Identification of domains responsible for specific membrane transport and ligand specificity of the ACTH receptor (MC2R)
Identification of domains responsible for specific membrane transport and ligand specificity of the ACTH receptor (MC2R)
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DOI：
10.1016/j.mce.2010.02.032
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发表时间：
2010-06-10
影响因子：
4.1
通讯作者：
Klovins, Janis
中科院分区：
文献类型：
--
作者：
Fridmanis, Davids;Petrovska, Ramona;Klovins, Janis
促肾上腺皮质激素（ACTH）受体具有高度特异性的膜表达，仅限于肾上腺细胞；在其他细胞类型中，该多肽无法转运至细胞表面。与识别不同黑皮质素肽的黑皮质素受体家族 (MC1R-MC5R) 的其他进化相关成员不同，ACTHR (MC2R) 仅结合 ACTH。我们使用了涉及系统构建嵌合 ACTHR/MC4R 受体的诱变方法来鉴定决定 ACTHR 膜转运和 ACTH 结合选择性的结构域。通过用人 MC4R 的相应区域替换人 ACTHR 的选定结构域，创建了总共 15 个嵌合受体。我们开发了一种分析方法，通过共焦荧光显微镜准确定量与增强型绿色荧光蛋白融合的重组受体的细胞膜定位。还测试了嵌合受体结合 ACTH (1-24) 和黑素细胞刺激激素 (MSH) 类似物 NIe4 的能力。 DPhe7-α-MSH，并诱导 cAMP 反应 我们的结果表明，用 ACTHR 的同源片段替换 MC4R N 端片段显着降低了膜转运。我们还确定了位于第三和第四跨膜区的另一个信号作为 ACTHR 细胞内滞留的主要决定因素。此外，我们发现 ACTHR 的第四和第五跨膜结构域参与 ACTH 结合选择性。我们讨论了通过与黑皮质素 2 受体辅助蛋白 (MRAP) 相互作用绕过这些停滞信号的机制，以及决定 ACTHR 高配体结合特异性的可能机制。 (C) 2010 Elsevier Ireland Ltd. 保留所有权利。
The adrenocorticotropic hormone (ACTH) receptor has highly specific membrane expression that is limited to adrenal cells; in other cell types the polypeptide fails to be transported to the cell surface Unlike other evolutionarily related members of the melanocortin receptor family (MC1R-MC5R) that recognize different melanocortin peptides, ACTHR (MC2R) binds only ACTH. We used a mutagenesis approach involving systematic construction of chimeric ACTHR/MC4R receptors to identify the domains determining the selectivity of ACTHR membrane transport and ACTH binding In total 15 chimeric receptors were created by replacement of selected domains of human ACTHR with the corresponding regions of human MC4R. We developed an analytical method to accurately quantify cell-membrane localization of recombinant receptors fused with enhanced green fluorescent protein by confocal fluorescence microscopy The chimeric receptors were also tested for their ability to bind ACTH (1-24) and the melanocyte-stimulating hormone (MSH) analog, NIe4. DPhe7-alpha-MSH, and to induce a cAMP response Our results indicate that substitution of the MC4R N-terminal segment with the homologous segment of ACTHR significantly decreased membrane transport. We also identified another signal localized in the third and fourth transmembrane regions as the main determinant of ACTHR intracellular retention In addition, we found that the fourth and fifth transmembrane domains of the ACTHR are involved in ACTH binding selectivity We discuss the mechanisms involved in bypassing these arrest signals via an interaction with melanocortin 2 receptor accessory protein (MRAP) and the possible mechanisms that determine the high ligand-binding specificity of ACTHR. (C) 2010 Elsevier Ireland Ltd. All rights reserved.