Dutta Priyanka, Dargahi Leila, O'Connell Kara E, Bolia Ashini, Ozkan Banu, Sailer Andreas W, Dev Kumlesh K
Drug Development, School of Medicine, Trinity College Dublin, Ireland.
Department of Physics, Center for Biological Physics, Arizona State University, Tempe, AZ, USA.
Neurosci Lett. 2018 Apr 23;673:12-18. doi: 10.1016/j.neulet.2018.02.055. Epub 2018 Feb 26.
Parkin associated endothelin like receptor (PAELR) is G-protein coupled and ubiquitinated by parkin, promoting its degradation. In autosomal recessive Parkinson's disease, mutations in parkin lead to PAELR aggregation in the endoplasmic reticulum (ER), ER stress, neurotoxicity and cell death. We have identified previously that the protein kinase C interacting protein (PICK1) interacts with and regulates the expression and cell toxicity of PAELR. Here, we experimentally identify and provide in-silico modelling of a novel interaction between PAELR and GABARAPL2 (γ-aminobutyrate type A receptor associated protein like 2), which is an autophagosome-specific Ub-like protein implicated in vesicle trafficking and autophagy. We show that the family of GABARAPs interact with the carboxy terminal (ct) of PAELR and find the cysteine rich region (-CCCCCC-EEC) of ct-PAELR interacts with the GABA binding site of GABARAPL2. This interaction is modelled by in-slico analysis and confirmed using affinity chromatography, showing Myc-tagged GABARAPL2 is retained by a GST fusion of the ct-PAELR. We also demonstrate that transient transfection of GABARAPL2 in HEK293 cells reduces PAELR expression. This study supports the idea that protein levels of PAELR are likely regulated by a multitude of proteins including parkin, PICK1 and GABARAPL2 via mechanisms that include ubiquitination, proteasomal degradagtion and autophagy.
帕金相关内皮素样受体（PAELR）是一种G蛋白偶联受体，可被帕金泛素化，从而促进其降解。在常染色体隐性帕金森病中，帕金的突变会导致PAELR在内质网（ER）中聚集，引发内质网应激、神经毒性和细胞死亡。我们之前已经确定，蛋白激酶C相互作用蛋白（PICK1）与PAELR相互作用，并调节其表达和细胞毒性。在此，我们通过实验鉴定并提供了PAELR与GABARAPL2（γ-氨基丁酸A型受体相关蛋白样2）之间新型相互作用的计算机模拟模型，GABARAPL2是一种自噬体特异性的类泛素蛋白，参与囊泡运输和自噬过程。我们发现GABARAP家族与PAELR的羧基末端（ct）相互作用，并发现ct-PAELR富含半胱氨酸的区域（-CCCCCC-EEC）与GABARAPL2的GABA结合位点相互作用。这种相互作用通过计算机分析进行建模，并使用亲和色谱法得到证实，结果显示Myc标签的GABARAPL2被ct-PAELR的GST融合蛋白保留。我们还证明，在HEK293细胞中瞬时转染GABARAPL2可降低PAELR的表达。这项研究支持了这样一种观点，即PAELR的蛋白水平可能受到包括帕金、PICK1和GABARAPL2在内的多种蛋白质的调控，其调控机制包括泛素化、蛋白酶体降解和自噬。
