Molecular organization of EphA2 cell-cell communication complexes in human keratinocytes

Rosa Ventrella

doi:https://doi.org/10.21985/n2-pkzt-gc46

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Molecular organization of EphA2 cell-cell communication complexes in human keratinocytes

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Erythropoietin-producing human hepatocellular (Eph) receptors and their corresponding ephrin ligands are asymmetrically expressed at cell-cell contacts allowing for bidirectional signaling with forward signaling through the receptor expressing cell and reverse signaling through the ligand expressing cell. Eph receptors are the largest family of receptor tyrosine kinases (RTKs) in mammals, which allows for a vast array of signaling responses depending on which receptor and ligand are interacting and the cell type involved. In addition, when ephrin ligands are expressed on the same cell, Eph signaling is inhibited adding an additional mechanism of signal regulation. Most notably, Eph/ephrins have been shown to play important roles in cell sorting, boundary formation, and tissue morphogenesis. Misregulation of Eph/ephrins often lead to aberrant signaling pathways, leading to a variety of diseases. Specifically, in the epidermis, ephrin-A1 promotes keratinocyte differentiation and inhibits keratinocyte migration through EphA2. Alterations in this signaling axis is associated with inflammatory skin diseases such as psoriasis and non-melanoma skin cancer. Ephrin-A1 contains a glycosylphosphatidylinositol (GPI)-linked tail, which targets this ligand to specific membrane microdomains known as lipid rafts. Lipid raft domains are important for regulating the distribution of membrane proteins and are enriched in sterols, most notably cholesterol, making them thicker and less fluid than the surrounding membrane. Due to the importance of ephrin localization relative to its receptor, one would hypothesize that lipid rafts play an important role in the downstream signaling elicited by EphA2 and ephrin-A1; however, the ability of this receptor and ligand to localize to lipid raft domains and the mechanisms governing their localization patterns in keratinocytes is unknown. Additionally, due to the importance of EphA2 and ephrin-A1 in keratinocyte biology, it is likely that ephrin-A1 and EphA2 cellular distribution is important in controlling keratinocyte behaviors like differentiation and migration. In order to address these hypotheses, we used a combination of biochemical and imaging approaches to study the molecular organization of ephrin-A1 and EphA2 signaling complexes in a primary human keratinocyte culture model. We provide evidence that ephrin-A1-induced forward signaling promoted keratinocyte differentiation as assessed by tight junction proteins that are expressed during the final stages of epidermal differentiation. We also provide evidence that EphA2 is present in lipid raft domains along with ephrin-A1. The recruitment of EphA2 to lipid raft domains at cell-cell junctions was dependent on the unique properties of its transmembrane domain (TMD). Swapping the EphA2 TMD with a shorter and molecularly distinct TMD of the highly homologous EphA1 caused failure of this transmembrane mutant chimera to localize to cell-cell contacts, likely affecting its interaction with ephrin-A1. Correspondingly, this chimera increased ephrin-A1 expression levels and impaired the ability of keratinocytes to efficiently seal linear scratch wounds in an ephrin-A1-dependent manner. However, this chimera had minimal impact on calcium-induced keratinocyte differentiation. These findings suggest that cell-cell contact stabilization of EphA2 is not required to promote keratinocyte differentiation. Moreover, ephrin-A1 protein levels negatively regulate keratinocyte migration, but do not necessarily enhance keratinocyte migration. Collectively, these studies highlight a key role for the EphA2 TMD and its association with lipid rafts in modulating downstream signaling. Our data highlight the importance of EphA2 localization to lipid raft domains at cell-cell contacts in controlling ligand expression level. Specifically, these findings suggest the importance of the EphA2 TMD in regulating keratinocyte migration, which has relevance to cutaneous wound healing. These findings likely have broader implications for the understanding of Eph/ephrin and transmembrane receptor biology. It is likely that lipid rafts play important roles in organizing Eph/ephrin signaling networks at boundaries to control Eph/ephrin and other membrane receptor interactions that govern cell segregation and tissue patterning. In addition to modulating Eph/ephrin interactions, lipid rafts likely organize receptor crosstalk in different membrane regions. These discoveries will be important in understanding how lipid raft disruption or Eph/ephrin misregulation leads to abnormal signaling, resulting in loss of tissue homeostasis. Lastly, these outcomes show the significance of the TMD of RTKs in regulating downstream signaling pathways.

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