Enzyme-Linked Receptors

Recognize the significance of protein phosphorylation in regulating protein function and therefore cellular processes

Protein phosphorylation is, as its name suggests, the addition of a phosphate group to a protein. This often serves to turn the protein "on" or "off" as the addition of the phosphate group adds bulk and a negative charge, which might help other proteins and substances within the cell to interact with it.

Recall major groups of enzyme-linked receptors and their ligands

Probably the most common groups of enzyme-linked receptors that you will encounter are kinases (enzymes that phosphorylate other proteins). They come in two types: receptor serine/threonine kinases and receptor tyrosine kinases (RTKs), all named after the residues that they add phosphate groups to. They are both bound by various growth factors. RTKs tend to be bound by other ligands such as insulin. (The only example given as a ligand for receptor serine/threonine kinases is transforming growth factor TGF-β, but I'm sure there are more than just that one.)

Three other types of receptors that you need to know are cytokine receptors, receptor guanylate cyclases and receptor tyrosine phosphatases. Cytokine receptors bind molecules known as cytokines, which according to http://www.news-medical.net/health/What-are-Cytokines.aspx aid in cell-to-cell communication and cell movement during immune responses. (From what I've heard, I'm going to hear about cytokines way too much during Immunology next semester.) As for the other two? Receptor guanylate cyclase is like the guanine version of adenylate cyclase: it converts GTP into cyclic GMP (cGMP). They are activated in response to natriuretic peptides, which help get rid of excess Na⁺ and H₂O. Receptor tyrosine phosphatases remove phosphate from tyrosine residues and tend to exhibit constitutive activity, which means that they don't have to be bound to have an effect.

Describe the general structure and signalling pathways of kinase-linked receptors

Kinase-linked receptors are enzyme-linked receptors that come in two varieties: those with intrinsic kinase activity (i.e. they can add phosphate groups onto stuff all by themselves) and those without intrinsic kinase activity. Like many other receptors, they have three main parts: an extracellular N-terminal domain, a transmembrane domain and an intracellular C-terminal domain. Most enzyme-linked receptors (and kinase-linked receptors by extension) dimerise when bound, and this dimerisation is required for them to have an effect. Receptor tyrosine kinases (RTKs) are kinase-linked receptors with intrinsic kinase activity, whereas many cytokine receptors are kinase-linked receptors without this activity. I'm going to go through both of these cases separately.

Kinase-linked receptors with intrinsic kinase activity

When RTKs (which have intrinsic kinase activity) dimerise, the two monomers can phosphorylate each other in a process known as autophosphorylation or transphosphorylation. This allows recruitment of Grb2 (growth factor receptor bound protein 2). Grb2 then acts as GEF (guanine exchange factor) that removes GDP from the G-protein Ras, allowing GTP to bind. This activates Ras, which activates Raf, which is also known as MAPKK Kinase, or MAPKKK. This then phosphorylates Mek (MAPK Kinase/ MAPK), which phosphorylates MAP Kinase (MAPK), which then phosphorylates various transcription factors, which ultimately leads to gene transcription. (Raf, Mek and MAP Kinase are all serine/threonine kinases. Don't know how important that bit of info is, but it was in the slides.)

So, in short: Ligand binding -> Dimerisation -> Autophosphorylation -> Grb2 recruitment -> Ras activation -> Raf/MAPKKK -> Mek/MAPKK -> MAPK -> transcription factors -> ??? -> PROFIT!!!

Kinase-linked receptors without intrinsic kinase activity

Cytokine receptors lack intrinsic kinase activity, so they recruit another protein called Janus Kinase (JAK) to do their dirty work for them. JAK binds and phosphorylates another protein called STAT, which is a short way of saying Signal Transducer and Activator of Transcription. When STAT is phosphorylated, it can dimerise. STAT dimers can then stimulate gene transcription in the nucleus.

Describe the mechanisms of activation of receptor tyrosine kinases

In order to carry out their catalytic function, the active site of the receptor tyrosine kinases must be accessible. Sometimes, this active site must also be phosphorylated. Conversely, when a receptor tyrosine kinase is inactive, that is because the active site has been rendered inaccessible in some way.

The first mechanism in which this can happen is through the formation of an "activation loop," or "activation loop inhibition." The insulin receptor is an example of a receptor that does this. In its inactive form, a tyrosine residue projects into the active site as if it's meant to be the target of phosphorylation. This stabilises the configuration of the "active loop." When this activation loop inhibition is removed (by autophosphorylation of the tyrosine residues on each monomer), the receptor is activated.

The second mechanism is known as "juxtamembrane inhibition." In this mechanism, residues near the membrane ("juxta"- next to, "membrane"- membrane) interact with parts of the tyrosine kinase domain, including the active loop. Once again, when this inhibition is removed, the receptor is activated. KIT is an example of a receptor that uses this mechanism. (According to Google, KIT is another name for the mast/stem cell growth factor receptor.)

The third mechanism is known as "C-terminal tail inhibition." To my understanding, this is kinda similar to juxtamembrane inhibition, but this time it's residues near the C-terminal that are doing the trick, not the juxtamembrane residues. Tie2 is an example of a receptor that uses this mechanism. There seem to be a shitload of functions for this receptor, so I'm not even going to try and list all of them- hop over to http://www.phosphosite.org/proteinAction.action?id=1246 if you're really interested.

The fourth and final mechanism is allosteric activation. This mechanism requires two tyrosine kinases to work together: one as the activator and the other as the receiver. As the names suggest, the activator activates the receiver. EGF (epithelial growth factor) uses allosteric activation.

Describe the structure of insulin receptor, the signal transduction and effects associated with the activation of insulin receptor

Unlike other enzyme-linked receptors that I've written about so far, the insulin receptor does not dimerise when bound. This is because it naturally exists as a homodimer. Each monomer has an α subunit which is located entirely extracellularly, and a β subunit which pokes into the cell. These two monomers are linked by disulfide bonds (I'll write up a bit more about insulin in a later post for BIOC2001). Therefore, the overall structure of insulin can be represented by (αβ)₂.

Insulin exerts its effects in several different ways. Firstly, I'll have a look at the way in which it exerts its long-term effects. Insulin interacts with a protein called IRS, or insulin receptor substrate. This is an "adaptor" protein that interacts with Grb2, which interacts with Ras, Raf, Mek, MAP Kinase and finally with transcription factors. If you think that this looks familiar, you're right: as I mentioned before, this is a general pathway for many kinase-linked receptors with intrinsic kinase activity.

There are two pathways in which insulin exerts its short-term effects. Fortunately we don't have to know the details- just the names and what they do. Unfortunately, even the names are pretty long. The first pathway is the APS/c-CBL/CAP-mediated pathway, which creates targeting sites for GLUT4 storage vesicles (GSVs) on cell membrane. (Some quick definitions: targeting sites tell the vesicles where to go, and the vesicles here are carrying GLUT4 which is a glucose transporter.) The other pathway is the IRS/PI3K/Akt-mediated pathway, which causes the translocation and exocytosis of GSVs. This results in transporters being inserted into the membrane.

Phew! That was a long haul. There are still some things I'm not 100% sure on, such as the activation processes. But that's something I'll need to check up on another day.