Direct demonstration of NCAMcis-dimerization and inhibitory effect of palmitoylation using the BRET²technique

Abstract

a b s t r a c tBiological activity of the neural cell adhesion molecule (NCAM) depends on both adhesion and activation of intra-cellular signaling. Based on in vitro experiments with truncated extra-cellular domains, several models describing homophilic NCAM trans-and cis-interactions have been proposed. However, cis-dimerization in living cells has not been shown directly and the role of the cytoplasmic part in NCAM dimerization is poorly understood. Here, we used the bioluminescence resonance energy transfer (BRET 2 ) technique to directly demonstrate that full-length NCAM cishomodimerizes in living cells. Based on BRET 2 50 values we suggest that the intra-cellular part of NCAM inhibits cis-dimerization, an effect mainly dependent on the palmitoylation sites.

Structure of human ST8SiaIII sialyltransferase provides insight into cell-surface polysialylation

“…Alternatively, dimerization of ST8SiaIII may be required for specific binding of cognate and potentially oligomerized acceptor proteins. Interestingly, although ST8SiaIV appears to be monomeric in solution 33 , its major acceptor substrate NCAM has been shown to cisdimerize along its extracellular domain to facilitate trans interactions between cells 34 . The cisdimeric state might be affected by oligo or polySia modifications, owing to their antiadhesive properties 33 .…”

What does S‐palmitoylation do to membrane proteins?

“…Palmitoylation, however, can also negatively affect protein multimerization as was shown for the neural cell adhesion molecule 140 (NCAM140) that is essential for axonal growth. DHHC7‐dependent palmitoylation of NCAM140 leads to raft association but negatively regulates the hemophilic dimerization of extracellular NCAM domains in cis . Interestingly, neuritogenesis requires interplay between NCAMs and fibroblast growth factor receptor signaling, and fibroblast growth factor leads to the activation of DHHC7 that in turn enhances NCAM palmitoylation , further promoting axonal growth .…”

Setting Up a Bioluminescence Resonance Energy Transfer High throughput Screening Assay to Search for Protein/Protein Interaction Inhibitors in Mammalian Cells

“…Indeed, BRET superiority was shown by its ability to monitor PPI using endogenous level of protein expression (Couturier and Jockers, 2003; Pfleger and Eidne, 2003) and its consequent application to various live cell screening assays (Pfleger et al, 2007; Bacart et al, 2008; Kocan and Pfleger, 2011). Finally, using this method to screen for P2I2 is further supported as BRET is prone to disruption or modulation by co-expression of untagged interacting partner (Bacart et al, 2008; Ayoub and Pfleger, 2010; Kulahin et al, 2011) and by incubation with inhibitory peptides (Granier et al, 2004; Harikumar et al, 2006; Jarry et al, 2010) or inhibitory chemical compounds (Mazars and Fåhraeus, 2010; Corbel et al, 2011). …”