Adhesion is a crucial characteristic of epithelial cells to form barriers to pathogens and toxic substances from the environment. Epithelial cells attach to each other using intercellular junctions on the lateral membrane, including tight and adherent junctions, as well as the Na⁺,K⁺-ATPase. Our group has shown that non-adherent chinese hamster ovary (CHO) cells transfected with the canine β₁ subunit become adhesive, and those homotypic interactions amongst β₁ subunits of the Na⁺,K⁺-ATPase occur between neighboring epithelial cells. Ouabain, a cardiotonic steroid, binds to the α subunit of the Na⁺,K⁺-ATPase, inhibits the pump activity and induces the detachment of epithelial cells when used at concentrations above 300 nM. At nanomolar non-inhibiting concentrations, ouabain affects the adhesive properties of epithelial cells by inducing the expression of cell adhesion molecules through the activation of signaling pathways associated with the α subunit. In this study, we investigated whether the adhesion between β₁ subunits was also affected by ouabain. We used CHO fibroblasts stably expressing the β₁ subunit of the Na⁺,K⁺-ATPase (CHO β₁), and studied the effect of ouabain on cell adhesion. Aggregation assays showed that ouabain increased the adhesion between CHO β₁ cells. Immunofluorescence and biotinylation assays showed that ouabain (50 nM) increases the expression of the β₁ subunit of the Na⁺,K⁺-ATPase at the cell membrane. We also examined the effect of ouabain on the activation of signaling pathways in CHO β₁ cells, and their subsequent effect on cell adhesion. We found that cSrc is activated by ouabain and, therefore, that it likely regulates the adhesive properties of CHO β₁ cells. Collectively, our findings suggest that the β₁ subunit adhesion is modulated by the expression levels of the Na⁺,K⁺-ATPase at the plasma membrane, which is regulated by ouabain.

The β₂ subunit of Na⁺, K⁺-ATPase was originally identified as the adhesion molecule on glia (AMOG) that mediates the adhesion of astrocytes to neurons in the central nervous system and that is implicated in the regulation of neurite outgrowth and neuronal migration. While β₁ isoform have been shown to trans-interact in a species-specific mode with the β₁ subunit on the epithelial neighboring cell, the β₂ subunit has been shown to act as a recognition molecule on the glia. Nevertheless, none of the works have identified the binding partner of β₂ or described its adhesion mechanism. Until now, the interactions pronounced for β₂/AMOG are heterophilic cis-interactions. In the present report we designed experiments that would clarify whether β₂ is a cell–cell homophilic adhesion molecule. For this purpose, we performed protein docking analysis, cell–cell aggregation, and protein–protein interaction assays. We observed that the glycosylated extracellular domain of β₂/AMOG can make an energetically stable trans-interacting dimer. We show that CHO (Chinese Hamster Ovary) fibroblasts transfected with the human β₂ subunit become more adhesive and make large aggregates. The treatment with Tunicamycin in vivo reduced cell aggregation, suggesting the participation of N-glycans in that process. Protein–protein interaction assay in vivo with MDCK (Madin-Darby canine kidney) or CHO cells expressing a recombinant β₂ subunit show that the β₂ subunits on the cell surface of the transfected cell lines interact with each other. Overall, our results suggest that the human β₂ subunit can form trans-dimers between neighboring cells when expressed in non-astrocytic cells, such as fibroblasts (CHO) and epithelial cells (MDCK).

AMOG/β₂, the β₂ isoform of the sodium pump (Na⁺/K⁺-ATPase), functions as an adhesion molecule on glial cells, mediating critical neuron–astrocyte interactions during central nervous system (CNS) development. Despite its established role in glial adhesion, the neuronal receptor that partners with AMOG/β₂ remains unknown. This review examines the structural and functional properties of AMOG/β₂, including its capacity to form trans-dimers, both homophilic and potentially heterophilic—drawing comparisons with the β₁ subunit, a well-characterized adhesion molecule. By integrating computational modeling, in vitro data, and structural predictions, we explore how factors such as N-glycosylation and cis-membrane interactions influence β₂-mediated adhesion. We further consider candidate neuronal partners, including TSPAN31 and RTN4, and speculate on their potential roles in mediating heterophilic AMOG/β₂ interactions. Finally, we discuss the broader implications of AMOG/β₂ in neuron–glia communication, synaptic organization, neurodevelopment, and CNS disorders such as glioblastoma. Identifying the binding partner of AMOG/β₂ holds promise not only for understanding the molecular basis of CNS adhesion but also for uncovering novel mechanisms of neuroglial regulation in health and disease.

AMOG/β2, the β2 isoform of the sodium pump (Na⁺,K⁺-ATPase), functions as an adhesion molecule on glial cells, mediating critical neuron–astrocyte interactions during central nervous system (CNS) development. Despite its established role in glial adhesion, the neuronal receptor that partners with AMOG/β2 remains unknown. This review examines the structural and functional properties of AMOG/β2 including its capacity to form trans dimers, both homophilic and potentially heterophilic—drawing comparisons with the β1 subunit, a well-characterized adhesion molecule. By integrating computational modeling, in vitro data, and structural predictions, we explore how factors such as N-glycosylation and cis-membrane interactions influence β2-mediated adhesion. We further consider candidate neuronal partners, including TSPAN31 and RTN4, and speculate on their potential roles in mediating heterophilic AMOG/β2 interactions. Finally, we discuss the broader implications of AMOG/β2 in neuron-glia communication, synaptic organization, neurodevelopment, and CNS disorders such as glioblastoma. Identifying the binding partner of AMOG/β2 holds promise not only for understanding the molecular basis of CNS adhesion but also for uncovering novel mechanisms of neuroglial regulation in health and disease.