Chanda P K, Minchin M C, Davis A R, Greenberg L, Reilly Y, McGregor W H, Bhat R, Lubeck M D, Mizutani S, Hung P P
Department of Biotechnology and Microbiology, Wyeth-Ayerst Research, Philadelphia, Pennsylvania 19101.
Mol Pharmacol. 1993 Apr;43(4):516-20.
The functional significance of the conserved amino acids within transmembrane regions II and VII of the human 5-hydroxytryptamine (5-HT)1A receptor was analyzed by oligonucleotide-directed mutagenesis followed by transient expression of the mutated receptor genes in COS-1 cells. The substitution of a conserved asparagine at position 396 (transmembrane region VII) with either alanine, phenylalanine, or valine resulted in a receptor that did not bind the 5-HT1A agonist 8-hydroxy-2-(di-n-[3H]propylamino)tetralin. In contrast, replacement of Asn396 with glutamine did not affect agonist binding. In addition, serine residues at positions 391 and 393 (transmembrane domain VII) were changed to alanine. Changing the less conserved Ser391 to alanine had no effect on ligand binding. However, replacement of the conserved Ser393 with alanine reduced ligand binding by 86%. Replacement of a conserved aspartate at position 82 (transmembrane region II) with alanine also produced a receptor without detectable agonist binding. Protein immunoblotting detected receptor protein of approximately 51 kDa in both wild-type and mutant receptor-expressing cells, indicating that these mutations probably did not affect expression or processing of the protein. Importantly, the sequence of the human 5-HT1A receptor described in this paper differs from the published sequence [Nature (Lond.) 329:75-79 (1987)] in transmembrane region IV. The present sequence encodes a protein of 422 amino acids, instead of the 421-amino acid protein that has been described previously [Nature (Lond.) 329:75-79 (1987)], and has a change in the sequence in transmembrane region IV from ... RPRAL ... to ... RRAAA ..., which corresponds to the published sequence [J. Biol. Chem. 265:5825-5832 (1990)] of the rat 5-HT1A receptor. Moreover, conversion of the transmembrane region IV sequence of the present clone to that of the published sequence by site-directed mutagenesis abolished ligand binding to the receptor.
通过寡核苷酸定向诱变，随后在COS-1细胞中瞬时表达突变的受体基因，分析了人5-羟色胺（5-HT）1A受体跨膜区II和VII内保守氨基酸的功能意义。将位于396位（跨膜区VII）的保守天冬酰胺分别替换为丙氨酸、苯丙氨酸或缬氨酸，导致受体无法结合5-HT1A激动剂8-羟基-2-（二-n-[3H]丙基氨基）四氢萘。相反，用谷氨酰胺替换Asn396不影响激动剂结合。此外，将位于391和393位（跨膜结构域VII）的丝氨酸残基替换为丙氨酸。将保守性较低的Ser391替换为丙氨酸对配体结合没有影响。然而，用丙氨酸替换保守的Ser393使配体结合减少了86%。将位于82位（跨膜区II）的保守天冬氨酸替换为丙氨酸也产生了一种无法检测到激动剂结合的受体。蛋白质免疫印迹法在野生型和表达突变受体的细胞中均检测到约51 kDa的受体蛋白，表明这些突变可能不影响该蛋白的表达或加工。重要的是，本文所述的人5-HT1A受体序列在跨膜区IV与已发表的序列[《自然》（伦敦）329:75-79（1987）]不同。目前的序列编码一个422个氨基酸的蛋白质，而不是先前描述的421个氨基酸的蛋白质[《自然》（伦敦）329:75-79（1987）]，并且跨膜区IV的序列从……RPRAL……变为……RRAAA……，这与大鼠5-HT1A受体已发表的序列[《生物化学杂志》265:5825-5832（1990）]相对应。此外，通过定点诱变将本克隆的跨膜区IV序列转换为已发表的序列，消除了配体与受体的结合。
