸修饰的受体仍保持功能，而精氨酸327 - 精氨酸328突变形式在高达10 microM的浓度下对NT完全不敏感。就精氨酸327 - 精氨酸328突变而言，观察到的对NT不敏感可能是该突变蛋白剧烈构象改变的结果。相比之下，似乎位于受体第一细胞外环中的天冬氨酸139和精氨酸143残基可能直接参与受体与神经降压素的相互作用。
