Institute for Human Genetics, University Medical Center Hamburg Eppendorf, Martinistrasse 52, 20246, Hamburg, Germany.
Institute of Integrative Biology, University of Liverpool, Liverpool, UK.
Mol Neurobiol. 2024 Feb;61(2):693-706. doi: 10.1007/s12035-023-03611-5. Epub 2023 Sep 1.
Members of the Shank family of postsynaptic scaffold proteins (Shank1-3) link neurotransmitter receptors to the actin cytoskeleton in dendritic spines through establishing numerous interactions within the postsynaptic density (PSD) of excitatory synapses. Large Shank isoforms carry at their N-termini a highly conserved domain termed the Shank/ProSAP N-terminal (SPN) domain, followed by a set of Ankyrin repeats. Both domains are involved in an intramolecular interaction which is believed to regulate accessibility for additional interaction partners, such as Ras family G-proteins, αCaMKII, and cytoskeletal proteins. Here, we analyze the functional relevance of the SPN-Ank module; we show that binding of active Ras or Rap1a to the SPN domain can differentially regulate the localization of Shank3 in dendrites. In Shank1 and Shank3, the linker between the SPN and Ank domains binds to inactive αCaMKII. Due to this interaction, both Shank1 and Shank3 exert a negative effect on αCaMKII activity at postsynaptic sites in mice in vivo. The relevance of the SPN-Ank intramolecular interaction was further analyzed in primary cultured neurons; here, we observed that in the context of full-length Shank3, a closed conformation of the SPN-Ank tandem is necessary for proper clustering of Shank3 on the head of dendritic spines. Shank3 variants carrying Ank repeats which are not associated with the SPN domain lead to the atypical formation of postsynaptic clusters on dendritic shafts, at the expense of clusters in spine-like protrusions. Our data show that the SPN-Ank tandem motif contributes to the regulation of postsynaptic signaling and is also necessary for proper targeting of Shank3 to postsynaptic sites. Our data also suggest how missense variants found in autistic patients which alter SPN and Ank domains affect the synaptic function of Shank3.
突触后支架蛋白 Shank 家族成员（Shank1-3）通过在兴奋性突触的突触后密度（PSD）内建立许多相互作用，将神经递质受体与树突棘中的肌动蛋白细胞骨架连接起来。大 Shank 同种型在其 N 端携带一个高度保守的结构域，称为 Shank/ProSAP N 端（SPN）结构域，后面是一组锚蛋白重复序列。这两个结构域都参与了一个分子内相互作用，据信这种相互作用调节了其他相互作用伙伴（如 Ras 家族 G 蛋白、αCaMKII 和细胞骨架蛋白）的可及性。在这里，我们分析了 SPN-Ank 模块的功能相关性；我们表明，活性 Ras 或 Rap1a 与 SPN 结构域的结合可以差异调节 Shank3 在树突中的定位。在 Shank1 和 Shank3 中，SPN 和 Ank 结构域之间的接头与非活性 αCaMKII 结合。由于这种相互作用，Shank1 和 Shank3 在体内的小鼠突触后部位对 αCaMKII 活性均产生负向影响。我们进一步在原代培养神经元中分析了 SPN-Ank 分子内相互作用的相关性；在这里，我们观察到在全长 Shank3 的情况下，SPN-Ank 串联的封闭构象对于 Shank3 在树突棘头部的正确聚类是必要的。与 SPN 结构域不相关的 Shank3 变体携带的锚蛋白重复序列导致突触后簇在树突干上的异常形成，而不是在棘状突起上的簇。我们的数据表明，SPN-Ank 串联基序有助于调节突触后信号转导，对于 Shank3 正确靶向突触后部位也是必要的。我们的数据还表明，在自闭症患者中发现的改变 SPN 和 Ank 结构域的错义变异如何影响 Shank3 的突触功能。
