Han B, Tashjian A H
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts, USA.
Biochemistry. 1995 Oct 17;34(41):13412-22. doi: 10.1021/bi00041a019.
To test the hypothesis that pGlu of the thyrotropin-releasing hormone (TRH, pGlu-His-ProNH2) binds to Asn289 in the third extracellular loop (EL3) of its receptor through a hydrogen bonding interaction, we converted Asn289 to Asp (N289D mutant) and measured the potencies of TRH and Pro1TRH for the wild-type and mutant receptors. TRH was 100 times less potent for the N289D receptor than for the wild-type. In contrast, Pro1TRH, which has a protonated proline in place of the pGlu of TRH, was 10 times more potent for the N289D receptor than for the wild-type. A similar result was obtained when Asn289 was converted to Glu, while the potency of Pro1TRH did not change when Asn289 was converted to Ala, confirming that the increased potency of Pro1TRH for the N289D receptor was due to a charge interaction between Pro1TRH and the mutant receptor. These findings are inconsistent with a previous model indicating a direct interaction of the pGlu of TRH with Asn110 in the third transmembrane helix of the receptor (Perlman et al. (1994) J. Biol. Chem. 269, 23383-23386). When Asn110 was converted to Asp (N110D mutant), unlike the N289D receptor, the potency of Pro1TRH for the N110D receptor was decreased by > 10-fold rather than increased. Therefore, a direct interaction of Asn110 with the pGlu of TRH could not be supported by our experiments. We propose a new model in which the pGlu of TRH binds to Asn289 in EL3 and conclude that, unlike catecholamines which bind completely within the transmembrane domain of their receptors, this tripeptide binds, at least in part, to the extracellular domain of its receptor.
为了验证促甲状腺激素释放激素（TRH，焦谷氨酸-组氨酸-脯氨酰胺）中的焦谷氨酸通过氢键相互作用与受体的第三个细胞外环（EL3）中的天冬酰胺289结合这一假说，我们将天冬酰胺289转换为天冬氨酸（N289D突变体），并测定了TRH和Pro1TRH对野生型和突变型受体的效力。TRH对N289D受体的效力比对野生型受体低100倍。相比之下，Pro1TRH（其脯氨酸质子化，取代了TRH的焦谷氨酸）对N289D受体的效力比对野生型受体高10倍。当将天冬酰胺289转换为谷氨酸时，得到了类似的结果，而当将天冬酰胺289转换为丙氨酸时，Pro1TRH的效力没有变化，这证实了Pro1TRH对N289D受体效力的增加是由于Pro1TRH与突变型受体之间的电荷相互作用。这些发现与之前的一个模型不一致，该模型表明TRH的焦谷氨酸与受体第三个跨膜螺旋中的天冬酰胺110直接相互作用（Perlman等人，（1994年）《生物化学杂志》269，23383 - 23386）。当将天冬酰胺110转换为天冬氨酸（N110D突变体）时，与N289D受体不同，Pro1TRH对N110D受体的效力降低了10倍以上，而不是增加。因此，我们的实验不支持天冬酰胺110与TRH的焦谷氨酸直接相互作用。我们提出了一个新模型，其中TRH的焦谷氨酸与EL3中的天冬酰胺289结合，并得出结论，与完全结合在其受体跨膜结构域内的儿茶酚胺不同，这种三肽至少部分地与其受体的细胞外结构域结合。
