Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden.
Biochem Biophys Res Commun. 2010 Nov 26;402(4):801-7. doi: 10.1016/j.bbrc.2010.10.122. Epub 2010 Oct 30.
A single serine point mutation (S374A) in the adenosine A(2A) receptor (A(2A)R) C-terminal tail reduces A(2A)R-D(2)R heteromerization and prevents its allosteric modulation of the dopamine D(2) receptor (D(2)R). By means of site directed mutagenesis of the A(2A)R and synthetic transmembrane (TM) α-helix peptides of the D(2)R we further explored the role of electrostatic interactions and TM helix interactions of the A(2A)R-D(2)R heteromer interface. We found evidence that the TM domains IV and V of the D(2)R play a major role in the A(2A)R-D(2)R heteromer interface since the incubation with peptides corresponding to these domains significantly reduced the ability of A(2A)R and D(2)R to heteromerize. In addition, the incubation with TM-IV or TM-V blocked the allosteric modulation normally found in A(2A)R-D(2)R heteromers. The mutation of two negatively charged aspartates in the A(2A)R C-terminal tail (D401A/D402A) in combination with the S374A mutation drastically reduced the physical A(2A)R-D(2)R interaction and lost the ability of antagonistic allosteric modulation over the A(2A)R-D(2)R interface, suggesting further evidence for the existence of an electrostatic interaction between the C-terminal tail of A(2A)R and the intracellular loop 3 (IL3) of D(2)R. On the other hand, molecular dynamic model and bioinformatic analysis propose that specific AAR, AQE, and VLS protriplets as an important motive in the A(2A)R-D(2L)R heteromer interface together with D(2L)R TM segments IV/V interacting with A(2A)R TM-IV/V or TM-I/VII.
腺嘌呤 A2A 受体（A2AR）C 端尾部的单个丝氨酸点突变（S374A）可减少 A2AR-D2R 异源二聚体的形成，并阻止其对多巴胺 D2 受体（D2R）的变构调节。通过 A2AR 的定点突变和 D2R 的合成跨膜（TM）α螺旋肽，我们进一步探讨了 A2AR-D2R 异源二聚体界面静电相互作用和 TM 螺旋相互作用的作用。我们发现，D2R 的 TM 结构域 IV 和 V 在 A2AR-D2R 异源二聚体界面中起着重要作用，因为与这些结构域相对应的肽的孵育显著降低了 A2AR 和 D2R 异源二聚体的能力。此外，TM-IV 或 TM-V 的孵育阻断了通常在 A2AR-D2R 异源二聚体中发现的变构调节。在 A2AR C 端尾部（D401A/D402A）中的两个带负电荷的天冬氨酸突变与 S374A 突变相结合，大大降低了 A2AR-D2R 的物理相互作用，并失去了在 A2AR-D2R 界面上对抗性变构调节的能力，进一步证明了 A2AR C 端尾部和 D2R 细胞内环 3（IL3）之间存在静电相互作用。另一方面，分子动力学模型和生物信息学分析表明，特定的 AAR、AQE 和 VLS 三联体与 D2R 的 TM 片段 IV/V 一起作为 A2AR-D2L 异源二聚体界面中的一个重要动力，与 A2AR 的 TM-IV/V 或 TM-I/VII 相互作用。
