Lecture 1 2025 - Cell Biology PDF

The cell is the basic structural, functional, and biological unit of all known living organisms. Cells are the smallest unit of life that can replicate independently. All known types of organisms are capable of: response to stimuli Able to respond to their environm...

The cell is the basic structural, functional, and biological unit of all known living organisms. Cells are the smallest unit of life that can replicate independently. All known types of organisms are capable of: response to stimuli Able to respond to their environment, to do so, you have specialized sensors called receptors that allow you to mount a response to a stimulus. 8  response to stimuli leads to post translational modifications (PTM) What are the major types of PTM In order to respond to a stimulus a receptor is activated by something in the environment, this often elicits a PTM. PTMs are modifications that occur to a protein that alter its function in some way. There are many major types of PTMs, phosphorylation, nitration, acetylation, methylation etc. they modify proteins to activate or deactivate the protein are how the cell responds to the extracellular stimulus. We are going to talk about a class of receptors called RTKs 9  Receptor Tyrosine Kinases (RTKs) Directly phosphorylate specific tyrosines on themselves and on a set of intracellular signaling proteins. Ligands are either soluble (i.e. epidermal growth factor) or membrane-bound (i.e. ephrins) 60 genes encode human RTKs classified into 20 subfamilies Ligand Binding domain They are a family of receptors but they all function in the same way. Terms highlighted in red will be recurring throughout the course and you need to understand the meaning of. 10  The three most commonly phosphorylated amino acids in eukaryotes. Tyrosine phosphorylation is involved in signal transduction. Serine Threonine Phosphorylation occurs on hydroxyl groups (red circles) Tyrosine The phosphorylation occurs on the hydroxyl groups of the amino acids, we are talking about RTKs so when I mention the phosphorylation of the tyrosine residues this is what I am referring to (DO NOT MEMORIZE STRUCTURE) 11  Signal Always binds on extracellular side Leads to phosphorylation of Tyrosine side chains in cytosol Figure 15-52 Molecular Biology of the Cell (© Garland Science 2008) Here are some of the RTK family membranes, we have an extracellular side and an intracellular side. The extracellular side contains the ligand binding domain, this is where the ligand will bind to and activate the receptor, it is what responds to the extracellular environment. The intracellular side is the effector side, it effects a protein on the inside of the cell and will lead to a signal cascade that will cause alterations within the cell. It is a receptor that is able to phosphorylate itself, which means it must contain a kinase domain, which is on the intracellular side highlighted in red here. If you take a look at the structure of these receptors there is usually some things you can infer about their function, for instance, receptors that contain the immunoglobulin domains are likely to respond to something to do with the immune system that is going to generate immunoglobulins. Cysteine rich domains are able to be modified easily, for instance, it can respond to oxidative stress and be oxidized, which can change its ability to respond to its ligands. This kinase insert breaks up the kinase domain which can alter its activity as well 12  Table 15-4 Molecular Biology of the Cell (© Garland Science 2008) We are going to focus on the molecular details of these pathways but here you can see each of these receptors and pathways have big picture effects. 13  How do extracellular ligands transmit a signal on the other side of the plasma membrane?  For RTKs, ligand binding causes receptor dimerization and cross-phosphorylation called trans-autophosphorylation 14  Usually exist as monomers brought together by ligand binding IGF: Dimerization brings kinase domains closer together, they Phosphorylate each other EGF: Conformational Change upon binding activates kinase domain Figure 15-53a Molecular Biology of the Cell (© Garland Science 2008) Generalized cartoon to represent all RTKs Ligand is soluble. Each RTK subunit has a tyrosine kinase domain, in order to activate, these domains need to come close enough together to phosphorylate each other. There are 2 ways this is regulated, 1- pictured here: ligand binding brings the RTKs closer together so the kinase domains can phosphorylate each other, this leads to increased kinase activity and will lead to phosphorylation of other tyrosines creating Phosopho-tyrosine residues on the intracellular side of the RTK The other way, like with EGF, here the binding of the ligand to the receptor triggers a conformational change and this is what will activate the kinase domain of the receptor. This will lead to phosphorylation of other tyrosines generating phospho- tyrosine residues on the intracellular side of the RTK 15  The requirement for receptor dimerization can be exploited in a laboratory setting to investigate the function of an RTK Cells are transfected with DNA encoding a mutant form of the receptor that can dimerize but not phosphorylate Transfected means that we have delivered an artificial construct to the cells, so now these cells are expressing the receptor that has this mutation. 16  Figure 15-53b Molecular Biology of the Cell (© Garland Science 2008) One subunit has normal and one has a mutant TKD. If we expressed mutant form in this cell how is there normal versions as well? Now when the ligand binds, it brings the 2 subunits together, you do not get phosphorylation because you cannot cross phosphorylate because only one of them has an TKD. This is called a dominant negative mutation, because it only takes one mutant subunit to have the mutated effect. In the absence of one functioning receptor, the other receptor cannot function, result- receptor signalling is turned off. You would have to have high enough expression of your mutant receptor to turn off signalling completely, this would depend on the promotor you had in your DNA construct that you transfected into the cell. 17  Trans-autophosphorylation contributes to RTK activation in two ways: increases the kinase activity of the enzyme and creates high-affinity docking sites for intracellular signaling proteins Once bound, a signaling protein may become activated by: 1) itself becoming phosphorylated on tyrosines (eg IGF and IRS1), 2) binding alone may induce a conformational change 3) simply bringing it near the next protein in the signaling pathway 1- increasing the kinase activity of the enzyme: allows it to phosphorylate other residues/molecules 2- can recruit adaptor proteins that will induce a signal cascade 18  Figure 15-54 Molecular Biology of the Cell (© Garland Science 2008) This is a fully active RTK, The ligand has bound, brought the 2 subunits close together to allow for trans-auto phosphorylation, that will lead to phosphorylation of tyrosine residues which with then act as docking stations for adaptor molecules. Here we have recruited 3 different adaptor proteins (1, 2 and 3) all of which are bound to phospho-tyrosine residues and can effect changes in the cell in different ways. The phospho-tyrosine residue will always bind to some domain in specific proteins, this domain is called an SH2 domain 19  These intracellular signaling proteins have varied structure and function, however they usually share highly conserved phosphotyrosine binding domains SH2 (SRC Homology) domains or PTB (phosphotyrosine binding) domains. Through these domains, these signaling proteins can bind to activated RTKs, but also to other phosphorylated intracellular signaling proteins. Many signaling proteins contain other interaction domains (SH3 domains – binds proline rich motifs) that allow them to specifically interact with other proteins in the signaling process. Any phospho-tyrosine can recruit any protein with an SH2 domain, this can result in the formation of a protein complex to lead to a signalling pathway. 20  Figure 15-55a Molecular Biology of the Cell (© Garland Science 2008) Here we have an example of the PDGF receptor, it has one of the split tyrosine kinase domains, meaning you will get phosphorylation at different sites. The top set of phospho-tyrosine residues will recruit to and bind to SH2 domains (in red), it will recruit PI-3K. The middle ones will recruit GAP proteins and the bottom will recruit PLC. You will notice that all of these proteins also have SH3 domains (in blue) which means they can bind other proteins that have proline rich regions. What you see here is a common feature- you start with a signal stimulus – ligand binding and then this signal is amplified, it recruits 3 different protein complexes, all of which are capable of recruiting and interacting with more proteins. These signals get further amplified and you will get the effect of cell proliferation or migration etc. 21  Not all proteins that bind to RTKs via SH2 domains act to relay the signal onwards. c-Cbl protein decreases the signaling process by covalently adding a single ubiquitin (monoubiquitylation) to one or more sites on the RTK. Monoubiquitylation promotes endocytosis and degradation of RTKs by targeting to clathrin-coated vesicles and, ultimately, to lysosomes. Mono-UB targets the RTK so it is endocytosed in clathrin vesicles and it has 1 of 2 fates- to be recycled to the PM or to be targeted for degradation in the lysosome. Poly-UB, targets proteins for degradation directly via the proteosome So c-Cbl is an inhibitor of this signalling pathway, as it turns the pathway down or off 22  Figure 15-29 Molecular Biology of the Cell (© Garland Science 2008) But why? You have a receptor bound to a signalling molecule that is causing certain effects within the cell, but it is time for the signal to be turned off, the receptor can be internalized, once the pH changes in the endosome the ligand will dissociate from the receptor and the receptor can be recycled back to the PM this would be when the cell wants downregulate the response but not permanently turn it off. If it wants to stop signalling all together the receptor/ligand complex can be targeted to the lysosome for degradation 23  Mutations that inactivate c-Cbl-dependent RTK down-regulation caused prolonged RTK signaling leading to cancer. RTK endocytosis does not always result in decreased signaling. The nerve growth factor (NGF) ligand bound to its RTK can travel in an endosome along the nerve axon to signal to the cell body. When you inhibit an inhibitor of growth you almost always get cancer. Remember c- Cbl is an inhibitor of this RTK pathway, so when we inhibit the inhibitor we increase the signalling which leads to increase growth. This is because axons are very long, to promote signalling over distance this can occur- the activated RTK can actually be moved within the cell to other areas. 24  Adaptor proteins are composed almost entirely of SH2 and SH3 domains. They couple activated RTKs to important signaling proteins that do not have their own SH2 domains, such as Ras. IGFR insulin =RAS-GEF, then stimulates inactive Ras Figure 15-22 Molecular Biology of the Cell (© Garland Science 2008) Let’s Build! We have talked about SH2 and SH3 domains. The SH2 domain binds to the phospho-tyrosine residues and the SH3 domain binds to other proteins that contain proline rich regions, this builds the scaffold which eventually activates a signalling cascade in the cell. Adaptor proteins are made up of mostly SH2 and SH3 domains. They couple an active RTK to important signalling molecules that do not have their own SH2 domains. An example here is RAS. Here we have the IGF receptor, the 2 dimers come together, and undergo trans-auto phosphorylation, this results in increased kinase activity and further phosphorylation of tyrosine residues. These phospho-tyrosine residues act as docking sites for proteins (IRS1 in this case) which will recruit adaptor proteins that contain SH2 domains, like Grb2, these adaptor proteins also contain SH3 domains which allow them to recruit with and interact with other proteins to start a signal cascade and elicit a cellular response. Still building.. Up here we have a PH domain, it bind to a specific set of lipids, these molecules here on the inside of the PM are from the inositide family, they function to keep this complex docked at the PM. Scaffold proteins bind the complex to the cytoskeleton. So now we have added the connection to the intracellular highway to this activated receptor which brings in the element altering movement within the cell. We have also brought in a protein called SOS, it is a Ras- GEF, which means it is able to stimulate an inactive Ras. So far we have anchored an active RTK to the PM, to the cytoskeleton and allowed for the activation of the Ras pathway which is a family of GTPases, that now give us more ability to signal and communicate within the cell. 25  The Ras superfamily consists of various families of monomeric GTPases, but only Ras and Rho relay signals from cell-surface receptors. Ras containing covalently attached lipid groups that anchor them to the cytoplasmic face of the plasma membrane. Ras are often involved in signaling to the nucleus for activating gene expression to stimulate proliferation and differentiation. There are 2 end results that we are trying to get to when an RTK is activated, the cell needs to be able to move – needs to link to the cytoskeleton and need to be able to alter gene expression. Ras is one of the ways these tasks can be achieved. 26  Ras function as molecular switches, cycling between GTP-bound (active) and GDP-bound (inactive). Two classes of signaling proteins influence the transition between these two states: Ras-GEFs and Ras-GAPs. 27  RTKs activate Ras primarily by activating a Ras-GEF, but also by inactivating a Ras-GAP. Activator- exchange factor, exchanges GDP for GTP Inhibitor-promotes hydrolysis of GTP to GDP Figure 15-19 Molecular Biology of the Cell (© Garland Science 2008) GTPase activating protein  confusing!! Guanine exchange factor 28  Adaptor proteins are composed almost entirely of SH2 and SH3 domains. They couple activated RTKs to important signaling proteins that do not have their own SH2 domains, such as Ras. IGFR insulin Figure 15-58 Molecular Biology of the Cell (© Garland Science 2008) We are still building on the same signalling pathway. Here is a specific example that is well studied in drosophila. Remember before when I said ligands can be membrane bound or soluble? This is an example of a membrane bound one. These are 2 neighbouring cells, one of the cell is expressing the activating ligand called BOSS, it turns on the RTK, the 2 monomoers come together, you get trans- autophosphorylation, leads to phosphorylation of the tyrosine residues which then act as docking stations for adaptor proteins containing SH2 domains, These adaptor proteins also have SH3 domains that will bind to other proteins that contain proline rich regions, in this example that protein is Sos which is a Ras-GEF, so what does it do? Activates the Ras pathway. We are going to keep building on this in the next couple lectures. 29 1) Signaling between cells usually results in the activation of protein _______. A) Lipases B) Kinases C) Proteases D) Nucleases 2) What does a SH2 domain bind to? A) Phosphorylated serines B) Complementary SH2 domains on other proteins C) Unphosphorylated transmembrane receptors D) Phosphorylated tyrosines 30 3) For the receptor tyrosine kinase (RTK)-Ras-MAP kinase pathway which is important for Drosophila eye development, what would be a direct functional consequence of mutating the SH3 domain of Drk? A) Drk would no longer bind and recruit Ras-GEF. B) Drk would no longer bind to phosphorylated tyrosine residues on activated receptor tyrosine kinases. C) The receptor tyrosine kinases would no longer be phosphorylated upon binding to signaling molecules. D) The receptor tyrosine kinases would no longer dimerize upon binding to signaling molecules. E) The receptor tyrosine kinases would no longer bind to signaling molecules 31