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

Abstract

Electrical synapses in the mammalian central nervous system (CNS) are increasingly recognized as highly complex structures for mediation of neuronal communication, both with respect to their capacity for dynamic short‐ and long‐term modification in efficacy of synaptic transmission and their multimolecular regulatory and structural components. These two characteristics are inextricably linked, such that understanding of mechanisms that contribute to electrical synaptic plasticity requires knowledge of the molecular composition of electrical synapses and the functions of proteins associated with these synapses. Here, we provide evidence that the key component of gap junctions that form the majority of electrical synapses in the mammalian CNS, namely connexin36 (Cx36), directly interacts with the related E3 ubiquitin ligase proteins Ligand of NUMB protein X1 (LNX1) and Ligand of NUMB protein X2 (LNX2). This is based on immunofluorescence colocalization of LNX1 and LNX2 with Cx36‐containing gap junctions in adult mouse brain versus lack of such coassociation in LNX null mice, coimmunoprecipitation of LNX proteins with Cx36, and pull‐down of Cx36 with the second PDZ domain of LNX1 and LNX2. Furthermore, cotransfection of cultured cells with Cx36 and E3 ubiquitin ligase‐competent LNX1 and LNX2 isoforms led to loss of Cx36‐containing gap junctions between cells, whereas these junctions persisted following transfection with isoforms of these proteins that lack ligase activity. Our results suggest that a LNX protein mediates ubiquitination of Cx36 at neuronal gap junctions, with consequent Cx36 internalization, and may thereby contribute to intracellular mechanisms that govern the recently identified modifiability of synaptic transmission at electrical synapses.

Regulation of gap junction intercellular communication by connexin ubiquitination: physiological and pathophysiological implications

“…Through pull-down assays, LNX1 and LNX2 were found to directly interact with Cx36 via its carboxy terminus. Co-transfection of N2A cells with Cx36-eCFP and LNX1 caused a strong loss of Cx36-eCFP-containing gap junctions, whereas co-transfection with Cx36-eCFP and an LNX1 mutant that lacked E3 ubiquitin ligase activity did not affect Cx36-eCFP gap junction levels [125]. A similar loss of gap junctions was observed in response to overexpression of LNX2, whereas a catalytically inactive form of LNX2 did not affect gap junction levels.…”

“…In addition to the above-mentioned E3 ubiquitin ligases, another member of the NEDD4 family, WWP1, has been shown to regulate Cx43 gap junctions in cardiomyocytes [124] and the E3 ubiquitin ligases LNX1 and -2 (ligand of NUMB Protein-X1 and -2) were recently found to have important roles in controlling the level of Cx36 gap junctions in the central nervous system [125], as is further discussed below.…”

“…In addition to the RING domain, LNX1 and -2 contain four PDZ domains through which they interact with their substrate proteins [159,160]. Recently, Nagy and colleagues identified LNX1 and -2 as important regulators of Cx36 gap junctions in neurons [125]. LNX1 and LNX2 were found to partly colocalize with neuronal gap junctions formed by Cx36 in rodent brain, as well as in N2A cells transfected with Cx36enhanced cyan fluorescence protein (Cx36-eCFP).…”

Role of Connexins 30, 36, and 43 in Brain Tumors, Neurodegenerative Diseases, and Neuroprotection

“…In rodent brains, E3 ubiquitin ligases LNX1 and LNX2 localize at neuronal GJ formed by Cx36 [106]. Similarly, in the neuroblastoma cell line N2A transfected with a fluorescently labeled Cx36, both LNX1 and LNX2 interact with the Cx36 CT [106]. A significant reduction in neuronal Cx36 GJ was observed as a response to Cx36 ubiquitination.…”

“…Similarly, lysine residues can be post-translationally modified by the small ubiquitin-like modifier (SUMO) family of proteins that are involved in multiple cellular processes like transcription, translation, cellular transport, cell growth, and programmed death [105]. In rodent brains, E3 ubiquitin ligases LNX1 and LNX2 localize at neuronal GJ formed by Cx36 [106]. Similarly, in the neuroblastoma cell line N2A transfected with a fluorescently labeled Cx36, both LNX1 and LNX2 interact with the Cx36 CT [106].…”