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. 2020 Apr;10(4):e01587.
doi: 10.1002/brb3.1587. Epub 2020 Feb 28.

Comparison of SynCAM1/CADM1 PDZ interactions with MUPP1 using mammalian and bacterial pull-down systems

Martina Baliova  1 , Frantisek Jursky  1
Affiliations

Comparison of SynCAM1/CADM1 PDZ interactions with MUPP1 using mammalian and bacterial pull-down systems

Martina Baliova et al. Brain Behav. 2020 Apr.

Abstract

Background: 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).

Methods: 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.

Results: 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.

Conclusions: 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.

Keywords: MUPP1; PDZ; SynCAM1/CADM1; protein interaction; pull-down assay.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Interaction of the CBD‐EKKEYFI fusion protein containing the CADM1 PDZ motif (EKKEYFI) with GST proteins fused with MUPP1 PDZ domains 1–13 (GST‐MP‐PDZ) (a). Interaction of the GST protein containing the whole cytosolic C‐terminal region of CADM1 (GST‐CADM1C) with the CBD protein fused in‐frame with certain MUPP1 PDZ domains (CBD‐MP‐PDZ) (b). Part c shows the end of the CADM1 protein aligned with the GST‐CADM1C fusion protein used in the pull‐down assay (c). The positions of the transmembrane domain, FERM binding 4.1B domain, PDZ binding motif, and the GST fusion tag are highlighted
Figure 2
Figure 2
Stabilization of the GST fusion protein interaction with glutathione sepharose with certain PDZ interactions of the CBD‐EKKEYFI protein. The glutathione elution efficiency of GST fusion proteins containing domains 2, 3, and 4 decreased following interaction with the CBD‐EKKEYFI protein (a, left) when compared to the domains that previously interacted with only the interacting buffer (a, right). The relative elution efficiency is quantified in (b). The elution deficiency of domain 2 was absent when the interacting protein CBD‐EKKEYFI was truncated to CBD‐EKKE (c). A comparison of glutathione‐ (GSH) and SDS‐eluted samples suggests that the elution deficiency was caused by the stabilization of the fusion protein on the glutathione resin (d). A similar stabilization effect is observed when the whole N‐terminal domain, including the first five domains of MUPP1 (GST‐PDZ1‐5), is used (e). The effect is dependent on the presence of the ‐YFI PDZ motif. The PDZ interaction and stabilization effect of several domains is supported by its presence in the construct GST‐PDZ1‐5‐M2 with mutated PDZ domain 2. Filled and open arrows indicate the position of CBD‐EKKEYFI and CBD‐EKKE proteins
Figure 3
Figure 3
The interaction of the CADM1 PDZ motif containing the protein GlyT2Nrcf1‐EKKEYFI (marked by filled arrow), with GST proteins fused to the MUPP1 PDZ domains (a). The interaction was not observed when the GLG motif in PDZ domain 2 was replaced with GRG (marked as ‐PDZ2‐M2) (b). Similarly, the YFI‐deleted GlyT2Nrcf1‐EKKE protein did not interact when the whole N‐terminal domain, including the first five domains of MUPP1 (GST‐PDZ1‐5), was used for the interaction (c). On the other site, GST‐PDZ1‐5‐M2, whose PDZ2 function was disrupted by GLG‐GRG mutation still interacted with GlyT2Nrcf1‐EKKEYFI protein, supporting its interaction with other domains which is part of the GST‐PDZ1‐5‐M2 protein (c). The filled arrow indicates the position of both GlyT2Nrcf1‐EKKEYFI and GlyT2Nrcf1‐EKKE proteins
Figure 4
Figure 4
Comparison of the PDZ interaction of the MUPP1 domains with the CADM1 EKKEYFI PDZ motif fused with the CBD protein and the GlyT2Nrcf1 protein. Note that while the specificity of the interaction with MUPP1 domains is identical for both protein fusion partners, the interaction affinity differs significantly when the highly structured CBD fusion partner is replaced by the unstructured GlyT2Nrcf1 fusion partner. The samples were eluted with SDS buffer to prevent the observed effect of different elution efficiencies of glutathione. Data represent the mean ± SEM of three independent experiments
Figure 5
Figure 5
Interaction of Triton X‐100‐solubilized synaptosomal CADM1 with MUPP1 PDZ domains 1–6. GST fusion proteins fused in‐frame with PDZ domains 1–6 immobilized on glutathione resin interacted with the Triton X‐100 extract of mouse synaptosomes. Following washing, interaction complexes were eluted with 20 mM glutathione, resolved on 7.5% polyacrylamide gel, and immunoblotted with anti‐CADM1 antibodies (a). GST fusion proteins were visualized with anti‐GST antibodies (b)
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