CD82 palmitoylation site mutations at Cys5+Cys74 affect EGFR internalization and metabolism through recycling pathway

Abstract

Tetraspanin CD82 often participates in regulating the function of epidermal growth factor receptor (EGFR) and hepatocyte growth factor receptor (c-Met). Palmitoylation is a post-translational modification that contributes to tetraspanin web formation and affects tetraspanin-dependent cell signaling. However, the molecular mechanisms by which CD82 palmitoylation affects the localization and stability of EGFR and c-Met have not yet been elucidated. This study focuses on the expression and distribution of EGFR and c-Met in breast cancer as well as the related metabolic pathways and molecular mechanisms associated with different CD82 palmitoylation site mutations. The results show that CD82 with a palmitoylation mutation at Cys5+Cys74 can promote the internalization of EGFR. EGFR is internalized and strengthened by direct binding to CD82 with the tubulin assistance and located at the recycling endosome. After studying the recycling pathway marker proteins Rab11a and FIP2, we found that formation of the EGFR/CD82/Rab11a/FIP2 complex promotes the internalization and metabolism of EGFR through the recycling pathway and results in the re-expression of EGFR and CD82 on the cell membrane.

Keywords: CD82; EGFR; Rab11a; palmitoylation; tetraspanin.

Figure1

The expression and localization of EGFR and c-Met with different palmitoylation site mutations in CD82(A) Schematic diagram of mutations at different palmitoylation sites of CD82. "+" represents mutation and "-" represents no mutation. (B) and (C) Western blot analysis results confirmed EGFR, CD82 and c-Met expressions in the cytoplasm and cytomembrane after mutations at different palmitoylation sites of CD82. (D) and (E) The expressions of CD82 and EGFR in the total protein after different mutations at palmitoylation sites of CD82. (F) The levels of CD82 after the transformation of different mutants of CD82 determined by qPCR.

Figure2

The interaction and co-localization of CD82 and EGFR(A) After the mutations at different palmitoylation sites of CD82, the immunoblotting results showed the physical interaction relationship between CD82 and Lamp1, Rab7a and Rab11a. (B) CD82 could directly interact with EGFR in the CD82 palmitoylation Cys5+Cys74 sites combined mutation group, WT group and normal control group, and protein expression was also found in the input group. (C) EGFR in the CD82 palmitoylation Cys5+Cys74 sites combined mutation group, WT group and normal control group, the IP results showed that EGFR can directly bind to CD82, but not to Lamp1, Rab7a or Rab11a. In the input group, CD82 palmitoylationCys5+Cys74 sites combined mutation group, WT group and normal control group all had protein expression. (D) and (E) When the CD82 palmitoylation sites Cys5+Cys74 were mutated, co-localization of CD82 and EGFR separately with Lamp1, Rab11a and Rab7a. Scale bar=25 μm.

Figure3

EGFR was metabolized through the recycling endosome pathway by forming a complex(A) Western blot analysis and (B) immunofluorescence assay were used to detect the expression of EGFR in the total protein with different inhibitors after CD82 palmitoylation sites Cys5+Cys74 were mutated. Scale bar=25 μm. (C) After the combined mutation of the CD82 palmitoylation sites Cys5+Cys74, the tubulin inhibitor nocodazole (66 μM, 4 min) was added to detect the expression of EGFR. The upper row is the nocodazole group, the bottom row is the control group. Scale bar=25 μm. (D) FIP2 in the CD82 palmitoylation sites Cys5+Cys74 combined mutation group, WT group and normal control group, the IP results showed that FIP2 can directly bind to Rab11a and CD82. In the input group, the CD82 palmitoylation sites Cys5+Cys74 combined mutation group, WT group and normal control group all had protein expression.

Figure4

A mechanistic diagram of the effect of the tetraspanin CD82 palmitoylation site mutations on the expression and localization of EGFRWhen the CD82 palmitoylation sites Cys5+Cys74 of are mutated, the internalization ability of EGFR is enhanced. After internalization, most EGFR and CD82 enter the recycling endosome in a direct binding manner. This process requires the assistance of tubulin. CD82 can directly bind with Rab11a and FIP2 to form an EGFR/CD82/Rab11a/FIP2 complex, which is metabolized by the recycling endosome pathway to re-express EGFR and CD82 on the cell membrane surface. The remaining small part of the EGFR/CD82 complex is degraded in the lysosome, and this metabolic process does not go through the late endosome pathway.