Abstract:
The glucagon-like peptide 1 receptor (GLP-1R) is a validated clinical target for the treatment of type 2 diabetes and obesity. Unlike most G protein-coupled receptors (GPCRs), the GLP-1R undergoes an atypical mode of internalisation that does not require β-arrestins. While differences in GLP-1R trafficking and β-arrestin recruitment have been observed between clinically used GLP-1R agonists, the role of G protein-coupled receptor kinases (GRKs) in affecting these pathways has not been comprehensively assessed. In this study, we quantified the contribution of GRKs to agonist-mediated GLP-1R internalisation and β-arrestin recruitment profiles using cells where endogenous β-arrestins, or non-visual GRKs were knocked out using CRISPR/Cas9 genome editing. Our results confirm the previously established atypical β-arrestin-independent mode of GLP-1R internalisation and revealed that GLP-1R internalisation is dependent on the expression of GRKs. Interestingly, agonist-mediated GLP-1R β-arrestin 1 and β-arrestin 2 recruitment were differentially affected by endogenous GRK knockout with β-arrestin 1 recruitment more sensitive to GRK knockout than β-arrestin 2 recruitment. Moreover, individual overexpression of GRK2, GRK3, GRK5 or GRK6 in a newly generated GRK2/3/4/5/6 HEK293 cells, rescued agonist-mediated β-arrestin 1 recruitment and internalisation profiles to similar levels, suggesting that there is no specific GRK isoform that drives these pathways. This study advances mechanistic understanding of agonist-mediated GLP-1R internalisation and provides novel insights into how GRKs may fine-tune GLP-1R signalling.
摘要:
胰高血糖素样肽1受体(GLP-1R)是治疗2型糖尿病和肥胖症的有效临床靶标。与大多数G蛋白偶联受体(GPCRs)不同,GLP-1R经历不需要β-抑制素的非典型内在化模式.虽然已观察到临床使用的GLP-1R激动剂之间的GLP-1R运输和β-抑制素募集的差异,G蛋白偶联受体激酶(GRKs)在影响这些途径中的作用尚未得到全面评估.在这项研究中,我们量化了GRKs对激动剂介导的GLP-1R内化和β-抑制素募集谱的贡献,使用内源性β-抑制素,或者使用CRISPR/Cas9基因组编辑敲除非视觉GRK。我们的结果证实了先前建立的GLP-1R内化的非典型β-抑制蛋白非依赖性模式,并揭示了GLP-1R内化依赖于GRK的表达。有趣的是,激动剂介导的GLP-1Rβ-抑制素1和β-抑制素2募集受到内源性GRK敲除的不同影响,β-抑制素1募集对GRK敲除比β-抑制素2募集更敏感。此外,在新产生的GRK2/3/4/5/6HEK293细胞中个别过表达GRK2、GRK3、GRK5或GRK6,拯救的激动剂介导的β-抑制素1募集和内化谱达到相似水平,这表明没有特定的GRK同工型驱动这些途径。这项研究推进了对激动剂介导的GLP-1R内化的机制理解,并提供了GRKs如何微调GLP-1R信号的新见解。