Data are means SEM of six independent experiments. analysis show that deletion of neither the N- nor the C-terminal intracellular domains interferes with the recognition of Gpm6a by the function-blocking antibody directed against the extracellular part of Gpm6a. Expression levels of both truncation mutants were not A-769662 affected but we observed decrease in the amount of both truncated proteins on cell surface suggesting that the incapacity of the Gpm6a lacking C-terminus to induce filopodium formation is not due to the lower amount of Gpm6a on cell surface. Following colocalization assays shows that deletion of the C- but not the N-terminus diminishes the association of Gpm6a with clathrin implying involvement of clathrin-mediated trafficking events. Next, using comprehensive alanine scanning A-769662 mutagenesis of the C-terminus we identify K250, K255, and E258 as the key residues for the formation of filopodia by Gpm6a. Substitution of these charged residues with alanine also diminishes the amount of Gpm6a on cell surface and in case of K255 and E258 leads to the lower amount of total expressed protein. Subsequent bioinformatic analysis of Gpm6a amino acid sequence reveals that highly conserved and functional residues cluster preferentially within the C- and not within the N-terminus and that K250, K255, and E258 are predicted as part of sorting signals of transmembrane proteins. Altogether, our results provide evidence that filopodium outgrowth induced by Gpm6a requires functionally critical residues within the C-terminal cytoplasmic tail. expression levels or sequence. Downregulation of mRNA levels has been shown in the hippocampus of depressed suicide victims (Fuchsova et al., 2015) and the association of gene with schizophrenia (Boks et al., Rabbit Polyclonal to ZP4 2008; Ma et al., 2018), bipolar disorders (Greenwood et al., 2012), and claustrophobia (El-Kordi et al., 2013) has been described. On the other hand, duplication of gene leading to the higher expression of has been connected to learning disability and anomalies in the behavior (Gregor et al., 2014) suggesting the importance of accurate expression of for cognitive function. In several animal models, chronic stress, an agent critically involved in the etiology of depression, alters expression levels of Gpm6a and this effect is counteracted by treatment with antidepressants (Alfonso et al., 2004a,b; Cooper et al., 2009; Monteleone et al., 2014). The roles of Gpm6a in the nervous system are incompletely comprehended. However, there is abundant evidence for its participation in filopodium formation, neurite extension, synaptogenesis (Lagenaur et al., 1992; Mukobata et al., 2002; Alfonso et al., 2005; Michibata et al., 2008; Zhao et al., 2008; Fuchsova et al., 2009; Brocco et al., 2010; Huang et al., 2011; Scorticati et al., 2011; Formoso et al., 2015; Mita et al., 2015), neuronal differentiation of human stem cells (Michibata et al., 2008) and PC12 cells (Mukobata et al., 2002), as well as in determination of neuronal polarity during neurite formation in neuronal development (Honda et al., 2017; Ito et al., 2018). In addition, Gpm6a has been shown to interact with the micro-opioid receptor [and with a number of other G protein-coupled receptors (GPCRs)] and to facilitate receptor endocytosis and recycling (Wu et al., 2007; Liang et al., 2008). Endocytic sorting and recycling of Gpm6a A-769662 involves clathrin-dependent pathway and affects neuronal synapses (Garcia et al., 2017). Overexpression of Gpm6a in rat hippocampal neurons or in cells of neuronal (N2a, PC12) as well as non-neuronal (COS7) origin leads to the vast formation of filopodia, while decrease of endogenous Gpm6a expression by siRNA reduces filopodium density (Alfonso et al., 2005). Filopodia are slender protrusions of plasma membrane filled with actin filaments that underlie many major morphogenetic events in the nervous system (Mattila and Lappalainen, 2008; Gallo, 2013). They are required to initiate extension of neurites and their ramification (Dent et al., 2007). They are also present in neuronal growth cones where they guide axons and dendrites (Gallo and Letourneau, 2004), and filopodia in dendrites function as precursors of spines (Sekino et al., 2007) that create postsynaptic regions of most excitatory synapses. The mechanism that mediates formation of filopodia by Gpm6a is not fully understood. The localization of Gpm6a in lipid microdomains as well as Src kinases and MAPK activity have been reported to participate (Scorticati et al., 2011). More recently, it has been shown that the actin regulator Coronin 1a associates with Gpm6a in rat hippocampal neurons and facilitates the formation of filopodia by Gpm6a and Rac1/Pak1 signaling pathway has been shown to be involved (Fuchsova et al., 2015; Alvarez Julia et al., 2016). In the present study our objective was to determine the regions of Gpm6a molecule that.