Florey Institute of Neuroscience and Mental Health and Florey Department of Neuroscience and Mental Health Parkville Victoria Australia.
Department of Biochemistry and Molecular Biology The University of Melbourne Parkville Victoria Australia.
Pharmacol Res Perspect. 2019 Jul 30;7(4):e00513. doi: 10.1002/prp2.513. eCollection 2019 Aug.
Relaxin family peptide 1 (RXFP1) is the receptor for relaxin a peptide hormone with important therapeutic potential. Like many G protein-coupled receptors (GPCRs), RXFP1 has been reported to form homodimers. Given the complex activation mechanism of RXFP1 by relaxin, we wondered whether homodimerization may be explicitly required for receptor activation, and therefore sought to determine if there is any relaxin-dependent change in RXFP1 proximity at the cell surface. Bioluminescence resonance energy transfer (BRET) between recombinantly tagged receptors is often used in GPCR proximity studies. RXFP1 targets poorly to the cell surface when overexpressed in cell lines, with the majority of the receptor proteins sequestered within the cell. Thus, any relaxin-induced changes in RXFP1 proximity at the cell surface may be obscured by BRET signal originating from intracellular compartments. We therefore, utilized the newly developed split luciferase system called HiBiT to specifically label the extracellular terminus of cell surface RXFP1 receptors in combination with mCitrine-tagged receptors, using the GABA heterodimer as a positive control. This demonstrated that the BRET signal detected from RXFP1-RXFP1 proximity at the cell surface does not appear to be due to stable physical interactions. The fact that there is also no relaxin-mediated change in RXFP1-RXFP1 proximity at the cell surface further supports these conclusions. This work provides a basis by which cell surface GPCR proximity and expression levels can be specifically studied using a facile and homogeneous labeling technique such as HiBiT.
松弛素家族肽 1（RXFP1）是松弛素肽激素的受体，具有重要的治疗潜力。与许多 G 蛋白偶联受体（GPCR）一样，据报道 RXFP1 形成同源二聚体。鉴于松弛素对 RXFP1 的复杂激活机制，我们想知道同源二聚化是否明确需要受体激活，因此试图确定在细胞表面是否存在与松弛素相关的 RXFP1 接近度的变化。在 GPCR 接近研究中，经常使用重组标记受体之间的生物发光共振能量转移（BRET）。当在细胞系中过表达时，RXFP1 靶向细胞表面的效率很差，大多数受体蛋白被隔离在细胞内。因此，细胞表面 RXFP1 接近度的任何松弛素诱导变化都可能被源自细胞内隔室的 BRET 信号所掩盖。因此，我们利用称为 HiBiT 的新开发的分裂荧光素系统，专门标记细胞表面 RXFP1 受体的细胞外末端，同时使用 mCitrine 标记的受体，使用 GABA 异二聚体作为阳性对照。这表明从细胞表面 RXFP1-RXFP1 接近度检测到的 BRET 信号似乎不是由于稳定的物理相互作用。事实上，在细胞表面，RXFP1-RXFP1 接近度也没有松弛素介导的变化，这进一步支持了这些结论。这项工作为使用简便、均相的标记技术（如 HiBiT）特异性研究细胞表面 GPCR 接近度和表达水平提供了基础。
