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Cadherin family proteins play a central role in epithelial and endothelial cell-cell adhesion. The dynamic regulation of cell adhesion is achieved in part through endocytic membrane trafficking pathways that modulate cadherin cell surface levels. Here, we define the role for various MARCH family ubiquitin ligases in the regulation of cadherin degradation. We find that MARCH2 selectively downregulates VE-cadherin, resulting in loss of adherens junction proteins at cell borders and a loss of endothelial barrier function. Interestingly, N-cadherin is refractory to MARCH ligase expression, demonstrating that different classical cadherin family proteins are differentially regulated by MARCH family ligases. Using chimeric cadherins, we find that the specificity of different MARCH family ligases for different cadherins is conferred by the cadherin transmembrane domain. Further, juxta-membrane lysine residues are required for cadherin degradation by MARCH proteins. These findings expand our understanding of cadherin regulation and highlight a new role for mammalian MARCH family ubiquitin ligases in differentially regulating cadherin turnover., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Seo et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Cilia-related proteins are believed to be involved in a broad range of cellular processes. Retinitis pigmentosa GTPase regulator interacting protein 1-like (RPGRIP1L) is a ciliary protein required for ciliogenesis in many cell types, including epidermal keratinocytes. Here we report that RPGRIP1L is also involved in the maintenance of desmosomal junctions between keratinocytes. Genetically disrupting the Rpgrip1l gene in mice caused intraepidermal blistering, primarily between basal and suprabasal keratinocytes. This blistering phenotype was associated with aberrant expression patterns of desmosomal proteins, impaired desmosome ultrastructure, and compromised cell-cell adhesion in vivo and in vitro. We found that disrupting the RPGRIP1L gene in HaCaT cells, which do not form primary cilia, resulted in mislocalization of desmosomal proteins to the cytoplasm, suggesting a cilia-independent function of RPGRIP1L. Mechanistically, we found that RPGRIP1L regulates the endocytosis of desmogleins such that RPGRIP1L-knockdown not only induced spontaneous desmoglein endocytosis, as determined by AK23 labeling and biotinylation assays, but also exacerbated EGTA- or pemphigus vulgaris IgG-induced desmoglein endocytosis. Accordingly, inhibiting endocytosis with dynasore or sucrose rescued these desmosomal phenotypes. Biotinylation assays on cell surface proteins not only reinforced the role of RPGRIP1L in desmoglein endocytosis, but also suggested that RPGRIP1L may be more broadly involved in endocytosis. Thus, data obtained from this study advanced our understanding of the biological functions of RPGRIP1L by identifying its role in the cellular endocytic pathway., Competing Interests: The authors have declared that no competing interests exist.

Strong intercellular adhesion is critical for tissues that experience mechanical stress, such as the skin and heart. Desmosomes provide adhesive strength to tissues by anchoring desmosomal cadherins of neighboring cells to the intermediate filament cytoskeleton. Alterations in assembly and disassembly compromise desmosome function and may contribute to human diseases, such as the autoimmune skin blistering disease pemphigus vulgaris (PV). We previously demonstrated that PV auto-antibodies directed against the desmosomal cadherin desmoglein 3 (Dsg3) cause loss of adhesion by triggering membrane raft-mediated Dsg3 endocytosis. We hypothesized that raft membrane microdomains play a broader role in desmosome homeostasis by regulating the dynamics of desmosome assembly and disassembly. In human keratinocytes, Dsg3 is raft associated as determined by biochemical and super resolution immunofluorescence microscopy methods. Cholesterol depletion, which disrupts rafts, prevented desmosome assembly and adhesion, thus functionally linking rafts to desmosome formation. Interestingly, Dsg3 did not associate with rafts in cells lacking desmosomal proteins. Additionally, PV IgG-induced desmosome disassembly occurred by redistribution of Dsg3 into raft-containing endocytic membrane domains, resulting in cholesterol-dependent loss of adhesion. These findings demonstrate that membrane rafts are required for desmosome assembly and disassembly dynamics, suggesting therapeutic potential for raft targeting agents in desmosomal diseases such as PV.

Pemphigus vulgaris (PV) is an autoimmune epidermal blistering disease caused by autoantibodies directed against the desmosomal cadherin desmoglein-3 (Dsg3). Significant advances in our understanding of pemphigus pathomechanisms have been derived from the generation of pathogenic monoclonal Dsg3 antibodies. However, conflicting models for pemphigus pathogenicity have arisen from studies using either polyclonal PV patient IgG or monoclonal Dsg3 antibodies. In the present study, the pathogenic mechanisms of polyclonal PV IgG and monoclonal Dsg3 antibodies were directly compared. Polyclonal PV IgG cause extensive clustering and endocytosis of keratinocyte cell surface Dsg3, whereas pathogenic mouse monoclonal antibodies compromise cell-cell adhesion strength without causing these alterations in Dsg3 trafficking. Furthermore, tyrosine kinase or p38 MAPK inhibition prevents loss of keratinocyte adhesion in response to polyclonal PV IgG. In contrast, disruption of adhesion by pathogenic monoclonal antibodies is not prevented by these inhibitors either in vitro or in human skin explants. Our results reveal that the pathogenic activity of polyclonal PV IgG can be attributed to p38 MAPK-dependent clustering and endocytosis of Dsg3, whereas pathogenic monoclonal Dsg3 antibodies can function independently of this pathway. These findings have important implications for understanding pemphigus pathophysiology, and for the design of pemphigus model systems and therapeutic interventions.