Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin.
Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin
Mol Pharmacol. 2024 Nov 18;106(6):287-297. doi: 10.1124/molpharm.124.000942.
-arrestins are multifaceted adaptor proteins that mediate G protein-coupled receptor (GPCR) desensitization, internalization, and signaling. It is emerging that receptor-specific determinants specify these divergent functions at GPCRs, yet this remains poorly understood. Here, we set out to identify the receptor determinants responsible for -arrestin-mediated regulation of the chemokine receptor C-X-C motif chemokine receptor 5 (CXCR5). Using bioluminescence resonance energy transfer, we show that -arrestin1 and -arrestin2 are dose-dependently recruited to CXCR5 by its cognate ligand C-X-C motif chemokine ligand 13 (CXCL13). The carboxy-terminal tail of CXCR5 contains several serine/threonine residues that can be divided into three discrete phospho-site clusters based on their position relative to transmembrane domain 7. Mutagenesis experiments revealed that the distal and medial phospho-site clusters, but not the proximal, are required for agonist-stimulated -arrestin1 or -arrestin2 recruitment to CXCR5. Consistent with this, we provide evidence that the distal and medial, but not proximal, phospho-site clusters are required for receptor desensitization. Surprisingly, the individual phospho-site clusters are not required for agonist-stimulated internalization of CXCR5. Further, we show that CXCL13-stimulated CXCR5 internalization and ERK1/2 phosphorylation, but not desensitization, remain intact in human embryonic kidney 293 cells lacking -arrestin1 and -arrestin2. Our study provides evidence that -arrestins are recruited to CXCR5 and are required for desensitization but are dispensable for internalization or signaling, suggesting that discrete receptor determinants specify the divergent functions of -arrestins. SIGNIFICANCE STATEMENT: C-X-C motif ligand 13 (CXCL13) and C-X-C motif chemokine receptor 5 (CXCR5) are important in the immune system and are linked to diseases, yet regulation of CXCR5 signaling remains poorly understood. We provide evidence that a phospho-site cluster located at the extreme distal carboxyl-terminal tail of the receptor is responsible for -arrestin recruitment and receptor desensitization. -arrestins are not required for CXCL13-stimulated internalization or signaling, indicating that -arrestins perform only one of their functions at CXCR5 and that discrete receptor determinants specify the divergent functions of -arrestins.
-arrestins 是多功能衔接蛋白，可介导 G 蛋白偶联受体 (GPCR) 的脱敏、内化和信号转导。目前新兴的观点认为，受体特异性决定因素在 GPCR 上指定了这些不同的功能，但对此仍知之甚少。在这里，我们着手确定负责-arrestin 介导的趋化因子受体 C-X-C 基序趋化因子受体 5 (CXCR5) 调节的受体决定因素。使用生物发光共振能量转移，我们表明-arrestin1 和 -arrestin2 可被其同源配体 C-X-C 基序趋化因子配体 13 (CXCL13) 剂量依赖性地募集到 CXCR5。CXCR5 的羧基末端尾部包含几个丝氨酸/苏氨酸残基，根据它们相对于跨膜域 7 的位置，可以分为三个离散的磷酸化位点簇。突变实验表明，远端和中间的磷酸化位点簇，但不是近端的磷酸化位点簇，是激动剂刺激-arrestin1 或 -arrestin2 募集到 CXCR5 所必需的。与此一致，我们提供的证据表明，远端和中间，但不是近端，磷酸化位点簇是受体脱敏所必需的。令人惊讶的是，单个磷酸化位点簇对于激动剂刺激的 CXCR5 内化不是必需的。此外，我们表明，在缺乏-arrestin1 和 -arrestin2 的人胚肾 293 细胞中，CXCL13 刺激的 CXCR5 内化和 ERK1/2 磷酸化，但不是脱敏，仍然完整。我们的研究提供的证据表明-arrestins 被募集到 CXCR5 并需要脱敏，但对于内化或信号转导是可有可无的，这表明离散的受体决定因素指定了-arrestins 的不同功能。
C-X-C 基序趋化因子配体 13 (CXCL13) 和 C-X-C 基序趋化因子受体 5 (CXCR5) 在免疫系统中很重要，与疾病有关，但 CXCR5 信号转导的调节仍知之甚少。我们提供的证据表明，位于受体极远端羧基末端尾部的磷酸化位点簇负责-arrestin 的募集和受体脱敏。-arrestins 对于 CXCL13 刺激的内化或信号转导不是必需的，这表明-arrestins 在 CXCR5 上只执行其功能之一，并且离散的受体决定因素指定了-arrestins 的不同功能。
