The effects of CD148 Q276P/R326Q polymorphisms in A431D epidermoid cancer cell proliferation and epidermal growth factor receptor signaling

Abstract

Background CD148 is a transmembrane protein tyrosine phosphatase that is expressed in multiple cell types. Previous studies have shown that CD148 dephosphorylates growth factor receptors and their signaling molecules, including EGFR and ERK1/2, and negatively regulates cancer cell growth. Furthermore, research of clinical patients has shown that highly linked CD148 gene polymorphisms, Gln276Pro (Q276P) and Arg326Gln (R326Q), are associated with an increased risk of several types of cancer. However, the biological effects of these missense mutations have not been studied. Aim We aimed to determine the biological effects of CD148 Q276P/R326Q mutations in cancer cell proliferation and growth factor signaling, with emphasis on EGFR signaling. Methods CD148 forms, wild‐type (WT) or Q276P/R326Q, were retrovirally introduced into A431D epidermoid carcinoma cells that lacks CD148 expression. The stable cells that express comparable levels of CD148 were sorted by flow cytometry. A431D cells infected with empty retrovirus was used as a control. CD148 localization, cell proliferation rate, EGFR signaling, and the response to thrombospondin‐1 (TSP1), a CD148 ligand, were assessed by immunostaining, cell proliferation assay, enzyme‐linked immunosorbent assay, and Western blotting. Results Both CD148 forms (WT, Q276P/R326Q) were distributed to cell surface and all three cell lines expressed same level of EGFR. Compared to control cells, the A431D cells that express CD148 forms showed significantly lower cell proliferation rates. EGF‐induced EGFR and ERK1/2 phosphorylation as well as cell proliferation were also significantly reduced in these cells. Furthermore, TSP1 inhibited cell proliferation in CD148 (WT, Q276P/R326Q)‐expressing A431D cells, while it showed no effects in control cells. However, significant differences were not observed between CD148 WT and Q276P/R326Q cells. Conclusion Our data demonstrates that Q276P/R326Q mutations do not have major effects on TSP1‐CD148 interaction as well as on CD148's cellular localization and activity to inhibit EGFR signaling and cell proliferation.

Analysis of Receptor-Type Protein Tyrosine Phosphatase Extracellular Regions with Insights from AlphaFold

“…It has a homotrimeric structure of 450 kDa and Takahashi et al reported that it binds to the extracellular domain of CD148 with high affinity and specificity and increases CD148 phosphatase activity. Follow-on studies have shown that naturally occurring variations in the CD148 extracellular domain Q276P/R326Q mutations do not have major effects on TSP1-CD148 interaction, indicating that these residues are not involved [36,37]. In contrast with the soluble secreted TSP1, syndecans are cell-surface heparan sulfate proteoglycans that regulate many cellular functions.…”

Analysis of Receptor-type Protein Tyrosine Phosphatase Extracellular Regions with Insights from AlphaFold

“…It has a homotrimeric structure of 450 kDa and Takahashi et al reported that it binds to the extracellular domain of CD148 with high affinity and specificity and increases CD148 phosphatase activity. Follow-on studies have shown that naturally ocurring variations in the CD148 extracellular domain Q276P/R326Q mutations do not have major effects on TSP1-CD148 interaction indicating that these residues are not involved [36,37]. In contrast with the soluble secreted TSP1, syndecans are cell surface heparan sulfate proteoglycans that regulate many cellular functions.…”