Abstract
Neurobeachin (Nbea) is a multidomain scaffold protein abundant in the brain, where it is highly expressed during development. Nbea-null mice have severe defects in neuromuscular synaptic transmission resulting in lethal paralysis of the newborns. Recently, it became clear that Nbea is important also for the functioning of central synapses, where it is suggested to play a role in trafficking membrane proteins to both, the pre- and post-synaptic sites. So far, only few binding partners of Nbea have been found and the precise mechanism of their trafficking remains unclear. Here, we used mass spectrometry to identify SAP102, a MAGUK protein implicated in trafficking of the ionotropic glutamate AMPA- and NMDA-type receptors during synaptogenesis, as a novel Nbea interacting protein in mouse brain. Experiments in heterologous cells confirmed this interaction and revealed that SAP102 binds to the C-terminal part of Nbea that contains the DUF, PH, BEACH and WD40 domains. Furthermore, we discovered that introducing a mutation in Nbea’s PH domain, which disrupts its interaction with the BEACH domain, abolishes this binding, thereby creating an excellent starting point to further investigate Nbea-SAP102 function in the central nervous system.
Highlights
Neurobeachin (Nbea), a large (327 kDa), brain-enriched, multidomain protein is essential for synaptic transmission [1,2,3]
We show that Synapse Associated Protein 102 (SAP102), a scaffolding protein that has been implicated in trafficking of AMPA and NMDA receptors during synaptogenesis [20], binds to Nbea’s C-terminal part, and that this encompasses the DUF1088, Pleckstrin-Homology like domain (PH), BEACH and WD40 domains
Since the Nbea KO mice present a lethal phenotype, we first carried out 3 IPs on embryonic day 18 (E18) brain homogenates
Summary
Neurobeachin (Nbea), a large (327 kDa), brain-enriched, multidomain protein is essential for synaptic transmission [1,2,3]. Nbea knock-out (KO) mice lack spontaneous and reflexive movement (i.e. movement elicited by tail pinch) and die immediately after birth due to their inability to breathe [1,2]. This primary asphyxia is probably the result of the absence of evoked neurotransmitter release at neuromuscular junctions [1]. Altered miniature excitatory and inhibitory postsynaptic currents were reported in cultured hippocampal neurons from KO mice and cortical slices from heterozygous mice, accompanied by reduced numbers of spine-localized synapses [3]. In KO neurons excitatory presynaptic terminals are mostly on dendritic shafts instead of on spine heads and actin in these synapses is less enriched [3]
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