TRK Alterations

First we must distinguish whether we are talking about the gene or the protein that is coded by the gene. Generally, the gene name is given in italicized capital letters. The protein name can be in lower case letters, perhaps with the first letter capitalized if it is a proper name.  TRK1 or NTRK1 is a gene. TrkA is the protein product of this gene. When we discuss TRK alterations we must distinguish whether we are referring to the gene, the protein, or both. Alterations in the gene as well as the protein may occur at multiple levels of complexity.

Levels of complexity within the gene

Genes reside on chromosomes. Chromsosomes contain both DNA and protein. Histones make up the bulk of the protein in chromosomes.  Histones are spools on which DNA is wound and condensed. Winding and unwinding of DNA depends on post translational modification of N-terminal amino acids of histones. These modifications include acetylation, phosphorylation, and methylation. While these modifications should not be considered Trk alterations, they are part of the epigenetic phenomenon that impacts the presence of the translated Trk protein in the cell.

Cartoon showing DNA structural complexity of <em>TRK</em> gene and its relative impact on its protein function.

Figure 1. Levels of complexity of TRK, the gene, that may impact Trk functioning within the cell

Genes are not just the protein coding regions in the exons. Intervening sequences called introns are spliced out of the messenger RNA before it is translated into protein. Alterations in splicing of introns results in a variety of different protein coding “exons” in the message. The final outcome is alterations in the naturally occurring Trk protein. Some of the natural alterations called splice variants in Trk occur at this level.

Upstream (5’) of exons and introns are the promoter and the region of the gene coding for the 5’- UTR (untranslated) portion of the Trk message. The 5’-UTR is responsible for the organization of the mRNA translational machinery. Translation of the Trk kinase domain is regulated by the 5’ region of the fusion partner. The mRNA stability is governed by the 3’-UTR that contains microRNA binding sites as well as a polyA tail.

Levels of complexity within the protein

Four levels of complexity are generally considered to define protein structure. Primary structure is simply the amino acid sequence. Shown is an alignment of a portion of the extracellular domain of human, mouse, and chicken TrkA (Cole 2014). Representative fish and frog sequences were also presented in this publication as well as TrkB and TrkC sequences for each species. Conserved residues are shaded.  Note, there are single amino acid alterations in the TrkA sequence from one species of vertebrate to another.  Can we be sure that single amino acid  alterations in the human sequence of one of the Trks that coincidentally occur with cancer are actually driving the cancer?

Picture showing TrkA amino acid sequence complexity comparison in human, mouse, chicken, zebrafish and xenopus. In set shows beta and alpha configuration.

Figure 2 The first two levels of complexity in protein structure: primary and secondary structure.

The secondary structural motifs β-sheets are held together by hydrogen bonds between backbone amine nitrogens and carboxyl oxygens (arrows). R is any amino acid side chain. Note that the strands in β-sheets go in opposite directions. Another secondary structure motif, α-helices, are also held together by carboxyl oxygen and amine nitrogen hydrogen bonding between helical turns.

Tertiary structure of a protein is how the various structural motifs like β-sheets and α-helices fold together to make a functional protein. Electrostatic interactions between positively ( e.g. lysine) and negatively charged (e.g. aspartate) are one driving force.  Another driving force is to remove hydrophobic amino acid side chains to interior regions of the protein such they do not interact with water.  “Oil and water do not mix.”  When we see a very hydrophobic amino acid (e.g. tryptophan) in Trk substituted with a very charged amino acid (e.g. lysine) we have to wonder if this alteration might affect protein folding and therefore protein function. A small portion of the extracellular domain of TrkC, the leucine rich repeats (LRR) and one of the immunoglobin repeats, is given as one example of protein tertiary structure. Instead of showing the ribbons used to denote secondary structure, we are showing the surface of the protein that a small molecule in solution would “see.”   Another example of tertiary structure are some immunoglobin like repeats in the receptor protein tyrosine phosphatase σ (Cole 2014).

Pictures depicting TrkC protein structure and its interaction with receptor tyrosine phosphatase.

Figure 3 Higher order of protein structure: tertiary and quaternary.   Tertiary structure is the single protein. Extracellular domains of Trk interact with extracellular domains of a receptor tyrosine phosphatase  The interaction of the tertiary structures of two or more protein to form a combined structure is tertiary structure.

The quaternary structure is how the tertiary structure of one protein interacts with the tertiary structure of another protein. Our example of quaternary structure is some extracellular domains of the chicken version of TrkC in complex with PTPσ. Note from the surface plot that there really isn’t much room between the two interacting proteins. Most research has centered around cell-cell interactions of these domains in the synaptic cleft. Interaction off PTPσ with extracellular matrix components regulates how it may interact with extracellular domains of Trk. We will cover evidence for interaction of receptor PTP and Trk in the same cell in another blog.

Important information

Many different levels of alteration may affect Trk kinase function.  Perhaps the most all encompassing is the gene rearrangement in which the promoter and 5′-UTR end of a partner gene is fused to the kinase domain of Trk and the 3′-UTR portion of the TRK gene. These events not only impact transcription but also the control of kinase activity.  A TRK inhibitor clinical trial is accepting new patients.  Phase 1 results of this Trk treatment are available on line.

 

Reference

Coles CH, Mitakidis N, Zhang P, Elegheert J, Lu W, Stoker AW, Nakagawa T, Craig AM, Jones EY, Aricescu AR. (2014) Structural basis for extracellular cis and trans RPTPσ signal competition in synaptogenesis.  Nat Commun. 2014 Nov 11;5:5209. PubMed