Tumor-derived extracellular mutations of PTPRT /PTPrho are defective in cell adhesion

Abstract

Receptor protein tyrosine phosphatase T (PTPRT/PTPrho) is frequently mutated in human cancers including colon, lung, gastric, and skin cancers. More than half of the identified tumor-derived mutations are located in the extracellular part of PTPrho. However, the functional significance of those extracellular domain mutations remains to be defined. Here we report that the extracellular domain of PTPrho mediates homophilic cell-cell aggregation. This homophilic interaction is very specific because PTPrho does not interact with its closest homologue, PTPmu, in a cell aggregation assay. We further showed that all five tumor-derived mutations located in the NH(2)-terminal MAM and immunoglobulin domains impair, to varying extents, their ability to form cell aggregates, indicating that those mutations are loss-of-function mutations. Our results suggest that PTPrho may play an important role in cell-cell adhesion and that mutational inactivation of this phosphatase could promote tumor migration and metastasis.

Figure 1. Expression of PTPρ constructs in Sf9 cells

(A) Schematic diagram of PTPρ constructs. MAM domain (gray oval); Ig domain (pink circle); Juxtamembrane domain (yellow oval); Wedge domain (red circle); Phosphatase domain (green rectangles). (B) Western blot of lysates from Sf9 cells expressing the indicated PTPρ constructs. (C) Immunofluorescent staining of Sf9 cells expressing the indicated PTPρ proteins.

Figure 2. PTPρ mediates cell-cell aggregation

Sf9 cells were infected with indicated baculoviral constructs for 48 hrs. Cell aggregation assay was performed under low shear conditions and representative phase pictures were captured with a microscope.

Figure 3. PTPρ mediates homophilic cell-cell aggregation

(A) Sf9 cells were infected with GFP while another flask of Sf9 cells was co-infected with PTPρ and RFP viruses. Sf9 cells from the two flasks were mixed at a 1:1 ratio and a cell aggregation assay was performed. (B) One flask of Sf9 cells was co-infected with PTPμ and GFP viruses, while another flask of Sf9 cells was co-infected with PTPρ and RFP viruses. Sf9 cells from the two flasks were mixed at 1:1 ratio and cell aggregation assay was performed. Images of cell aggregates were captured under phase, GFP and RFP fluorescent filters respectively.

Figure 4. Expression of tumor-derived PTPρ mutants in Sf9 cells

(A) Schematic diagram of PTPρ mutations located in the MAM and Ig domain. (B) Western blot of lysates from Sf9 cells expressing with indicated PTPρ mutations. (C) Immunofluorescent staining of Sf9 cells expressing the indicated PTPρ mutant proteins.

Figure 5. Tumor-derived mutations of PTPρ are defective in cell-cell aggregation

Cell aggregation assay of Sf9 cells expressing either wild-type or the indicated mutation of PTPρ was performed under low shear conditions. Representative phase pictures were captured with a microscope.

Figure 6. Quantification of the cell-cell aggregation assays

The percentage of aggregated Sf9 cells for each indicated protein was calculated from three independent experiments. Student t.tests were performed between wild-type PTPρ and each of the mutants (asterisk indicated that the P < 0.01).

Figure 7. Cell Surface Protein expression of PTPμ and the PTPρ mutant proteins

The cell surface proteins were labeled with Sulfo-NHS-SS-Biotin. The biotinylated proteins were isolated on NeutrAvidin Gel. The bound proteins were eluted, run on SDS-PAGE gels, transferred to nitrocellulose and immunoblotted with antibodies to PTPμ (SK18) or the V5 tag. The SK18 antibody recognizes the cytoplasmic domain of both PTPμ and PTPρ.