Abstract
BackgroundSynaptic 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).MethodsWe used in vitro pull‐down systems based on the bacterial and mammalian extracts to study SynCAM1/CADM1 PDZ interactions with MUPP1 at various conditions.ResultsSo 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.ConclusionsWe 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.
Highlights
| INTRODUCTIONThe members of the cell adhesion molecule (CADM) family are type I transmembrane proteins that have been independently identified by several investigators, and they have acquired several different names, such as Igsf (Ig-like spermatogenic factor; Gomyo et al, 1999), TSLC (tumor suppressor in lung cancer; Kuramochi et al, 2001), Necl (Nectin-like molecules; Takai, Irie, Shimizu, Sakisaka, & Ikeda, 2003), Ra175 (Urase, Soyama, Fujita, & Momoi, 2001), SgIGSF (spermatogenic immunoglobulin superfamily; Wakayama, Ohashi, Mizuno, & Iseki, 2001; Watabe, Ito, Koma, & Kitamura, 2003), and SynCAM (synaptic cell adhesion molecule; Biederer et al, 2002)
The members of the cell adhesion molecule (CADM) family are type I transmembrane proteins that have been independently identified by several investigators, and they have acquired several different names, such as Igsf4 (Ig-like spermatogenic factor; Gomyo et al, 1999), TSLC, Necl (Nectin-like molecules; Takai, Irie, Shimizu, Sakisaka, & Ikeda, 2003), Ra175 (Urase, Soyama, Fujita, & Momoi, 2001), SgIGSF, and SynCAM
We report the pull-down of Triton X-100 solubilized native synaptosomal cell adhesion molecule 1 (CADM1) protein interacting solely with MUPP1 domain 2
Summary
The members of the cell adhesion molecule (CADM) family are type I transmembrane proteins that have been independently identified by several investigators, and they have acquired several different names, such as Igsf (Ig-like spermatogenic factor; Gomyo et al, 1999), TSLC (tumor suppressor in lung cancer; Kuramochi et al, 2001), Necl (Nectin-like molecules; Takai, Irie, Shimizu, Sakisaka, & Ikeda, 2003), Ra175 (Urase, Soyama, Fujita, & Momoi, 2001), SgIGSF (spermatogenic immunoglobulin superfamily; Wakayama, Ohashi, Mizuno, & Iseki, 2001; Watabe, Ito, Koma, & Kitamura, 2003), and SynCAM (synaptic cell adhesion molecule; Biederer et al, 2002). C739A(H246N) and A755C(Y251S), were previously found in the CADM1 gene of male Caucasian ASD patients and their family members (Zhiling et al, 2008). These mutations impaired CADM1 function and altered synaptogenesis in neurons (Fujita et al, 2010), which underlies the possible involvement of CADM1 in certain conditions of ASD pathogenesis. The cytoplasmic tail is highly conserved and contains two short protein–protein interaction domains, namely a juxta membrane protein 4.1 binding motif (FERM domain binding) and a C-terminal type II PDZ binding motif (Biederer, 2006). We report the pull-down of Triton X-100 solubilized native synaptosomal CADM1 protein interacting solely with MUPP1 domain 2
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