Melanocortin-2 receptor accessory protein MRAP forms antiparallel homodimers
Melanocortin-2 receptor accessory protein MRAP forms antiparallel homodimers
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DOI：
10.1073/pnas.0708916105
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发表时间：
2007-12-18
影响因子：
11.1
通讯作者：
Hinkle, Patricia M.
中科院分区：
文献类型：
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作者：
Sebag, Julien A.;Hinkle, Patricia M.
黑皮质素-2（MC 2）受体辅助蛋白（MRAP）是G蛋白偶联的MC 2受体转运至质膜所必需的。MRAP具有单一跨膜结构域，其作用机制和结构尚不清楚。在这里，我们表明，MRAP显示一个以前未表征的拓扑结构。MRAP的N-和C-末端上的表位以相当的水平定位在CHO细胞的外表面上。使用针对N-和C-末端MRAP肽的抗体，我们证明了内源性MRAP的两端都面向肾上腺细胞的外部。近一半的MRAP在单个内源性N-末端糖基化位点被糖基化，当天然糖基化位点被C-末端结构域中的一个取代时，超过一半的MRAP被糖基化。在膜两侧具有潜在糖基化位点的突变体MRAP是单一糖基化的，但不是双重糖基化的，这表明MRAP不是单调的。差异标记的MRAP的免疫共沉淀确定MRAP是二聚体。通过选择性免疫沉淀细胞表面的MRAP在一个或另一个方向，我们表明，MRAP同源二聚体是反平行的，并形成一个稳定的复合物与MC 2受体。在没有MRAP的情况下，MC 2受体被捕获在内质网中，但在MRAP的情况下，MC 2受体被糖基化并定位在质膜上，在那里它响应于ACTH而发出信号。MRAP特异性地起作用，因为它不增加黑皮质素、β 2-肾上腺素能或TSH释放激素受体的表面表达。MRAP是第一个被鉴定为具有反平行同源二聚体结构的真核细胞膜蛋白。
The melanocortin-2 (MC2) receptor accessory protein (MRAP) is required for trafficking of the G protein-coupled MC2 receptor to the plasma membrane. The mechanism of action and structure of MRAP, which has a single transmembrane domain, are unknown. Here, we show that MRAP displays a previously uncharacterized topology. Epitopes on both the N- and C-terminal ends of MRAP were localized on the external face of CHO cells at comparable levels. Using antibodies raised against N- and C-terminal MRAP peptides, we demonstrated that both ends of endogenous MRAP face the outside in adrenal cells. Nearly half of MRAP was glycosylated at the single endogenous N-terminal glycosylation site, and over half was glycosylated when the natural glycosylation site was replaced by one in the C-terminal domain. A mutant MRAP with potential glycosylation sites on both sides of the membrane was singly but not doubly glycosylated, suggesting that MRAP is not monotopic. Coimmunoprecipitation of differentially tagged MRAPs established that MRAP is a dimer. By selectively immunoprecipitating cell surface MRAP in one or the other orientation, we showed that MRAP homodimers are antiparallel and form a stable complex with MC2 receptor. In the absence of MRAP, MC2 receptor was trapped in the endoplasmic reticulum, but with MRAP, the MC2 receptor was glycosylated and localized on the plasma membrane, where it signaled in response to ACTH. MRAP acted specifically, because it did not increase surface expression of melanocortin, beta 2-adrenergic, or TSH-releasing hormone receptors. MRAP is the first eukaryotic membrane protein identified with an antiparallel homodimeric structure.