Transmembrane Domains Of Receptor Tyrosine Kinases Research Articles

Nuclear receptor tyrosine kinase transport and functions in cancer.

Signaling functions of plasma membrane-localized receptor tyrosine kinases (RTKs) have been extensively studied after they were first described in the mid-1980s. Plasma membrane RTKs are activated by extracellular ligands and cellular stress stimuli, and regulate cellular responses by activating the downstream effector proteins to initiate a wide range of signaling cascades in the cells. However, increasing evidence indicates that RTKs can also be transported into the intracellular compartments where they phosphorylate traditional effector proteins and non-canonical substrate proteins. In general, internalization that retains the RTK's transmembrane domain begins with endocytosis, and endosomal RTK remains active before being recycled or degraded. Further RTK retrograde transport from endosome-Golgi-ER to the nucleus is primarily dependent on membranes vesicles and relies on the interaction with the COP-I vesicle complex, Sec61 translocon complex, and importin. Internalized RTKs have non-canonical substrates that include transcriptional co-factors and DNA damage response proteins, and many nuclear RTKs harbor oncogenic properties and can enhance cancer progression. Indeed, nuclear-localized RTKs have been shown to positively correlate with cancer recurrence, therapeutic resistance, and poor prognosis of cancer patients. Therefore, understanding the functions of nuclear RTKs and the mechanisms of nuclear RTK transport will further improve our knowledge to evaluate the potential of targeting nuclear RTKs or the proteins involved in their transport as new cancer therapeutic strategies.

Investigation of Transmembrane Helix Hetero-Dimerization by Combinatorial Peptide Libraries and High-Throughput Measurement of FÖRSTER Resonance Energy Transfer in Liposomes

Receptor tyrosine kinases (RTKs) are membrane proteins containing an N-terminal extracellular ligand-binding domain, a single helical transmembrane (TM) domain, and an intracellular catalytic kinase domain. Lateral dimerization driven in part by the TM domain is proposed to be a crucial intermediate step in signal transduction across the plasma membrane. Defects in this process such as over-dimerization are closely linked to unregulated signaling and disease. Therefore, dimerization inhibitors developed using the chemical-physical basis of RTK TM domain dimerization could be promising therapeutic agents. Here we present a first step towards this goal. We designed a rational combinatorial peptide library based on the TM domain of Neu, an RTK from the Erb-B/HER epidermal growth factor receptor family (EGFR) with a pathogenic V to E mutation in the TM domain. Förster Resonance Energy Transfer (FRET) in lipid vesicles was used as a method to probe the dimerization between the library members and mutant Neu sequence. To enable high-throughput screening in liposomes, we developed a novel multi-well FRET assay. FRET donor-labeled library members and acceptor-labeled Neu peptides were incorporated into lipid vesicles in a rapid high-throughput 96-well plate format. We are using this high throughput screen to identify inhibitors of Neu dimerization. Our findings will be discussed in a context of throughput and sensitivity along with a statistical analysis that takes into account the FRET that arises from random proximity of donors and acceptors in the bilayer.

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

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.

Synthesis and initial characterization of FGFR3 transmembrane domain: consequences of sequence modifications

Receptor Tyrosine Kinases (RTKs) conduct biochemical signals via lateral dimerization in the plasma membrane, and defects in their dimerization lead to unregulated signaling and disease. RTK transmembrane (TM) domains are proposed to play an important role in the process, underscored by the finding that single amino acids mutations in the TM domains can induce pathological phenotypes. Therefore, many important questions pertaining to the mode of signal transduction and the mechanism of pathology induction could be answered by studying the chemical–physical basis behind RTK TM domain dimerization and the interactions of RTK TM domains with lipids in model bilayer systems. As a first step towards this goal, here we report the synthesis of the TM domain of fibroblast growth factor receptor 3 (FGFR3), an RTK that is crucial for skeletal development. We have used solid phase peptide synthesis to produce two peptides: one corresponding to the membrane embedded segment and the naturally occurring flanking residues at the N- and C-termini (TM wt), and a second one in which the flanking residues have been substituted with diLysines at the termini (TM KK). We have demonstrated that the hydrophobic FGFR3 TM domain can be synthesized for biophysical studies with high yield. The protocol presented in the paper can be applied to the synthesis of other RTK TM domains. As expected, the Lys flanks decrease the hydrophobicity of the TM domain, such that TM KK elutes much earlier than TM wt during reverse phase HPLC purification. The Lysines have no effect on peptide solubility in SDS and on peptide secondary structure, but they abolish peptide dimerization on SDS gels. These results suggest that caution should be exercised when modifying RTK TM domains to render them more manageable for biophysical studies.