Sunday, February 19, 2012

Achondroplasia: following FGFR3 in the chondrocyte

Let’s start to see how things work in achondroplasia by reviewing how the fibroblast growth factor receptor 3 (FGFR3) is created, how it exerts its actions and how its fate is. The text may seem not exactly like a cookies’ recipe. However, talking a little bit about these topics will help the reader to understand why some potential approaches to treat achondroplasia are attractive and worth to be explored and others are not.

Before diving in the more technical parts, it will be useful to explain some terms and expressions being applied here:

Amino acids: these are molecules essential for life and when combined in predetermined sequences – imagine a sequence of bricks, one after another – they constitute the Proteins.
Cell matrix or interstitium: the medium inside the growth plate where the chondrocytes are located.
Cell membrane: this is the external cover of the cell, keeping the cell content safe and functioning as an in-and-out gate.
Cytoplasm: this is the medium inside the cell where the cell organelles and molecules lie and exert their functions.
Domains: the different parts of a protein. This expression may be used to denote a specific part of a protein bearing a distinctive pattern.
Nucleotides: these are the molecules that constitute the genetic code. There are four nucleotides and the combination of them produces all information needed to create and maintain life.
Protein: proteins are large molecules made of predetermined sequences of amino acids. When they cause chemical reactions, they are also called enzymes.

The FGFR family

FGFR3 pertains to a family of four similar proteins (named 1 to 4). These four proteins (or enzymes) are called receptors because they are located across the cell membrane, which makes one of their extremities being positioned outside the cell membrane (extracellular domain), in contact with the local environment (often called cell matrix or interstitium) and the other, inside the cell, exposed in the cytoplasm (intracellular or tyrosine kinase domain).

Their function is to work as communication channels between the matrix (or, in a broader vision, the body) and the cell. How does this communication work? It is through chemical contacts between locally circulating molecules or activators and these receptors. Messages are passed on in a chemical language. There are many other classes of receptors with distinct functions also placed across the cell membrane. These communication channels allow cells to easily respond to local changes in the external environment, the matrix. Although they have distinct functions, in many of them the way used to exert their actions is similar. We must save this information for later, because it has consequences.

The four FGFRs share a common basic structure and respond to the same activators (or, in the jargon, ligands) called fibroblast growth factors (FGFs). Compared to many other receptor proteins, the FGFR structure is quite simple. The extracellular part (or domain) is composed of three amino acid loops that resemble the structure ofimmunoglobulins (we know these structures by their popular name: antibodies). There is a transmembranedomain (naturally, the part of the protein placed across the cell membrane). And, finally, the intracellular domain, where lies the part of the protein which, when the receptor is turned on, will trigger series of intracellular reactions, commonly called signaling pathways or cascades. You can see a schematic figure of the FGFRs at Dr. Mohammadi’s website.

Now, let’s focus on the FGFR3 and the chondrocyte.

We will be talking about the steps FGFR3 makes from its production to its fate, with emphasis in its synthesis (production) and activation:  

  • Synthesis
  • Transport 
  • Activation 
  • Degradation
  • It reduces the speed of the cell multiplication and 
  • It slows the pace the cell get older (differentiate, becomes hypertrophic).
  • It reduces the cell proliferation rate
  • It reduces the cell maturation rhythm (differentiation, hypertrophy)

FGFR3 synthesis

Like any other protein, FGFR3 is produced (expressed) when its specific gene is read and the instructions it holds in the form of a chain of four molecules called nucleotides are translated to a chain of amino acids. In a very simplified model, each sequence of three nucleotides in the DNA sequence will give origin to the choice of one amino acid. You may read more about this here.

Simplifying again, let’s see the FGFR3 production sequence: the part of the DNA where lies the gene for FGFR3 is opened like a zipper. Then, tailor proteins, reading the nucleotide sequence, build a copy of the DNA guideline with free nucleotides available in the vicinity. The tailored DNA copy is called messenger RNA, or mRNA, and the process in which the gene is read is called transcription. The mRNA will leave the nucleus towards the ribosome, a small cell organelle.  There, the code in the mRNA will be read again and translated into amino acids, which will be assembled in a chain, creating the protein.

 In summary, the FGFR3 gene is read and copied through a process called transcription and the RNA copy is further read and translated into a chain of amino acids, the protein. You can learn more about gene transcription and protein expression watching this interesting animation.

The whole process, basically described here, is tightly regulated. There are many proteins and small pieces of other kinds of RNA molecules involved in the gene reading and in protein assembly, working as quality controls or checkpoints. They identify reading errors and correct them or send the defective product for degradation. Some of these small RNAs can also either start the gene frame reading or stop it, thus having a regulatory role (save this information for later). The system is not devoid of errors and many diseases and conditions are a result of failures in the quality control or when the defect is not identified as so, allowing changed proteins to become active. This is the case of the mutation of the FGFR3 in achondroplasia.

FGFR3 shaping and transport

After the amino acid sequence is built into a new protein, another chemical reaction starts. A group of proteins called chaperones make the ‘final arrangements’ in the new protein, putting it in the right shape, ready to be positioned across the cell membrane, which is a step mediated by an intracellular transport mechanism. In the case of FGFR3, the commonly involved chaperone is the one called HSP-90. If chaperones fail to finalize the protein, the new product usually is directed to degradation, too. Although carrying a composition error, the mutated FGFR3 keeps its functionality and is normally released for transport to the cell membrane. The transport system is a common path for many other proteins.

FGFR3 activation

FGFR3 is activated (turned on, like an electric switch) when a FGF binds to the extracellular domain of the receptor. In a reaction in which other matrix components also participate, the binding of the FGF will attract another molecule of FGFR3, which will be positioned with the first one in a structure called dimmer. The dimmer suffers conformational changes, we could say turnings, which will expose specific sites, like electric plugs, in the intracellular domain. When these plugs, called literally adenosine triphosphate (ATP) pockets, are exposed they attract phosphate carriers and the cascade begins: the signaling downstream pathway is activated through this process called phosphorylation. Several proteins are attracted to the FGFR3 activated tail, one becoming activated by the previous one and activating the next one: the cascade. Take a look in this article by Dr. William Horton, where there is a figure showing the cascade.
 There are two main different cascades turned on with the activation of FGFR3: the so called RAS-RAF-MAPK pathway and the STAT1 pathway. Let’s go here step-by-step, no rush.

The RAS and RAF proteins are extremely important to many biological processes. They are a kind of cell signal distributors lying just in the first steps of the signaling cascades of many receptor enzymes. In the case of FGFR3, the first important cascade they will trigger is the one headed by the mitogen activated protein kinase (MAPK) enzymes.

Once activated, RAF will ‘call’ the MAPK enzymes named MEKs (1 to 6). Which ones will be activated will depend on the origin of the signal upstream. For FGFR3, the most important are MEK1, MEK2, MEK3 and MEK6. MEK1 and MEK2 will then trigger a couple of enzymes called extracellular-signal regulated kinases (ERK1 and ERK2), while MEK3 and MEK6 will activate another enzyme called p38. The ERKs and p38 are the enzymes responsible for delivering the message from the matrix outside to the cell nucleus, where the response to the external signal will be generated, through the expression of some genes and/or the repression (inhibition) of others.

STAT1 (signal transducer and activator of transcription 1) is a cellular agent working directly in the nucleus in response to extracellular stimuli. When activated, it will tell the cell nucleus to reduce or stop the actions needed to let the cell multiply. In other words, it will reduce the cell proliferation rate.

ERKs and p38, in response to the signal initiated by FGFR3, will stimulate the nucleus to reduce the pace that chondrocyte is maturing (progressing to the hypertrophic state).
So, here are the main consequences of having the FGFR3 overactive in achondroplasia: 
FGFR3 Degradation

The last phase the FGFR3 passes through is the deactivation or degradation. You can have an excellent description of the process reading Dr. Horton’s Lab Blog in Growing Stronger: his group is working exactly with the steps involved in the metabolism (cleavage) of FGFR3. This is a field with still many questions to be answered but may bring one of the potential therapeutic solutions for achondroplasia.

We have made this complex trip through the chondrocyte to understand the machinery involved in the production of FGFR3. We also made a brief review of the main reactions FGFR3 causes in the chondrocyte. Now, we are ready to explore the potential approaches to counteract the defective FGFR3.

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