Comprehensive analysis of membrane protein interactions by using in vivo photo-cross-linking technology.

为了通过光交联方法研究gpcr与膜蛋白之间的体内相互作用，在哺乳动物细胞中，在受体活性修饰蛋白1 （RAMP1）的细胞外结构域的不同位置特异性地结合了一种可交联的非天然氨基酸。通过将细胞暴露在365 nm的光下，发现RAMP1变体（每个变体在特定位点含有非天然氨基酸）有效地与降钙素受体样受体交联，这是一种已知与RAMP1相互作用的GPCR。这些变异也能够与另一种GPCR，降钙素受体交联。这一观察结果表明，RAMP1直接与这些不同类型的GPCR结合，并且结合模式几乎没有变化，这取决于GPCR的类型。

To investigate the in vivo interaction between GPCRs and membrane proteins by a photo-cross-linking approach, a cross-linkable, non-natural amino acid was site-specifically incorporated at various positions of the extra-cellular domain of receptor-activity modified protein 1 (RAMP1) in mammalian cells. By exposing the cells to the light at 365 nm, the RAMP1 variants, each containing the non-natural amino acid at a specific site, were found to be efficiently cross-linked with the calcitonin receptor-like receptor, which is a GPCR known to interact with RAMP1. These variants were able to be cross-linked also with another GPCR, the calcitonin receptor. This observation revealed that RAMP1 binds to these different types of GPCRs directly, and the binding mode hardly changes, depending on the type of GPCR.