Chetwynd Stephen A, Ward Richard J, Milligan Graeme, Welch Heidi C E
Signalling Programme, The Babraham Institute, Babraham Research Campus, Cambridge, UK.
Centre for Translational Pharmacology, School of Molecular Biosciences, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, UK.
J Biol Chem. 2024 Dec;300(12):107940. doi: 10.1016/j.jbc.2024.107940. Epub 2024 Oct 28.
Norbin (Neurochondrin, NCDN) is a G protein-coupled receptor (GPCR) adaptor protein known for its importance in neuronal function. Norbin works by binding to numerous GPCRs, controlling their steady-state trafficking and sometimes their agonist-induced internalization, as well as their signaling. We recently showed that Norbin is expressed in neutrophils, limits the surface levels of the GPCRs C5aR1 and CXCR4 in neutrophils, and suppresses neutrophil-mediated innate immunity. Here, we identify C5aR1 and CXCR4 as direct Norbin interactors and used mice with myeloid-Norbin deficiency to investigate the role of Norbin in the trafficking of endogenous C5aR1 and CXCR4 in primary neutrophils by flow cytometry and cell fractionation. We show that Norbin mediates the agonist-induced internalization of C5aR1 through a β-arrestin-dependent mechanism and limits the recycling of internalized C5aR1 and CXCR4 back to the cell surface. Norbin does not control the constitutive internalization of C5aR1 and CXCR4 nor does it affect the agonist-induced internalization of CXCR4. Norbin suppresses C5aR1 signaling in mouse neutrophils by limiting the C5a-stimulated membrane translocation of Tiam1, Vav, and PKCδ, and activation of Erk and p38 Mapk pathways, as well as Gα-dependent reactive oxygen species production. Our study demonstrates how Norbin suppresses C5aR1 and CXCR4 function in neutrophils and increases our understanding of the mechanisms through which Norbin regulates GPCR trafficking generally, by identifying its importance in β-arrestin recruitment, β-arrestin dependent agonist-induced receptor internalization, and receptor recycling.
诺宾（神经软骨素，NCDN）是一种G蛋白偶联受体（GPCR）衔接蛋白，因其在神经元功能中的重要性而闻名。诺宾通过与多种GPCR结合发挥作用，控制它们的稳态运输，有时还控制它们的激动剂诱导的内化以及它们的信号传导。我们最近发现诺宾在中性粒细胞中表达，限制中性粒细胞中GPCRs C5aR1和CXCR4的表面水平，并抑制中性粒细胞介导的先天免疫。在这里，我们确定C5aR1和CXCR4为诺宾的直接相互作用分子，并使用髓系诺宾缺陷小鼠，通过流式细胞术和细胞分级分离来研究诺宾在原代中性粒细胞中内源性C5aR1和CXCR4运输中的作用。我们表明，诺宾通过β-抑制蛋白依赖性机制介导激动剂诱导的C5aR1内化，并限制内化的C5aR1和CXCR4再循环回到细胞表面。诺宾不控制C5aR1和CXCR4的组成型内化，也不影响激动剂诱导的CXCR4内化。诺宾通过限制C5a刺激的Tiam1、Vav和PKCδ的膜易位以及Erk和p38丝裂原活化蛋白激酶途径的激活，以及Gα依赖性活性氧的产生，抑制小鼠中性粒细胞中的C5aR1信号传导。我们的研究证明了诺宾如何抑制中性粒细胞中C5aR1和CXCR4的功能，并通过确定其在β-抑制蛋白募集、β-抑制蛋白依赖性激动剂诱导的受体内化和受体再循环中的重要性，增加了我们对诺宾一般调节GPCR运输机制的理解。
