LOWER BINDING AFFINITY IN SQUIRREL MONKEY GLUCOCORTICOID RECEPTORS

利用特定的细胞系，我们证实了糖皮质激素 新大陆灵长类动物的受体结合亲和力低于 人的受体(表观Kd，分别为20.9和4.3 nM)。作为一名 了解这种低亲和力的机制的第一步 我们用逆转录酶-聚合酶链式反应来克隆 松鼠猴的糖皮质激素受体，并比较了 从猫头猴身上获得的受体序列的序列 罗望子素和人体细胞。松鼠猴糖皮质激素受体 在核苷酸和氨基酸序列上大约97%相同 人类受体(Pub 2)。的配基结合域 松鼠猴糖皮质激素受体含有四种氨基酸 差异，所有这些都出现在猫头鹰猴子和棉毛猴身上 他马林受体。DNA结合区是完全保守的 在所有四种受体中。与人类的22个不同之处 在松鼠猴的N-末端区域发现了序列 受体。配基结合区中的所有取代都不存在 已知的影响其他基因结合亲和力的匹配突变 物种。以确定这些替换是否是 对于亲和力降低，松鼠猴和人类受体是 在TNT偶联网织红细胞裂解物系统中表达。表达式 人和松鼠、猴子和松鼠的糖皮质激素受体 将Phe774突变为Leu(F774L)的猴受体 很相似。当在TNT系统中表达时，松鼠猴和 人类糖皮质激素具有类似的、高亲和力的结合 地塞米松(表观KD，分别为5.9和4.3 nM)，而 松鼠猴F774L受体亲和力较低(明显 Kd，20.4 NM)。因此，配体结合域中的取代 松鼠猴糖皮质激素受体不能解释 松鼠猴体内这些受体的结合亲和力降低 细胞。

Using specific cell lines we confirmed that the glucocorticoid receptor from New World primates has a lower binding affinity than the human receptor (apparent Kd, 20.9 versus 4.3 nM, respectively). As a first step in understanding the mechanism of this lower affinity in New World primates, we used reverse transcriptase-PCR to clone the glucocorticoid receptor from squirrel monkey and have compared the sequence to receptor sequences obtained from owl monkey, cotton-top tamarin and human cells. The squirrel monkey glucocorticoid receptor is approximately 97% identical in nucleotide and amino acid sequence to the human receptor (pub 2). The ligand binding domain of the squirrel monkey glucocorticoid receptor contains four amino acid differences, all of which are present in owl monkey and cotton-top tamarin receptors. The DNA-binding domain is completely conserved among all four receptors. Twenty-two differences from the human sequence were found in the N-terminal region of the squirrel monkey receptor. None of the substitutions in the ligand binding domain matched mutations known to influence binding affinity in other species. To determine whether these substitutions were responsible for decreased affinity, squirrel monkey and human receptors were expressed in the TNT Coupled Reticulocyte Lysate System. Expressions of human and squirrel monkey glucocorticoid receptors and a squirrel monkey receptor in which Phe774 was mutated to Leu (F774L) were similar. When expressed in the TNT system, the squirrel monkey and human glucocorticoids had similar, high affinity binding for dexamethasone (apparent Kd, 5.9 and 4.3 nM, respectively), whereas the squirrel monkey F774L receptor had lower affinity binding (apparent Kd, 20.4 nM). Thus, substitutions in the ligand-binding domain of the squirrel monkey glucocorticoid receptor cannot account for the decreased binding affinity of these receptors in squirrel monkey cells.