Decreased Anxiety-Related Behaviour but Apparently Unperturbed NUMB Function in Ligand of NUMB Protein-X (LNX) 1/2 Double Knockout Mice

Abstract

Type of publicationArticle (peer-reviewed) AbstractNUMB is a key regulator of neurogenesis and neuronal differentiation that can be ubiquitinated and targeted for proteasomal degradation by ligand of numb proteinX (LNX) family E3 ubiquitin ligases. However, our understanding of LNX protein function in vivo is very limited. To examine the role of LNX proteins in regulating NUMB function in vivo, we generated mice lacking both LNX1 and LNX2 expression in the brain. Surprisingly, these mice are healthy, exhibit unaltered levels of NUMB protein and do not display any neuroanatomical defects indicative of aberrant NUMB function. Behavioural analysis of LNX1/LNX2 double knockout mice revealed decreased anxiety related behaviour, as assessed in the open field and elevated plus maze paradigms. By contrast, no major defects in learning, motor or sensory function were observed. Given the apparent absence of major NUMB dysfunction in LNX null animals, we performed a proteomic analysis to identify neuronal LNXinteracting proteins other than NUMB that might contribute to the anxiolytic phenotype observed. We identified and/or confirmed interactions of LNX1 and LNX2 with proteins known to have presynaptic and neuronal signalling functions, including the presynaptic active zone constituents ERC1, ERC2 and LIPRINαs (PPFIA1, PPFIA3), as well as the FBAR domain proteins FCHSD2 (nervous wreck homologue) and SRGAP2. These and other novel LNXinteracting proteins identified are promising candidates to mediate LNX functions in the central nervous system, including their role in modulating anxietyrelated behaviour.

E3 ubiquitin ligases LNX1 and LNX2 localize at neuronal gap junctions formed by connexin36 in rodent brain and molecularly interact with connexin36

“…Comparison of results with anti‐LNX1 ab200 versus ab247 after immunoblotting of these same lysates is shown in Figure b, which indicated that each of these antibodies have the capacity to detect both the full‐length isoform LNX1p80 and the shorter isoform LNX1p70. Immunoblotting of LNX1 and LNX2 in homogenates of brain is not shown due to difficulties in detection of these LNX isoforms in brain extracts, as also reported by others (Lenihan, Saha, Heimer‐McGinn, et al., ).…”

Ubiquitin ligase LNX1 is a major regulator of glycine recapture by the presynaptic transporter GlyT2

“…GlyT2 has been previously shown to interact with several presynaptic proteins, including syntaxin1 16 , Plasma Membrane Calcium ATPases PMCA2 and PMCA3 18 and sodium/potassium ATPase subunits α3 (α3NKA) and β2 (β2NKA) 17 . It is possible that LNX1 may act as a scaffold to facilitate the interaction of GlyT2 with the mentioned proteins, and this idea of a GlyT2-LNX1-PMCA/NKA multimolecular complex is supported by a recent proteomic study that found PMCA2 and β2NKA as candidate interacting partners of LNX1 32 .…”

“…This modular domain organization, unique to the LNX family, suggests that LNX members have a facility for ubiquitinating substrates containing protein binding motifs (PBM), which are recognized specifically through the different PDZ domains. LNX1 has been shown to localize to axons and presynaptic terminals 31 and it interacts with a set of presynaptic proteins including CAST 31 , PKCα 30,59 , ERC1, ERC2 and LIPRIN-αs 32 . LNX1 also interacts with NUMB 26,60 and c-Src 29 , which are also expressed in presynaptic terminals 61,62 , and it can ubiquitinate these substrates to target them for degradation 26,29,30 .…”

“…LNX1 presents two main isoforms, p70 and p80, with p70 not containing a RING-finger domain. LNX1 is mainly expressed in the nervous system 28 and presents four consecutive PDZ domains in its structure, which promote its interaction with many other neuronal substrates such as c-Src 29 , PKCα 30 or the presynaptic active zone proteins CAST 31 , ERC1, ERC2 and LIPRIN-αs 32 . Previous efforts to identify potential LNX1 substrates have used proteomic approaches to identify PDZ binding partners for each of the PDZ domains in LNX1 33,34 .…”

LNX1/LNX2 proteins: functions in neuronal signalling and beyond

“…The motions induced within the PDZ domain by the electric-field were postulated to reflect the conformational changes associated with ligand binding. Further analysis of these motions may provide insights into the mechanism of ligand binding by this PDZ domain and through comparison with the LNX1 PDZ2 structure may elucidate the structural basis for the differential binding of certain ligands to PDZ2 from LNX1 compared with LNX2 [13,14].…”