HA-MOP knockin mice express the canonical µ-opioid receptor but lack detectable splice variants

G protein-coupled receptors (GPCRs) are notoriously difficult to detect in native tissues. In an effort to resolve this problem, we have developed a novel mouse model by fusing the hemagglutinin (HA)-epitope tag sequence to the amino-terminus of the µ-opioid receptor (MOP). Although HA-MOP knock-in mice exhibit reduced receptor expression, we found that this approach allowed for highly efficient immunodetection of low abundant GPCR targets. We also show that the HA-tag facilitates both high-resolution imaging and immunoisolation of MOP. Mass spectrometry (MS) confirmed post-translational modifications, most notably agonist-selective phosphorylation of carboxyl-terminal serine and threonine residues. MS also unequivocally identified the carboxyl-terminal 387LENLEAETAPLP398 motif, which is part of the canonical MOP sequence. Unexpectedly, MS analysis of brain lysates failed to detect any of the 15 MOP isoforms that have been proposed to arise from alternative splicing of the MOP carboxyl-terminus. For quantitative analysis, we performed multiple successive rounds of immunodepletion using the well-characterized rabbit monoclonal antibody UMB-3 that selectively detects the 387LENLEAETAPLP398 motif. We found that >98% of HA-tagged MOP contain the UMB-3 epitope indicating that virtually all MOP expressed in the mouse brain exhibit the canonical amino acid sequence.

G蛋白偶联受体（G protein-coupled receptors, GPCRs）在天然组织中极难被检测到。为解决这一难题，我们开发了一种新型小鼠模型：将血凝素（hemagglutinin, HA）表位标签序列融合至μ阿片受体（µ-opioid receptor, MOP）的氨基端。尽管HA-MOP敲入小鼠的受体表达水平有所降低，但我们发现该方法可实现对低丰度GPCR靶标的高效免疫检测。我们还证实，HA标签可同时支持MOP的高分辨率成像与免疫分离。质谱法（Mass spectrometry, MS）验证了其翻译后修饰，最显著的是羧基端丝氨酸与苏氨酸残基的激动剂选择性磷酸化。质谱还明确鉴定出了经典MOP序列的组成部分——羧基端387LENLEAETAPLP398基序。出乎意料的是，对脑裂解物的质谱分析未检测到此前报道的、由MOP羧基端可变剪接产生的15种MOP亚型中的任何一种。为进行定量分析，我们使用经过充分验证的可特异性识别387LENLEAETAPLP398基序的兔单克隆抗体UMB-3开展了多轮连续免疫耗竭实验。结果发现，超过98%的带HA标签的MOP含有UMB-3表位，这表明小鼠脑内表达的几乎所有MOP均具备经典氨基酸序列。