The hydrophobic amino acids in putative helix 8 in carboxy-terminus of histamine H(3) receptor are involved in receptor-G-protein coupling.
The hydrophobic amino acids in putative helix 8 in carboxy-terminus of histamine H(3) receptor are involved in receptor-G-protein coupling.
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DOI：
10.1016/j.cellsig.2011.06.021
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发表时间：
2011-11
影响因子：
4.8
通讯作者：
Atsuo Kuramasu;J. Sukegawa;Takeya Sato;E. Sakurai;Takehiko Watanabe;T. Yanagisawa;K. Yanai
中科院分区：
文献类型：
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作者：
Atsuo Kuramasu;J. Sukegawa;Takeya Sato;E. Sakurai;Takehiko Watanabe;T. Yanagisawa;K. Yanai
使用缺失和丙氨酸取代的突变受体研究了人组胺 H3 受体羧基末端尾部中假定的螺旋 8 的功能作用。虽然羧基末端尾部的缺失不会降低总表达水平、表面表达或配体结合亲和力，但激动剂刺激的 cAMP 反应、[35S] GTPγS 结合和 MAPK 激活被完全消除。缺乏羧基末端尾部的受体也无法对反向激动剂硫哌丁胺作出反应，这表明羧基末端尾部通过改变G蛋白与受体的偶联来参与受体活性的调节。定点诱变揭示了假定的螺旋 8 中的疏水性氨基酸，例如位置 419 (F7.60) 和 423 (F7.64) 的苯丙氨酸或位置 426 (L7.67) 和 427 (L7.68) 的亮氨酸对于激动剂诱导的 H3 受体激活很重要。 F7.60 的取代还导致受体对反向激动剂的失活反应较小，这意味着存在可以激活或失活的中间构象。我们的结果表明，假定的螺旋 8 的疏水界面对于 H3 受体活性的调节很重要，可能是通过将螺旋稳定在质膜上来实现的。
Functional roles of putative helix 8 in the carboxy-terminal tail of the human histamine H3receptor were investigated using deleted and alanine-substituted mutant receptors. While the deletion of the carboxy-terminal tail did not decrease the total expression level, surface expression, or ligand binding affinity, the agonist-stimulated cAMP response, [35S] GTPγS binding, and MAPK activation were totally abolished. The receptor lacking the carboxy-terminal tail also failed to respond to an inverse agonist, thioperamide, suggesting that the carboxy-terminal tail is involved in the regulation of receptor activity by changing G-protein coupling with the receptor. Site-directed mutagenesis revealed that hydrophobic amino acids in the putative helix 8 such as phenylalanines at position 419 (F7.60) and 423 (F7.64) or leucines at 426 (L7.67) and 427 (L7.68) were important for the agonist-induced activation of H3receptor. Substitution of F7.60 also resulted in a receptor that was less responsive to inactivation by the inverse agonist, implying the existence of an intermediate conformation that can be either activated or inactivated. Our results suggest that hydrophobic interface of putative helix 8 is important for the regulation of H3receptor activity, presumably by stabilizing the helix to the plasma membrane.