Department of Pharmacology and Toxicology, School of Biomedical Sciences, University of Otago, Dunedin, New Zealand.
Department of Biology, Biotechnology Institute Thurgau (BITg) at the University of Konstanz, Kreuzlingen, Switzerland.
Br J Pharmacol. 2022 May;179(10):2223-2239. doi: 10.1111/bph.15748. Epub 2022 Feb 7.
The cannabinoid CB receptor (CB ) is a promising therapeutic target for modulating inflammation. However, little is known surrounding the mechanisms underpinning CB desensitisation and regulation, particularly the role of GPCR kinases (GRKs). Here, we evaluated the role of six GRK isoforms in β-arrestin recruitment to CB . Mutagenesis of several distal C-terminal aspartic acid residues was also performed in an attempt to delineate additional structural elements involved in the regulation of CB .
In CB -expressing HEK 293 cells, β-arrestin translocation was measured using real-time BRET assays. G protein dissociation BRET assays were performed to assess the activation and desensitisation of CB in the presence of β-arrestin 2.
Overexpression of GRK isoforms 1-6 failed to considerably improve translocation of either β-arrestin 1 or β-arrestin 2 to CB . Consistent with this, inhibition of endogenous GRK2/3 did not substantially reduce β-arrestin 2 translocation. Mutagenesis of C-terminal aspartic acid residues resulted in attenuation of β-arrestin 2 translocation, which translated to a reduction in desensitisation of G protein activation.
Our findings suggest that CB does not adhere to the classical GPCR regulatory paradigm, entailing GRK-mediated and β-arrestin-mediated desensitisation. Instead, C-terminal aspartic acid residues may act as phospho-mimics to induce β-arrestin activation. This study provides novel insights into the regulatory mechanisms of CB , which may aid in our understanding of drug tolerance and dependence.
大麻素 CB1 受体（CB1）是一种有前途的治疗靶点，可用于调节炎症。然而，对于支持 CB1 脱敏和调节的机制知之甚少，特别是 G 蛋白偶联受体激酶（GRK）的作用。在这里，我们评估了六种 GRK 同工型在 CB1 招募β-抑制蛋白中的作用。还对几个远端 C 末端天冬氨酸残基进行了突变，试图描绘参与 CB1 调节的其他结构元件。
在表达 CB1 的 HEK 293 细胞中，使用实时 BRET 测定法测量β-抑制蛋白 2 的转位。进行 G 蛋白解离 BRET 测定以评估在β-抑制蛋白 2 存在下 CB1 的激活和脱敏。
GRK 同工型 1-6 的过表达并没有显著改善β-抑制蛋白 1 或β-抑制蛋白 2 向 CB1 的转位。与此一致，抑制内源性 GRK2/3 并没有显著减少β-抑制蛋白 2 的转位。C 末端天冬氨酸残基的突变导致β-抑制蛋白 2 的转位减弱，这转化为 G 蛋白激活脱敏的减少。
我们的发现表明，CB1 不遵循经典的 GPCR 调节范例，需要 GRK 介导和β-抑制蛋白 2 介导的脱敏。相反，C 末端天冬氨酸残基可能作为磷酸模拟物起作用，诱导β-抑制蛋白 2 的激活。这项研究为 CB1 的调节机制提供了新的见解，这可能有助于我们理解药物耐受性和依赖性。
