Ruehle Sabine, Wager-Miller James, Straiker Alex, Farnsworth Jill, Murphy Michelle N, Loch Sebastian, Monory Krisztina, Mackie Ken, Lutz Beat
Institute of Physiological Chemistry, University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany.
Department of Psychological and Brain Sciences, Gill Center for Biomolecular Science, Indiana University, Bloomington, Indiana, USA.
J Neurochem. 2017 Aug;142(4):521-533. doi: 10.1111/jnc.14099. Epub 2017 Jul 18.
Numerous studies have been carried out in the mouse model, investigating the role of the cannabinoid receptor type 1 (CB1). However, mouse CB1 (mCB1) receptor differs from human CB1 (hCB1) receptor in 13 amino acid residues. Two splice variants, hCB1a and hCB1b, diverging in their amino-termini, have been reported to be unique for hCB1 and, via different signaling properties, contribute to CB1 receptor physiology and pathophysiology. We hypothesized that splice variants also exist for the mCB1 receptor and have different signaling properties. On murine hippocampal cDNA, we identified two novel mCB1 receptor splice variants generated by splicing of introns with 117 bp and 186 bp in the N-terminal domain, corresponding to deletions of 39 or 62 amino acids, respectively. The mRNAs for the splice variants mCB1a and mCB1b are expressed at low levels in different brain regions. Western blot analysis of protein extracts from stably transfected HEK293 cells indicates a strongly reduced glycosylation because of the absence of two glycosylation sites in mCB1b. On-cell western analysis in these stable lines revealed increased internalization of mCB1a and mCB1b upon stimulation with the agonist WIN55,212-2 as compared to mCB1. Results also point toward an increased affinity to SR141716 for mCB1a, as well as slightly enhanced inhibition of neurotransmission compared to mCB1. In mCB1b, agonist-induced MAPK phosphorylation was decreased compared to mCB1 and mCB1a. Identification of mouse CB1 receptor splice variants may help to explain differences found between human and mouse endocannabinoid systems and improve the understanding of CB1 receptor signaling and trafficking in different species.
已经在小鼠模型中开展了大量研究，以探究1型大麻素受体（CB1）的作用。然而，小鼠CB1（mCB1）受体与人类CB1（hCB1）受体在13个氨基酸残基上存在差异。据报道，两种剪接变体hCB1a和hCB1b在其氨基末端有所不同，是hCB1所特有的，并且通过不同的信号特性，对CB1受体的生理和病理生理过程有影响。我们推测mCB1受体也存在剪接变体，并且具有不同的信号特性。在小鼠海马cDNA上，我们鉴定出两种新的mCB1受体剪接变体，它们是由内含子在N端结构域剪接产生的，长度分别为117 bp和186 bp，分别对应于39个或62个氨基酸的缺失。剪接变体mCB1a和mCB1b的mRNA在不同脑区中低水平表达。对稳定转染的HEK293细胞的蛋白质提取物进行的蛋白质印迹分析表明，由于mCB1b中缺少两个糖基化位点，其糖基化程度大幅降低。在这些稳定细胞系中进行的细胞表面蛋白质印迹分析显示，与mCB1相比，用激动剂WIN55,212-2刺激后，mCB1a和mCB1b的内化增加。结果还表明，mCB1a对SR141716的亲和力增加，并且与mCB1相比，对神经传递的抑制作用略有增强。在mCB1b中，与mCB1和mCB1a相比，激动剂诱导的MAPK磷酸化降低。鉴定小鼠CB1受体剪接变体可能有助于解释在人类和小鼠内源性大麻素系统之间发现的差异，并增进对不同物种中CB1受体信号传导和转运的理解。
