Department of Physiology, Faculty of Medical and Health Sciences, University of Auckland, Auckland 1142, New Zealand.
J Mol Endocrinol. 2013 Mar 18;50(2):217-27. doi: 10.1530/JME-12-0220. Print 2013 Apr.
Human melanocortin 2 receptor accessory protein 1(hMRAPa) is essential for human melanocortin 2 receptor (hMC2R)-regulated adrenal steroidogenesis. hMRAPa enhances hMC2R N-linked glycosylation and maturation, promotes hMC2R cell surface expression and enables ACTH to bind and activate the MC2R. However, hMRAPa is predicted to have functions beyond its critical role in hMC2R activity. It is more widely expressed than the hMC2R and it has been shown to co-immunoprecipitate with all other hMCR subtypes and other G-protein-coupled receptors, when these are co-expressed with each receptor in heterologous cells. The physiological relevance of hMRAPa interactions with these receptors is unknown. We hypothesised that hMRAPa could influence post-translational processing and maturation of these receptors, similar to its actions on the hMC2R. Here we used co-immunoprecipitation and western blotting techniques to characterise effects of hMRAPa-FLAG co-expression on the maturation of each HA-tagged hMCR subtype and the HA-tagged human calcitonin receptor-like receptor (hCL), co-expressed in HEK293 cells. While hMRAPa-FLAG interacted with all five HA-hMCR subtypes and the HA-hCL, it only altered HA-hMC4R molecular mass. This altered HA-hMC4R molecular mass was due to a change in endoglycosidase H-resistant complex N-linked glycosylation, which we observed for HA-hMC4R in both intracellular and cell surface fractions. This effect was specific to the HA-hMC4R as hMRAPa did not alter the molecular mass of any of the other receptors that we examined. In conclusion, the specific effects of hMRAPa on hMC4R molecular mass and complex N-linked glycosylation provide evidence in support of a role for MRAPα in hMC4R functions.
人类黑素皮质素 2 受体辅助蛋白 1（hMRAPa）对于人类黑素皮质素 2 受体（hMC2R）调节肾上腺类固醇生成是必不可少的。hMRAPa 增强 hMC2R 的 N-连接糖基化和成熟，促进 hMC2R 细胞表面表达，并使 ACTH 结合并激活 MC2R。然而，hMRAPa 的功能超出了其在 hMC2R 活性中的关键作用。它的表达比 hMC2R 更广泛，并且已经显示与所有其他 hMCR 亚型和其他 G 蛋白偶联受体共免疫沉淀，当这些受体与异源细胞中的每个受体共表达时。hMRAPa 与这些受体相互作用的生理相关性尚不清楚。我们假设 hMRAPa 可以影响这些受体的翻译后加工和成熟，类似于其对 hMC2R 的作用。在这里，我们使用共免疫沉淀和 Western blot 技术来描述 hMRAPa-FLAG 共表达对每个 HA 标记的 hMCR 亚型和 HA 标记的人类降钙素受体样受体（hCL）成熟的影响，这些受体在 HEK293 细胞中共表达。虽然 hMRAPa-FLAG 与所有五个 HA-hMCR 亚型和 HA-hCL 相互作用，但它仅改变了 HA-hMC4R 的分子质量。这种改变的 HA-hMC4R 分子质量是由于内糖苷酶 H 抗性复合物 N-连接糖基化的变化引起的，我们在细胞内和细胞表面部分都观察到了 HA-hMC4R 的这种变化。这种效应是特异性的，因为 hMRAPa 没有改变我们研究的任何其他受体的分子质量。总之，hMRAPa 对 hMC4R 分子质量和复杂 N-连接糖基化的特定影响为 MRAPα 在 hMC4R 功能中的作用提供了证据。
