Mascia Fabrizio, Klotz Lisa, Lerch Judith, Ahmed Mostafa H, Zhang Yan, Enz Ralf
Institut für Biochemie (Emil-Fischer-Zentrum), Universität Erlangen-Nürnberg, Erlangen, Germany.
Department of Medicinal Chemistry, Virginia Commonwealth University, Richmond, Virginia, USA.
J Neurochem. 2017 May;141(4):577-591. doi: 10.1111/jnc.14021. Epub 2017 Apr 12.
The excitability of the central nervous system depends largely on the surface density of neurotransmitter receptors. The endocannabinoid receptor 1 (CB R) and the metabotropic glutamate receptor mGlu R are expressed pre-synaptically where they reduce glutamate release into the synaptic cleft. Recently, the CB R interacting protein cannabinoid receptor interacting protein 1a (CRIP1a) was identified and characterized to regulate CB R activity in neurons. However, underlying molecular mechanisms are largely unknown. Here, we identified a common mechanism used by CRIP1a to regulate the cell surface density of two different types of G-protein coupled receptors, CB R and mGlu R. Five amino acids within the CB R C-terminus were required and sufficient to reduce constitutive CB R endocytosis by about 72% in the presence of CRIP1a. Interestingly, a similar sequence is present in mGlu R and consistently, endocytosis of mGlu R depended on CRIP1a, as well. Docking analysis and molecular dynamics simulations identified a conserved serine in CB R (S468) and mGlu R (S894) that forms a hydrogen bond with the peptide backbone of CRIP1a at position R82. In contrast to mGlu R, the closely related mGlu R splice-variant carries a lysine (K894) at this position, and indeed, mGlu R endocytosis was not affected by CRIP1a. Chimeric constructs between CB R, mGlu R, and mGlu R underline the role of the identified five CRIP1a sensitive amino acids. In summary, we suggest that CRIP1a negatively regulates endocytosis of two different G-protein coupled receptor types, CB R and mGlu R.
中枢神经系统的兴奋性很大程度上取决于神经递质受体的表面密度。内源性大麻素受体1（CB R）和代谢型谷氨酸受体mGlu R在突触前表达，它们可减少谷氨酸释放到突触间隙中。最近，已鉴定并表征了与CB R相互作用的蛋白大麻素受体相互作用蛋白1a（CRIP1a），其可调节神经元中的CB R活性。然而，其潜在的分子机制在很大程度上尚不清楚。在此，我们鉴定出CRIP1a用于调节两种不同类型G蛋白偶联受体CB R和mGlu R细胞表面密度的共同机制。在存在CRIP1a的情况下，CB R C末端的五个氨基酸是必需且足以将组成型CB R内吞作用降低约72%。有趣的是，mGlu R中也存在类似序列，并且一致地，mGlu R的内吞作用也依赖于CRIP1a。对接分析和分子动力学模拟确定了CB R（S468）和mGlu R（S894）中的一个保守丝氨酸，该丝氨酸与CRIP1a第82位的肽主链形成氢键。与mGlu R不同，密切相关的mGlu R剪接变体在该位置携带赖氨酸（K894），实际上，mGlu R的内吞作用不受CRIP1a影响。CB R、mGlu R和mGlu R之间的嵌合构建体强调了所鉴定的五个对CRIP1a敏感的氨基酸的作用。总之，我们认为CRIP1a负向调节两种不同类型G蛋白偶联受体CB R和mGlu R的内吞作用。
