From the Institute for Pharmacology, Center for Physiology and Pharmacology, Medical University of Vienna, Waehringerstrasse 13a, A-1090 Vienna, Austria.
J Biol Chem. 2013 Oct 4;288(40):28831-44. doi: 10.1074/jbc.M113.464776. Epub 2013 Aug 21.
The adenosine A2A receptor is a prototypical rhodopsin-like G protein-coupled receptor but has several unique structural features, in particular a long C terminus (of >120 residues) devoid of a palmitoylation site. It is known to interact with several accessory proteins other than those canonically involved in signaling. However, it is evident that many more proteins must interact with the A2A receptor, if the trafficking trajectory of the receptor is taken into account from its site of synthesis in the endoplasmic reticulum (ER) to its disposal by the lysosome. Affinity-tagged versions of the A2A receptor were expressed in HEK293 cells to identify interacting partners residing in the ER by a proteomics approach based on tandem affinity purification. The receptor-protein complexes were purified in quantities sufficient for analysis by mass spectrometry. We identified molecular chaperones (heat-shock proteins HSP90α and HSP70-1A) that interact with and retain partially folded A2A receptor prior to ER exit. Complex formation between the A2A receptor and HSP90α (but not HSP90β) and HSP70-1A was confirmed by co-affinity precipitation. HSP90 inhibitors also enhanced surface expression of the receptor in PC12 cells, which endogenously express the A2A receptor. Finally, proteins of the HSP relay machinery (e.g. HOP/HSC70-HSP90 organizing protein and P23/HSP90 co-chaperone) were recovered in complexes with the A2A receptor. These observations are consistent with the proposed chaperone/coat protein complex II exchange model. This posits that cytosolic HSP proteins are sequentially recruited to folding intermediates of the A2A receptor. Release of HSP90 is required prior to recruitment of coat protein complex II components. This prevents premature ER export of partially folded receptors.
腺苷 A2A 受体是一种典型的视紫红质样 G 蛋白偶联受体，但具有几个独特的结构特征，特别是一个长的 C 端（超过 120 个残基），没有棕榈酰化位点。除了那些经典的信号转导相关蛋白之外，它还已知与其他几种辅助蛋白相互作用。然而，如果考虑到受体的运输轨迹，从内质网（ER）中合成的部位到溶酶体中的处理，显然有更多的蛋白质必须与 A2A 受体相互作用。用亲和标签标记的 A2A 受体在 HEK293 细胞中表达，通过基于串联亲和纯化的蛋白质组学方法鉴定驻留在 ER 中的相互作用伙伴。受体蛋白复合物被纯化到足够的数量，可通过质谱进行分析。我们鉴定了分子伴侣（热休克蛋白 HSP90α 和 HSP70-1A），它们在 A2A 受体离开 ER 之前与部分折叠的 A2A 受体相互作用并保留它。A2A 受体与 HSP90α（而不是 HSP90β）和 HSP70-1A 之间的复合物形成通过共亲和沉淀得到证实。HSP90 抑制剂还增强了内源性表达 A2A 受体的 PC12 细胞中受体的表面表达。最后，HSP 接力机制的蛋白质（例如 HOP/HSC70-HSP90 组织蛋白和 P23/HSP90 共伴侣）与 A2A 受体一起被回收。这些观察结果与提出的伴侣/外套蛋白复合物 II 交换模型一致。该模型假设细胞质 HSP 蛋白依次被募集到 A2A 受体的折叠中间体。在募集外套蛋白复合物 II 成分之前，需要释放 HSP90。这可以防止部分折叠的受体过早地从 ER 中输出。
