GPR160 is not a receptor of anorexigenic cocaine- and amphetamine-regulated transcript peptide
GPR160 is not a receptor of anorexigenic cocaine- and amphetamine-regulated transcript peptide
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DOI：
10.1016/j.ejphar.2023.175713
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发表时间：
2023-04-16
影响因子：
5
通讯作者：
Maletinska, Lenka
中科院分区：
文献类型：
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作者：
Freitas-Lima, Leandro Ceotto;Pacesova, Andrea;Maletinska, Lenka
可卡因和安非他明调节转录肽（CARTp）是一种抑制食欲的神经肽，其受体尚未公开。之前，我们报道了CART（61-102）与嗜铬细胞瘤PC12细胞的特异性结合，其中CART（61-102）亲和力和每个细胞的结合位点数量对应于配体-受体结合。最近，约斯滕等人。指定孤儿GPR160为CARTp受体，因为GPR160抗体消除了CART（55-102）和外源性CART（55-102）在KATOIII细胞中与GPR160共免疫沉淀诱导的神经性疼痛和厌食作用。由于没有直接证据表明 CARTp 是 GPR160 的配体，我们决定通过测试 CARTp 对 GPR160 受体的亲和力来验证这一假设。我们研究了 PC12 细胞中的 GPR160 表达，因为已知它是特异性结合 CARTp 的细胞系。此外，我们检查了具有高内源性 GPR160 表达的 THP1 细胞以及 GPR160 转染细胞系 U2OS 和 U-251 MG 中的特异性 CARTp 结合。在PC12细胞中，GPR160抗体不与125I-CART(61-102)或125I-CART(55-102)竞争特异性结合，并且未检测到GPR160 mRNA表达和GPR160免疫反应性。此外，尽管通过荧光免疫细胞化学（ICC）检测到GPR160，THP1细胞并未表现出任何125I-CART（61-102）或125I-CART（55-102）特异性结合。最后，尽管通过荧光 ICC 检测到了 GPR160，但在转染 GPR160 的细胞系 U2OS 和 U-251 MG 中没有检测到 125I-CART(61-102) 或 125I-CART(55-102) 特异性结合，因为它们的 GPR160 内源表达可以忽略不计。我们的结合研究清楚地表明 GPR160 不能成为 CARTp 的受体。需要进一步研究来鉴定真正的 CARTp 受体。
Cocaine-and amphetamine-regulated transcript peptide (CARTp) is an anorexigenic neuropeptide whose re-ceptor is undisclosed. Previously, we reported the specific binding of CART(61-102) to pheochromocytoma PC12 cells, where CART(61-102) affinity and the number of binding sites per cell corresponded to ligand-receptor binding. Recently, Yosten et al. designated orphan GPR160 as the CARTp receptor, because the GPR160 antibody abolished neuropathic pain and anorexigenic effects induced by CART(55-102) and exogenous CART(55-102) coimmunoprecipitated with GPR160 in KATOIII cells. As no direct evidence that CARTp is a ligand for GPR160 has been described, we decided to verify this hypothesis by testing CARTp affinity to the GPR160 receptor. We investigated the GPR160 expression in PC12 cells since it is cell line known to specifically bind CARTp. Moreover, we examined the specific CARTp binding in THP1 cells, with high endogenous GPR160 expression and GPR160-transfected cell lines U2OS and U-251 MG. In PC12 cells, the GPR160 antibody did not compete for specific binding with 125I-CART(61-102) or with 125I-CART(55-102), and GPR160 mRNA expression and GPR160 immunoreactivity were not detected. Moreover, THP1 cells did not show any 125I-CART(61-102) or 125I-CART(55-102) specific binding despite GPR160 detection by fluorescent immunocytochemistry (ICC). Finally, no 125I-CART(61-102) or 125I-CART(55-102) specific binding in the GPR160-transfected cell lines U2OS and U-251 MG, selected due to their negligible endogenous expression of GPR160, was detected, despite the detection of GPR160 by fluorescent ICC. Our binding studies clearly demonstrated that GPR160 cannot be a receptor for CARTp. Further studies are needed to identify true CARTp receptors.