Specific inhibition of a pathogenic receptor tyrosine kinase by its transmembrane domain

Abstract

The transmembrane (TM) domains of receptor tyrosine kinases (RTKs) are believed to be important players in RTK signal transduction. However, the degree of specificity and promiscuity of RTK TM domain lateral interactions in mammalian membranes has not been assessed in detail in the literature. A technique to probe the occurrence of interactions between TM domains and their biological significance is to evaluate the propensity for formation of heterodimers of a full-length RTK and its TM domain. Here we examine if the inhibition of two RTK pathogenic mutants, Neu/V664E and FGFR3/A391E, can be achieved by the TM domains of Neu, Neu/V664E, FGFR3 and FGFR3/A391E. We show that the TM domain of Neu/V664E specifically inhibits the phosphorylation of full-length Neu/V664E, while the wild-type Neu TM domain does not. In addition, Neu/V664E TM domain does not affect the phosphorylation levels of full-length FGFR3/A391E. The results suggest that TM domain peptides could be exploited in the future for the development of specific inhibitors of mutant RTKs.

Figure 1. Plasmids encoding Neu and FGFR3 TM domains

(A) The amino acid sequences of FGFR3, FGFR3/A391E, Neu and Neu/V664E TM domains. The amino acids in the hydrophobic hydrocarbon core - embedded domain are underlined. The mutant amino acids (E) are shown in italic. (B) Structure of the constructed genes, containing the FGFR3 signal peptide at the N-terminus, a VSV-G tag for probing expression, and the TM domains.

Figure 2

Immunofluorescent staining of the TM domains in HEK 293 cells. (A). FGFR3 TM domain. B. FGFR3/A391E TM domain. (C). Neu TM domain. (D). Neu/V664E TM domain. Intact cells were stained with anti-VSV-G antibodies, followed by Alexa Fluor 647 goat anti rabbit IgG (H+L). Images were acquired with a Nikon confocal laser scanning microscope. Neu TM and Neu/V664E TM express at the cell surface with higher efficiency, only a small fraction of cells express FGFR3 and FGFR3/A391E TM domains in their plasma membrane. These results, along with results shown in Figure 3 suggest that the FGFR3 TM domains are not trafficked efficiently to the cell surface. Similar results were observed for B104-1-1 cells (not shown).

Figure 3

Western Blot analysis of the expression of the TM domains in B104-1 cells (A) and HEK 293 cells (B). Cells were transfected with the same amount (1 µg of plasmid per well) of pcDNA3.1+ plasmids encoding the TM domain of FGFR3 (lane 1), FGFR3/A391E (lane 2), Neu (lane 3) and Neu /V664E (lane 4). Cells were lysed 24 hours after transfection. The proteins were separated by SDS-PAGE gel and detected with anti-VSV-G antibodies. The expression of the four TM constructs is comparable.

Figure 4

Western blot analysis of the TM domains after chemical cross-linking. B104-1-1 cells were transfected with plasmids encoding the TM domains of FGFR3 (lane 1), FGFR3/A391E (lane 2), Neu (lane 3) and Neu/V664E (lane 4). Twenty-four hours after transfection, cells were subjected to cross-linking, followed by lysis and Western blotting. Dimeric bands were observed for all constructs. The cross-linker used was Ethylene glycol succinimidyl succinate (EGS), a membrane permeable linker that cross-links proteins within all cellular membranes [42].

Figure 5

Effect of the TM domains on the expression and phosphorylation of Neu/V664E in stable cells. Results shown are average of six sets of independent experiments. Values in cells transfected with empty pcDNA 3.1 vectors are assigned as one. The phosphorylation of Neu/V664E in stable cell lines can be specifically inhibited by the TM domain of Neu/V664E.

Figure 6

Effect of the TM domains on the expression and phosphorylation of FGFR3/A391E in stable cells. Results shown here are average of three sets of independent experiments. Values in cells transfected with empty pcDNA 3.1 vectors are assigned as one. None of the TM domains could decrease the phosphorylation of FGFR3/A391E.