Trk juxta membrane mutations

The Trk juxta membrane domain is coming out of the shadows and maybe telling us something about Trk fusion proteins

In many structural reviews of the Trk family of receptor tyrosine kinases, the extracellular domains that bind growth factors are given their own symbols. The transmembrane and kinase domains are usually denoted with boxes and maybe ovals, respectively. The Trk juxta membrane region is usually left with an enigmatic gray or black line and largely ignored.  Are we right to ignore this region?

  • Medicinal chemists are interested in the juxtamembrane region of TrkA as a means of inhibiting neurons involved in chronic pain.  PubMed
  • Further down the pipeline, a phospho- peptide from the juxtamembrane region of TrkB has received interest  for its ability to bind to the phosphotyrosine binding domain  of the adaptor/docking protein FSR2α. PubMed This peptide is nothing unique to TrkB, but rather a generic motif in receptor tyrosine kinases.
  • If the juxtamembrane region regulates the kinase domain or the access of the substrate to said kinase domain, then surely TRK fusions occurring near this region will have consequences.

The juxtamembrane region of the generic receptor tyrosine kinase

Toffalini and Demoulin (2010)  reviewed hematopoietic  malignancy activating mutations in receptor tyrosine kinases (RTK).  They divided their review into RTK protein domains, including the juxtamembrane region.  The platelet derived growth factor  receptor was one of the kinases they reviewed.  Even though the protein product of the ETV6-NTRK3 gene rearrangement can be associated with leukemias, it didn’t make the list.  In some of the cancers associated with the ETV6-NTRK3 gene rearrangement we have covered, the translated protein lacks the juxtamembrane region of TrkC.  This trend seems consistent with a recent publication describing  pediatric mesenchymal tumors with the ETV6-NTRK3 gene rearrangement

Cartoon showing Trk extracellular ligand binding region

Figure 1. The stereotypical receptor tyrosine kinase. Ligand binding induced dimerization results in phosphorylation of the activation loop and its movement from the active site.  Also note movement of juxtamembrane region.

 

Toffalini F, Demoulin JB.(2010) New insights into the mechanisms of hematopoietic cell transformation by activated receptor tyrosine kinases. Blood. 116(14):2429-37. PubMed

TrkA, a juxtamembrane region in neurons that sense pain

The NTRK1 gene rearrangements we have reviewed on a sister site occur to the N-terminus of the of the transmembrane  α-helix:  BCAN-NTRK1 (V418), LMNA-NTRK1 (V418), NFASC-NTRK1 (T400), and TPM3-NTRK1 (D399).  The fusion partners are not membrane proteins.  If the hydrophobic trans membrane helix of TrkA is not in the membrane, it might interact with what we are learning to be a regulatory  region of TrkA.  Arrows mark some not so conservative substitutions in the juxtamembrane region, Figure 2.  Note that many are substitution of a hydrophilic amino acid for a hydrophobic amino acid. The R508W arginine to tryptophan substitution is particularly interesting.

Bar diagram showing juxtamembrane region of Trk A

Figure 2.  The juxtamembrane region of Trk A.  A. sequence information from UniProt.org B. sequence and cancer associations from COSMIC

We may know more about the juxtamembrane of TrkA than any other Trk family member, not because of signaling molecules that dock to the tyrosine autophosphorylation site (Fig 1A), but because of the role of Trk A mutations in congenital insensitivity to pain, anhidrosis (CIPA).  Very selective inhibition of TrkA is being sought as a targeted a means of eliminating chronic pain. The small molecule inhibitor in Figure 3 is not shown to emphasize the interaction of the C-terminus of the juxtamembrane region and he rest of he TrkA kinase domain. The juxta membrane region is deep red progressing to the C-terminus of the kinase domain, deep indigo.

 Ribbon diagrams of two views of the structure of TrkA along with crystal structure

Figure 3. A portion of the juxtmembrane region of Trk A and the kinase domain. Left: Ribbon diagrams of two views of the structure.  Right: Surface renderings of the same views of the structure.

Figure 4 is another way of looking at the juxtamembrane region with the kinase domain.  Panel 4A is the usual rainbow color scheme going from red (N-terminus) to the C-terminus (deep indigo).  Panel 4B is the structure in a ribbon diagram with the most hydrophobic amino acids  (e.g. tryptophan) colored red to the most hydrophilic amino acids (e.g. arginine) colored green.  Note that the Arg508 on the surface relative to a Phe576 in the kinase domain (Panel 4B).  Such a substitution in Fig 2 could alter the orientation of the entire juxtamembrane region.  Would the responsiveness to dimerization and the activation loop follow?  In most cases such mutations result in insensitivity to pain.

ribbon diagram of TrkA activation loop with diagram showing hydrophobic and hydrophilic regions

Figure 4 Additional views of the activation loop of Trk A   A. The rainbow view from the N-terminus (red) to the C-terminus (indigo). B. The same ribbon diagram colored according to hydrophobicity. C. surface plot colored according to hydrophobicity.

 

Trk B, the juxtamembrane region examined as a generic signaling molecule docking site

Because of variable splicing, keeping track of sequences in Trk B is challenging.  We will ignore splice variants and focus on the canonical Trk B sequence for now.  The single tyrosine in the juxtamembrane region that is phosphorylated in the  process of receptor dimerization/autophosphorylation has attracted some interest.  Many signaling molecules have phosphotyrosine binding (PTB) motifs.

Bar diagram of juxtamembrane region of Trk B

Figure 5. The juxtamembrane region of Trk B A. sequence information from UniProt.org B. sequence and cancer associations from COSMIC C. sequence information from Zheng 2014.

 

Zeng and coworkers (2014) solved an NMR structure  of the phosphotyrosine binding domain (PTB)  of fibroblast growth factor receptor substrate 2 (FRS2α ) bound to a peptide from the juxtamembrane region of TrkB.

Zeng L, Kuti M, Mujtaba S, Zhou MM. (20104) Structural insights into FRS2α PTB domain recognition by neurotrophin receptor TrkB. Proteins. 82(7):1534-41. PubMed

One of the goals of this study was to define a high affinity phospho- peptide resembling the  stereotypical RTK docking site (Fig 1), so as to test small molecule inhibitors of FRS2α  as potential cancer therapies.  The translated product of the QKI-TRK2 gene contains the transmembrane region of TrkB as well as the juxtamembrane region.

Ribbon diagrams showing the TrkB peptide (blue) and the FRS2a PTB domain

Figure 6. Trk B juxtamembrane structure, one peptide interacting with a signaling adapter protein receptor.  A.  Ribbon diagrams showing the TrkB peptide (blue) and the FRS2a PTB domain.  B.  The same structure colored from most hydrophobic (red) to most hydrophilic (green).  C. β-strands of the TrkB peptide in a surface rendering of FRS2a colored according to surface charge.

 

It should be noted that this high affinity peptide is retained in the fusion protein product of the QKI-TRK2 gene rearrangement.

Trk C, Trk B  Déjà vu

Some similarities exist between the juxta membrane region of Trk C and that of Trk B, the splice variations in Trk B not withstanding, (Figure 7A).   Regions of cancer associated mutations from the COSMIC database (Figure 7B) are also shown.  An alignment between the two domains demonstrates regions of high homology as well  as sequence divergence (Figure 7C.)  Two Trk C regions prone to mutations were chosen from Fig 7B and referenced to the alignment between Trk C and Trk B.

COSMIC database cancer associated Trk C mutations in the juxtamembrane region shown with alignment between the juxtamembrane of Trk B and Trk C

Figure 7.   The Trk C juxtamembrane region A. UniProt domains of Trk C with Trk B for comparison B. COSMIC database cancer associated Trk C mutations in the juxtamembrane region. C. An alignment between the juxtamembrane of Trk B and Trk C.

 

The green boxed region is highly conserved between Trk B and Trk C.  One would speculate that these mutations are more likely to disrupt protein function. On the other hand, the red box region is highly divergent between the two proteins.   As for TrkC fusion proteins, the myosin5-TrkB fusion protein contains the transmembrane and juxtamembrane regions of TrkB.  This is in contrast to the protein product of ETV6-TRK3 that contains no transmembrane domain and intracellular juxtamembrane domain..

The juxtamembrane region and Trk signaling

One our Trk fusion sister site we presented typical Trk signaling that included (1) PLCγ, RAS, and PI3K.  Taking a closer look at the the mutations within the juxtamembrane region brought to light  subtle nuances that may affect some but not all Trk fusions.  The cartoons in Figure 8 are not to be taken literally, just as tool for forming new hypotheses.

Cartoon showing basics of Trk signaling binding to the autophosphorylated tyrosine in the juxtamembrane region

Figure 8. Trk signaling A. The basics of Trk signaling B.  FRS2 binding to the autophosphorylated tyrosine in the juxtamembrane region.

 

Do the fusion partners of Trk fusions play a role in recruiting signaling molecules?  This appears to be the case for the TEL-TkC fusion protein’s recruitment of IRS-1.

Important Information

Use of a Trk juxtamembrane  phosphopeptide to treat cancer or to guide the development of a small molecule is an exciting possibility.   The caveat is that not all cancer driving Trk gene rearrangements have the juxtamembrane region.  The Trk phosphopeptides may be useful for fibroblast growth factor receptor driven cancers.  They may be useful in conjunction with specific Trk kinase inhibitor for which there is an open clinical trial for patients with Trk gene rearrangement driven cancers.