Laboratory of Neurobiology, Institute of Molecular Biology, Slovak Academy of Sciences, Bratislava, Slovakia.
Brain Behav. 2020 Apr;10(4):e01587. doi: 10.1002/brb3.1587. Epub 2020 Feb 28.
Synaptic cell adhesion molecule 1 (SynCAM1) also known as cell adhesion molecule 1 (CADM1) is a transmembrane cell adhesion protein that operates in a variety of physiological and pathological cellular contexts, and its interaction with the PDZ signalling protein MUPP1 have been previously implicated in autism spectrum disorder (ASD).
We used in vitro pull-down systems based on the bacterial and mammalian extracts to study SynCAM1/CADM1 PDZ interactions with MUPP1 at various conditions.
So far, the investigated interaction of SynCAM1/CADM1 with MUPP1 has been mostly attributed to an unspecified region of MUPP1 PDZ domains 1-5 or exclusively to domain 2, using a yeast two-hybrid system. We also confirmed the single interaction of native synaptosomal CADM1 with PDZ domain 2. However, in this work, using recombinant proteins overexpressed in bacteria, we found an in vitro pull-down conditions in which all first five domains and, to a much lesser extent, MUPP1 domains 7 and 11 significantly interacted with the whole C-terminal domain of SynCAM1/CADM1. These PDZ interactions were confirmed by a pull-down assay using the last seven amino acids of the SynCAM1/CADM1 PDZ motif and using two fusion partners. Multiple interactions were additionally replicated using the continuous N-terminal MUPP1 protein fragment, which included first five PDZ domains, containing either intact or mutated domain 2.
We hypothesize that multiple interactions might exist in vivo, representing transient low-affinity interactions or alternative binding sites on MUPP1 when domain 2 is occupied or occluded by the interaction with other ligands. This newly identified interactions extend the potential genetic mutations, possibly affecting SynCAM1/CADM1/MUPP1 function. Possible reasons for the absence of some of the identified CADM1 PDZ interactions in mammalian extracts are discussed.
突触细胞粘附分子 1（SynCAM1）也称为细胞粘附分子 1（CADM1），是一种跨膜细胞粘附蛋白，在多种生理和病理细胞环境中发挥作用，其与 PDZ 信号蛋白 MUPP1 的相互作用先前已被牵连到自闭症谱系障碍（ASD）中。
我们使用基于细菌和哺乳动物提取物的体外下拉系统，在各种条件下研究 SynCAM1/CADM1 与 MUPP1 的 PDZ 相互作用。
到目前为止，使用酵母双杂交系统，已经研究了 SynCAM1/CADM1 与 MUPP1 的相互作用，主要归因于 MUPP1 PDZ 结构域 1-5 的未指定区域，或者专门归因于结构域 2。我们还证实了天然突触 CADM1 与 PDZ 结构域 2 的单一相互作用。然而，在这项工作中，使用在细菌中过表达的重组蛋白，我们发现了一种体外下拉条件，其中所有前五个结构域，并且在较小程度上，MUPP1 结构域 7 和 11 与 SynCAM1/CADM1 的整个 C 端结构域显著相互作用。这些 PDZ 相互作用通过下拉测定得到证实，该测定使用 SynCAM1/CADM1 PDZ 基序的最后七个氨基酸，并使用两个融合伴侣。使用包含前五个 PDZ 结构域的连续 N 端 MUPP1 蛋白片段，包括完整或突变的结构域 2，复制了多种相互作用。
我们假设，在体内可能存在多种相互作用，代表当结构域 2 被与其他配体的相互作用占据或封闭时，瞬时低亲和力相互作用或替代的 MUPP1 结合位点。这种新发现的相互作用扩展了可能影响 SynCAM1/CADM1/MUPP1 功能的潜在遗传突变。讨论了在哺乳动物提取物中缺失一些鉴定出的 CADM1 PDZ 相互作用的可能原因。
