The tetraspanin CD82 can regulate the occurrence, development and metastasis of most tumors, and has the effect of inhibiting tumor metastasis[11-13]. CD82 can also regulate the distribution of proteins on the cell membrane by interacting with other molecules. When the protein undergoes palmitoylation modification, the protein located in the cytoplasm has the ability to anchor on the cell membrane, and the protein located on the cell membrane will promote the signal transport process and stably maintain normal physiological functions[3, 14]. Palmitoylation contributes to the formation of the tetraspanin protein network and cell signal transduction[15, 16]. Studies have shown that when the palmitoylation mutation of CD82 occurs, it can promote the recombination of dynein in the microdomain and also affect the formation of cadherin binding protein complex. CD82 palmitoylation mutation can change the composition of the cell membrane and stabilize the expression and activation of protein kinase C. When palmitoylation is inhibited, the stability of protein kinase C on the membrane is reduced, and the activation of the ERK1/2 signaling pathway is also affected weakened.
The EGFR signaling pathway is regulated by many factors, and different ligands can transmit different signals. CD82 can weaken the EGF/EGFR induction signal and inhibit tumor metastasis and spread. Studies have shown that CD82 can regulate the ligand-induced ubiquitination of EGFR, but before EGFR is activated, CD82 can regulate it. CD82 can also reduce the formation of EGFR dimers, but the mechanism is still unclear . In epithelial tumor cells, CD82 can promote the endocytosis and desensitization effect of EGFR. In gastric cancer cells, CD82 can be inhibited by miR-197, which increases the activated EGFR and enhances tumor cell invasion and metastasis .
Up to now, the effect of CD82 palmitoylation mutation on the expression, location and metabolism of EGFR and c-Met in breast cancer cells remains to be further studied. In this experiment, by constructing different CD82 palmitoylation mutants to explore the influence of different site mutations on the expression, distribution and metabolic pathways of EGFR and c-Met. When the Cys5+Cys74 site of CD82 is combined with palmitoylation mutation, the internalization ability of EGFR is strengthened, and more EGFR cannot be stably expressed on the cell membrane, but transferred from the cell membrane to the cytoplasm. CD82 palmitoylation mutations cannot affect the expression and localization of c-Met, which may also indicate that CD82 has regulation of c-Met in tumor cells requires the participation of other post-translational modifications or the assistance of other signaling molecules, which are still to be studied in the future. Therefore, in the follow-up, we will mainly discuss the interaction relationship and molecular mechanism of the combined palmitoylation mutation at Cys5+Cys74 of CD82 and EGFR.
We further researched and found that there was no significant difference in the expression of EGFR in the total protein of different CD82 palmitoylation mutants. This shows that palmitoylation mutations at different sites of CD82 cannot change the total content of EGFR in cells, but can change its distribution position in cells. At the same time, for CD82 itself, when the Cys5+Cys74 site has a combined palmitoylation mutation, CD82 also changes its own distribution. It has no effect on the amount of self-expression.
Therefore, the following conjectures can be put forward. These internalized EGFR and CD82 may be (1) decomposed through certain metabolic pathways, such as lysosomal metabolic pathways. (2) Circulate metabolism through the circulatory pathway. (3) Metabolism through late endosomal pathway. Lamp1, Rab11a, and Rab7a are lysosomal markers, recycling endosome markers, and advanced endosomal markers respectively . After the Cys5+Cys74 site of CD82 combined with palmitoylation mutation, most of the CD82 and EGFR with enhanced internalization ability were located in the recycling endosome. That is to say, most of them are metabolized through the circulatory pathway, and a small part of CD82 and EGFR are metabolized through the lysosomal pathway.
Studies have pointed out that in small cell lung cancer A549, primary keratinocytes and human breast epithelial cells, CD82 can directly bind to EGFR and inhibit EGF-induced cell migration. CD82 does not affect the activation of EGFR, but the signal induced by EGF will be desensitized faster, and the process of weakening of this signal may be related to endocytosis . In this experiment, when CD82 palmitoylation Cys5+Cys74 sites are combined with mutations, the enhancement of EGFR internalization is also related to the direct binding of EGFR and CD82, and EGFR and CD82 are transferred into the cytoplasm in the form of direct interaction.
Endosomes can be transported along microtubules with the assistance of dynein. Studies have shown that endosomes containing CD133 can be recruited to the centrosome area through a dynein-based transport system . In this experiment, to further verify the specific molecular mechanism of EGFR internalization into the cytoplasm after the Cys5+Cys74 site of CD82 combined with palmitoylation mutations and metabolization through the circulatory pathway, the tubulin inhibitor Nocodazole was used to inhibit the aggregation of tubulin. Observe the expression of EGFR. From the immunofluorescence results, it can be seen that when Nocodazole is added, the expression of EGFR in the cytoplasm is reduced, and most of them are located on the cell membrane, and the internalization ability is weakened. Therefore, it can be inferred that after the Cys5+Cys74 site of CD82 is combined with palmitoylation mutation, EGFR is internalized under the binding of CD82, and this process also requires the assistance of tubulin.
Monensin is a polyether antibiotic that can change the pH of the environment by adjusting the Na/H converter, destroy the structure of related proteins, and inhibit the transport process of circulating endosomes to the cell membrane, Actinomycetes Ketone is a protein production inhibitor, which can inhibit the production of most proteins. Chloroquine is a selective inhibitor of lysosomes, and MG132 is an inhibitor of proteasomes . In order to further verify that the Cys5+Cys74 site of CD82 combined with palmitoylation mutations, EGFR is internalized and then metabolized through the circulatory pathway. In this experiment, monensin was selected as an inhibitor of the circulatory pathway. Inhibit the circulation pathway and observe the changes in the expression of EGFR. At the same time, cycloheximide was selected to inhibit the production of EGFR, and chloroquine was compared with MG132 as a control group. The results of immunoblotting and immunofluorescence show that when monensin is added, the effect of cycloheximide on the reduction of EGFR expression can be restored to a large extent. That is to say, when the circulatory pathway is inhibited, the metabolism of EGFR is blocked at this time, which reversely verifies that EGFR is metabolized through the circulatory pathway.
Rab11a belongs to the small molecule GTPase family Rab11 subfamily member , and can be used as a marker protein of the recycling endosome. The role of Rab11a in cancer progression has also been extensively studied. Studies have shown that Rab11a can promote the activation of the Wnt signaling pathway and further enhance the invasion ability of pancreatic cancer. Rab11a has many effect factors, which play a regulatory role in different signal pathways. The most important one is Rab11-FIPs. Rab11-FIPs can be divided into three subtypes. The first type of subtype includes: FIP2, Rab coupling protein (RCP) and Rab11 interacting protein (Rip11), the second type of subtype includes: FIP3 and FIP4, and the third type of subtype: FIP1 . The first subtypes often specifically bind to Rab14 in a GTP-dependent manner. FIP2 is one of the subfamily members of Rab11-FIPs, and plays an important regulatory role in the process of molecular recycling of the cell surface [30, 31]. Rab11a can recruit myosin Vb and cytoplasmic dynein through the effectors FIP2 and FIP3 . Insulin is a C-type lectin receptor. As a cargo molecule, it can be recovered through the endosomal circulation pathway in the Rab11a-positive membranous region. Studies have shown that the myosin Vb/Rab11a/Rab11-FIP2 signaling pathway is involved in the endosomal recovery process of islet protein. In the late stage of recovery, it can assist the connection and fusion of islet protein and plasma membrane.
When the Cys5+Cys74 sites of CD82 were combined with palmitoylation mutations, CD82 and Rab11a were immunoprecipitated with FIP2 respectively. The results confirmed that FIP2 can directly bind to Rab11a and CD82. Therefore, it can be inferred that CD82, Rab11a and FIP2 can form a complex to assist the re-recovery of EGFR on the cell membrane. At the same time, CD82 will also be expressed on the cell membrane again, as shown in Figure 6.
However, this experiment only explored the metabolic pathways of EGFR after the Cys5+Cys74 sites of CD82 combined with palmitoylation mutations. The metabolic pathways of other mutants and the mechanism of CD82 internalization enhancement are still unclear. Whether the EGFR and CD82 located in the lysosome are completely degraded remains to be verified.