Synthetic analogs of the carboxyl-terminus of beta-thyrotropin: the importance of basic amino acids in receptor binding activity.
Synthetic analogs of the carboxyl-terminus of beta-thyrotropin: the importance of basic amino acids in receptor binding activity.
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β-促甲状腺素羧基末端的合成类似物：碱性氨基酸在受体结合活性中的重要性。
DOI：
10.1021/bi00156a032
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发表时间：
1992
期刊：
影响因子：
2.9
通讯作者：
Morris,JC
中科院分区：
文献类型：
--
作者：
Leinung,MC;Bergert,ER;McCormick,DJ;Morris,JC
1992年7月30日收到的修订版摘要：以前，使用合成肽策略，我们确定了人0-促甲状腺激素（0 TSH）的四个不同区域负责TSH与TSH受体的相互作用。这四个区域中最有效的是亚基的羧基末端，由肽序列0101 - 112表示，其在放射受体测定中抑制放射性标记的TSH与受体的结合，IC 50约为100 µ。在目前的研究中，我们系统地用丙氨酸取代了0101 - 112区域的天然氨基酸，并确定了该跨度内哪些残基对TSH与其受体的结合活性是重要的。与天然序列相比，用丙氨酸取代Lys101、Asn103、Tyr104、Cys105、Lys107和Lys110各自引起活性的显著下降，而在其余位置的取代几乎没有或没有影响。由于这些残基中的三个在生理pH下带正电荷，我们假设这种电荷对序列的结合活性可能很重要。我们通过合成两个系列的类似物来修改该区域的电荷特性，其中在丙氨酸取代研究中鉴定的残基在每个位置用Arg、D-Lys和D-Arg取代。此外，我们还合成了一系列含有碱性残基的类似物，这些类似物可以是在序列0101 - 112中添加或取代非碱性残基。Arg，D-Lys和D-Arg取代Lys101，Lys107和Lys110对活性几乎没有影响;然而，在0101 - 112序列中包含额外的碱性残基显著增强了该区域的抑制活性。用Ala、Ser、Lys或D-Lys取代Cys105导致活性显著降低。为了确定该区域的活性是否是序列特异性的，而是由于该区域的氨基酸组成，我们逆转并扰乱了另外一系列肽中的氨基酸残基。序列颠倒的肽和四个序列混乱的肽具有与天然肽相同的活性，这表明该区域的氨基酸组成和净电荷比其特定序列更重要。我们的结论是，TSH的101 - 112区的净正电荷是代表这部分激素的肽的抑制活性的一个重要因素。通过加入碱性残基来增强电荷，可增加TSH与其受体相互作用的效力。促甲状腺激素（TSH）是糖蛋白激素家族的一员，该家族还包括促黄体激素（LH）、促卵泡激素（FSH）和绒毛膜促性腺激素（CG）。这些蛋白质是异源二聚体，由一个亚基和一个受体特异性0亚基组成，亚基在一个物种内的所有四种激素中是相同的。因为α-亚基是共享的，所以α-亚基必须将受体和组织特异性赋予完整分子，作为α的内在性质或通过在α上诱导独特构象。然而，先前的研究表明，这两种亚基都与受体相互作用（Pierce & Parsons，
Revised Manuscript Received July 30, 1992 abstract: Previously, using a synthetic peptide strategy, we determined that four distinct regions of human 0-thyrotropin (0TSH) were responsible for interaction of TSH with the TSH receptor. The most potent of these four regions was the carboxyl-terminus of the subunit, represented by the peptide sequence 0101—112, which inhibited binding of radiolabeled 0TSH to receptor in radioreceptor assay with an IC50 of approximately 100 µ. In the current studies, we systematically substituted the native aminoacids in region 0101-112 with alanine, and we have determinedwhich residues within this span are important to the binding activity of TSH to its receptor. Substitution of Lys101, Asn103, Tyr104, Cys105, Lys107, and Lys110 with alanine each caused a significant fall in activity as compared to the native sequence, whereas substitution at the remaining positions had little or no effect. Because three of these residues are positively charged at physiologicpH, we hypothesized that this charge may be important to the binding activity of the sequence. We modified the charge characteristics of the region by synthesizing two series of analogs in which the residues identified in the alanine substitution studies were substituted with Arg, D-Lys, and D-Arg at each position. In addition, a series of analogs containing basicresidues, either added to or substituted for nonbasic residues in the sequence 0101-112, was synthesized. Substitution of Arg, D-Lys, and D-Argfor Lys101, Lys107, and Lys110 had little effect on activity; however, inclusion of additional basic residues in the 0101-112 sequence significantly enhanced the inhibitory activity of the region. Substitution of Ala, Ser, Lys or D-Lys for Cys105 resulted in marked reduction in activity. In order to determine if the activity of the region was sequence specificor, rather, due to the amino acid composition of the region, we reversed and scrambled the amino acid residues in an additional series of peptides. The peptide with reversed sequence as well as four peptides with scrambled sequence possessed equal activity to the native peptide, suggesting that the amino acid compositionand the net charge of the region is more important than its specific sequence. We concluded that a net positive charge of region 101-112 of 0TSH is an important factor in the inhibitory activity of peptides representing thisportion of the hormone. Enhancement of the charge, by addition of basic residues, may increase the potency of interaction of TSH withits receptor. However, positive charge is not all important, as removal of Cys105 results in marked loss in activity even though the resulting peptide may have overall significantly greater charge.Thyrotropin (TSH) is a member of the glycoprotein hormone family that also includes lutropin (LH), follitropin (FSH), and choriogonadotropin (CG). These proteins are heterodimers consisting of an-subunit, which is identical in all four hormones within a species, and a hormone-specific 0-subunit. Because the-subunit is shared, the 0-subunit must confer receptor and tissue specificity upon the intact molecule either as an intrinsic property of 0 or by inducing a unique conformation upon «. Prior work has shown, however, that both subunits interactwith the receptor (Pierce & Parsons,