Altered serotonin (5-HT) signaling is associated with multiple brain disorders, including major depressive disorder (MDD), obsessive-compulsive disorder (OCD), and autism spectrum disorder (ASD). The presynaptic, high-affinity 5-HT transporter (SERT) tightly regulates 5-HT clearance after release from serotonergic neurons in the brain and enteric nervous systems, among other sites. Accumulating evidence suggests that SERT is dynamically regulated in distinct activity states as a result of environmental and intracellular stimuli, with regulation perturbed by disease-associated coding variants. Our lab identified a rare, hypermorphic SERT coding substitution, Gly56Ala, in subjects with ASD, finding that the Ala56 variant stabilizes a high-affinity outward-facing conformation (SERT^∗) that leads to elevated 5-HT uptake in vitro and in vivo. Hyperactive SERT Ala56 appears to preclude further activity enhancements by p38α mitogen-activated protein kinase (MAPK) and can be normalized by pharmacological p38α MAPK inhibition, consistent with SERT Ala56 mimicking, constitutively, a high-activity conformation entered into transiently by p38α MAPK activation. We hypothesize that changes in SERT-interacting proteins (SIPs) support the shift of SERT into the SERT^∗ state which may be captured by comparing the composition of SERT Ala56 protein complexes with those of wildtype (WT) SERT, defining specific interactions through comparisons of protein complexes recovered using preparations from SERT^–/– (knockout; KO) mice. Using quantitative proteomic-based approaches, we identify a total of 459 SIPs, that demonstrate both SERT specificity and sensitivity to the Gly56Ala substitution, with a striking bias being a loss of SIP interactions with SERT Ala56 compared to WT SERT. Among this group are previously validated SIPs, such as flotillin-1 (FLOT1) and protein phosphatase 2A (PP2A), whose functions are believed to contribute to SERT microdomain localization and regulation. Interestingly, our studies nominate a number of novel SIPs implicated in ASD, including fragile X mental retardation 1 protein (FMR1) and SH3 and multiple ankyrin repeat domains protein 3 (SHANK3), of potential relevance to long-standing evidence of serotonergic contributions to ASD. Further investigation of these SIPs, and the broader networks they engage, may afford a greater understanding of ASD as well as other brain and peripheral disorders associated with perturbed 5-HT signaling.

血清素（5-HT）信号改变与多种脑部疾病有关，包括重度抑郁症（MDD），强迫症（OCD）和自闭症谱系障碍（ASD）。突触前的高亲和力5-HT转运蛋白（SERT）从大脑和肠神经系统中的血清素能神经元释放后，会紧紧调节5-HT的清除。越来越多的证据表明，由于环境和细胞内刺激，SERT在不同的活动状态下受到动态调节，并且受到与疾病相关的编码变体的干扰。我们的实验室在患有ASD的受试者中发现了一种罕见的，变态的SERT编码取代Gly56Ala，发现Ala56变体稳定了高亲和力的向外构象（SERT ^*），导致5-HT摄取增加 体外 和 体内。过度活跃的SERT Ala56似乎阻止了p38α丝裂原激活的蛋白激酶（MAPK）进一步的活性​​增强，并且可以通过药理上的p38αMAPK抑制作用进行标准化，这与SERT Ala56模仿性地构成为，通过p38αMAPK激活而暂时进入的高活性构象。我们假设SERT相互作用蛋白（SIP）的变化支持将SERT转变为SERT ^∗状态，这可以通过将SERT Ala56蛋白复合物与野生型（WT）SERT的复合物的组成进行比较，并通过比较使用来自SERT的制剂回收的蛋白质复合物^{– / –}（敲除； KO）小鼠。使用基于蛋白质组学的定量方法，我们确定了总共459个SIP，它们显示出SERT特异性和对Gly56Ala取代的敏感性，一个显着的偏差是与WT SERT相比，SIP与SERT Ala56的相互作用丧失了。在这一组中，先前已验证的SIP，例如弗洛林-1（FLOT1）和蛋白磷酸酶2A（PP2A），其功能被认为有助于SERT微域定位和调节。有趣的是，我们的研究提名了许多与ASD相关的SIP，包括脆弱的X智力低下1蛋白（FMR1）和SH3以及多个锚蛋白重复结构域蛋白3（SHANK3），这些可能与血清素能对ASD贡献的长期证据有关。对这些SIP及其参与的更广泛网络的进一步调查，