Cancer-derived mutations in the fibronectin III repeats of PTPRT/PTPrho inhibit cell-cell aggregation

Abstract

Abstract The receptor protein tyrosine phosphatase T PTPrho is the most frequently mutated tyrosine phosphatase in human cancer. PTPrho mediates homophilic cell-cell aggregation. In its extracellular region, PTPrho has cell adhesion molecule-like motifs, including a MAM domain, an immunoglobulin domain, and four fibronectin type III (FNIII) repeats. Tumor-derived mutations have been identified in all of these extracellular domains. Previously, the authors determined that tumor-derived mutations in the MAM and immunoglobulin domains of PTPrho reduce homophilic cell-cell aggregation. In this paper, the authors describe experiments in which the contribution of the FNIII repeats to PTPrho-mediated cell-cell adhesion was evaluated. The results demonstrate that deletion of the FNIII repeats of PTPrho result in defective cell-cell aggregation. Furthermore, all of the tumor-derived mutations in the FNIII repeats of PTPrho also disrupt cell-cell aggregation. These results further support the hypothesis that mutational inactivation of PTPrho may lead to cancer progression by disrupting cell-cell adhesion.

Figure 1

PTPρ deletion constructs are all expressed at the cell surface. Constructs containing deletions of one extracellular domain of PTPρ each were generated and expressed in Sf9 cells. Total cellular protein and cell surface protein were isolated and immunoblotted with the SK18 antibody.

Figure 2

Deletion of any of the extracellular domains of PTPρ disrupts cell-cell aggregation. Sf9 cells expressing deletion constructs of the individual extracellular domains of PTPρ were allowed to aggregate for 30 minutes prior to imaging. Scale bar equals 100μm. The percentage of Sf9 cell-cell aggregation for each deletion protein was calculated from a minimum of three experiments. An asterisk indicates a statistically significant reduction in aggregation (p<0.005).

Figure 3

PTPρ proteins containing tumor-derived mutations in their FNIII domains are expressed at the cell surface. Ten mutations in the FNIII domains of PTPρ were generated by site-directed mutagenesis and expressed in Sf9 cells. Total cellular and cell surface proteins were isolated and analyzed by immunoblotting with the SK18 antibody.

Figure 4

Tumor derived mutations in FNIII domains of PTPρ are defective in cell-cell aggregation. Sf9 cells expressing the mutated PTPρ proteins were allowed to aggregate for 30 minutes prior to imaging. Scale bar equals 100μm. The percentage of Sf9 cell-cell aggregation for each mutant protein was calculated from a minimum of three experiments. An asterisk indicates a statistically significant reduction in aggregation (p<0.008).

Figure 5

Structural modeling of the point mutations of PTPρ. The point mutations (indicated in red) evaluated in this manuscript were modeled onto the equivalent sites in the crystal structure of PTPμ. The PTPρ mutations likely alter cell-cell aggregation either by altering the three-dimensional topology and/or cis/trans interactions of PTPρ.