Shpakov A O, Shpakova E A, Tarasenko I I, Derkach K V
Tsitologiia. 2014;56(7):526-35.
The regulation of the specific activity of the thyroid gland is carried by thyroid-stimulating hormone (TSH) through TSH receptor (TSHR). This receptor is coupled to different types of G-proteins, including the G(s)-proteins, through which TSH stimulates the enzyme adenylyl cyclase (AC). As the application of TSH in medicine is limited, the development of selective regulators of TSHR with agonistic and antagonistic activity is carried out. One of the approaches to their creation is to develop the peptides corresponding to functionally important regions of TSHR which are located in its intracellular loops (ICL) and are involved in the binding and activation of G-proteins. We have synthesized peptide corresponding to the C-terminal region 612-627 of the third ICL of TSHR and its derivatives modified by palmitic acid residue (at the N- or the C-terminus) or by polylysine dendrimer (at the N-terminus), and studied their effect on the basal and TSH-stimulated AC activity in the membrane fraction isolated from the rat thyroid. The most active was peptide 612-627-K(Pal)A modified by palmitate at the C-terminus, where in TSHR the hydrophobic transmembrane region is located. At the micromolar concentrations the peptide increased AC activity and reduced the AC stimulating effect of TSH. The action of the 612-627-K(Pal)A has been directed onto TSHR homologous to it, as indicated by the following facts: 1) the inhibition of G(s)-protein, the downstream component of AC system, by treating the membranes with cholera toxin led to the blocking of peptide AC effect, 2) this effect was not detected in the tissues where no TSHR, 3) the peptide did not significantly affect the AC stimulating effects of hormones acting via other receptors. The unmodified peptide and the peptide with N-terminal dendrimer are far behind the 612-627-K(Pal)A in their ability to activate AC in the thyroid, while the peptide modified by palmitate at the N-terminus was inactive. At the same time, the peptide modified by dendrimer was comparable to the 612-627-K(Pal)A in the ability to inhibit the AC effect of TSH, but, although to a lesser extent that it decreased the AC effects of other hormones, demonstrating the low receptor specificity. Thus, these data point to the high efficiency of peptide 612-627-K(Pal)A, as a regulator of TSHR, and the prospects of creating the drugs based on it to control the thyroid functions in pathology.
甲状腺的特定活性调节由促甲状腺激素（TSH）通过促甲状腺激素受体（TSHR）来进行。该受体与不同类型的G蛋白偶联，包括G(s)蛋白，TSH通过G(s)蛋白刺激腺苷酸环化酶（AC）。由于TSH在医学上的应用有限，因此开展了具有激动和拮抗活性的TSHR选择性调节剂的研发。其制备方法之一是开发与TSHR功能重要区域相对应的肽段，这些区域位于其细胞内环（ICL）中，参与G蛋白的结合和激活。我们合成了与TSHR第三个ICL的C末端区域612 - 627相对应的肽及其经棕榈酸残基修饰（在N末端或C末端）或经聚赖氨酸树枝状大分子修饰（在N末端）的衍生物，并研究了它们对从大鼠甲状腺分离的膜部分中基础和TSH刺激的AC活性的影响。活性最高的是在C末端经棕榈酸修饰的肽612 - 627 - K(Pal)A，TSHR的疏水跨膜区域就位于此处。在微摩尔浓度下，该肽增加了AC活性并降低了TSH对AC的刺激作用。612 - 627 - K(Pal)A的作用针对与其同源的TSHR，以下事实表明了这一点：1）用霍乱毒素处理膜抑制AC系统的下游成分G(s)蛋白会导致肽对AC的作用受阻；2）在没有TSHR的组织中未检测到这种作用；3）该肽对通过其他受体起作用的激素的AC刺激作用没有显著影响。未修饰的肽和带有N末端树枝状大分子的肽在激活甲状腺AC的能力上远落后于612 - 627 - K(Pal)A，而在N末端经棕榈酸修饰的肽则无活性。同时，经树枝状大分子修饰的肽在抑制TSH对AC的作用方面与612 - 627 - K(Pal)A相当，但尽管程度较小，它也降低了其他激素的AC作用，表明其受体特异性较低。因此，这些数据表明肽612 - 627 - K(Pal)A作为TSHR调节剂具有高效性，以及基于它开发控制病理状态下甲状腺功能药物的前景。
