Growth Factors and Receptors Flashcards
Describe different methods of cell signalling
(A) Contact-dependent signaling requires cells to be in direct membrane-membrane contact
(B) Paracrine signaling depends on local mediators that are released into the extracellular space and act on neighboring cells.
(C) Synaptic signaling is performed by neurons that transmit signals electrically along their axons and release neurotransmitters at synapses, which are often located far away from the neuronal cell body.
(D) Endocrine signaling depends on endocrine cells, which secrete hormones into the bloodstream for distribution throughout the body. Many of the same types of signaling molecules are used in paracrine, synaptic, and endocrine signaling; the crucial differences lie in the speed and selectivity with which the signals are delivered to their targets.
Describe Cytokine Signalling
Cytokine signalling is usually paracrine, but can be contact-dependent, autocrine, or endocrine. It is not synaptic.
State Cytokine Receptor Families
- Tyrosine kinase receptors (such as RET)
- Serine-threonine kinase receptors
- Immunoglobulin-like receptors:
Class 1 cytokine receptors (such as LIFR/gp130)
Class 2 cytokine receptors
- Tumour necrosis factor-related
- G-protein coupled receptors
Simplified Signalling
- A simple intracellular signalling pathway activated by an extracellular signal molecule.
- The signal molecule usually binds to a receptor protein that is embedded in the plasma membrane of the target cell.
- The receptor activates one or more intracellular signalling pathways, involving a series of signalling proteins.
- Finally, one or more of the intracellular signalling proteins alters the activity of effector proteins and thereby the behaviour of the cell.
More Simplified Signalling
- A variety of protein messengers (growth factor ligands, light green rectangles, top) interact with a complex array of cell surface receptors, which transduce signals across the plasma membrane (gray) into the cytoplasm.
- There, a complex network of signaltransducing proteins processes these signals, funnels signals into the nucleus (bottom), and ultimately evokes a variety of biological responses (“output layer,” yellow rectangles, bottom). Many of the components of this circuitry, both at the cell surface and in the cell interior, are involved in cancer pathogenesis.
- This cartoon focuses on a small subset of the receptors—the EGF receptor and its cousins—that are displayed on the surfaces of mammalian cells.
- Receptors like these are the main topic of this chapter; the adaptors and signaling cascades will be covered in the next chapter.
- The X’s associated with the cytoplasmic domain of HER3 indicate the absence of detectable tyrosine kinase activity in contrast to the readily detectable kinase activity of the other three members of this family of receptors.
WHAT IS THE PRIMARY MECHANISM FOR
CYTOKINE/GROWTH FACTOR RECEPTOR ACTIVATION
?
LIGAND-MEDIATED OLIGOMERIZATION
Which Receptor Families are Activated via
Ligand-mediated Oligomerization
- Receptor Tyrosine Kinases
- Serine/Threonine Kinase Receptors
- Class I and Class II Cytokine Receptors
- Tumour Necrosis Factor Family
- Immunologlobulin Superfamily Receptors
Draw Ligand-mediated Oligomerization
Describe the Juxtaposition of the Cytoplasmic Domains
- By bringing the extracellular domains together, the ligand also brings the cytoplasmic domains together.
- The juxtaposition of the cytoplasmic domains triggers signal transduction.
- Both the proximity and the orientation of the cytoplasmic domains are likely to be important for optimal signaling.
Examples of Tyrosine Kinase Receptors
Only one or two members of each subfamily are indicated.
Note that in some cases, the tyrosine kinase domain is interrupted by a “kinase insert region” that is an extra segment emerging from the folded kinase domain. The functions of most of the cysteine-rich, immunoglobulin-like, and fibronectin-type-lll-like domains are not known.
Give examples of Cytokines Acting Through RTK
Typical Receptor Structure
A) The receptor for epidermal growth factor (EGF-R) is a complex protein with an extracellular domain (ectodomain, green), a transmembrane domain that threads its way through the plasma membrane (brown), and a cytoplasmic domain (red, blue). The ligand-binding domain is responsible for binding EGF. Similarity in amino acid sequences demonstrates that one region of the cytoplasmic domain (wide red rectangle) is related to a region in the Src oncoprotein (gray).
Explain Homology in Kinase Domains
(A) The receptor for epidermal growth factor (EGF-R) is a complex protein with an extracellular domain (ectodomain, green), a transmembrane domain that threads its way through the plasma membrane (brown), and a cytoplasmic domain (red, blue). The ligand-binding domain is responsible for binding EGF. Similarity in amino acid sequences demonstrates that one region of the cytoplasmic domain (wide red rectangle) is related to a region in the Src oncoprotein (gray).
(B) Comparison of the amino acid sequences (using the single-letter code) of the cytoplasmic domains of the EGF-R (labeled “ErbB” here) and Src revealed areas of sequence identity (green), suggesting that the EGF-R, like Src, emits signals by functioning as a tyrosine kinase. While the sequence identities seem to be quite scattered, the shared residues nonetheless indicate clear evolutionary relatedness (homology) between Src and ErbB. Yet other viral oncoproteins, such as Abl and Fes, some of whose sequences are also shown here, were found to share some sequence similarity with these two. The viral oncoproteins specified by the raf and mos oncogenes, which function as serine/threonine kinases, are seen to be more distantly related, sharing even fewer sequences with Src and the EGF-R. The dashes indicate amino acid residues that are missing in one protein but are present in one or more of its homologs; these have been introduced to maximize the sequence alignment between homologs.
What is a protein tyrosie kinase?
Protein-tyrosine kinases are enzymes that catalyze the transfer of the γ-phosphate of ATP to tyrosine residues of protein substrates.
Src can phosphorylate an anti-Src antibody revealing the activity of the oncogene
Protein kinases operate by removing the high energy γ phosphate group from ATP and attaching it to the hydroxyl groups in the side chains of serine, threonine, or tyrosine residues of substrate proteins.
(A) The antibody molecule that was used to immunoprecipitate Src molecules also happened to serve as a substrate for phosphorylation by this kinase. In cells, Src phosphorylates a wide range of protein substrates.
(B) This experiment revealed, for the first time, the biochemical activity associated with an oncoprotein. Normal rabbit serum (blue + signs) or serum from a rabbit bearing an RSV-induced, Src-expressing tumor (which contained antibodies against Src; red + signs) was used to immunoprecipitate cell lysates that were incubated with 32P-radiolabeled ATP. Lysates were prepared from uninfected chicken embryo fibroblasts (CEFs); CEFs infected with avian leukosis virus (ALV), which lacks a src oncogene; CEFs infected with wild-type (wt) RSV; or CEFs infected with a transformation-defective (td) mutant of RSV, which lacks a functional src gene. Only the combination of tumor-bearing rabbit serum and a lysate from wt RSV–infected CEFs yielded a strongly 32P-labeled protein that co-migrated with the precipitating antibody, indicating that the antibody molecule had become phosphorylated by the Src oncoprotein.
Autophosphorylation of EGFR: Phosphotyrosine is ligand-dependent
A) When human A431 epidermoid carcinoma cells, which greatly overexpress the EGF-R, are incubated in 32PO4-containing medium and then exposed to EGF, a radiolabeled protein can be immunoprecipitated with an anti-phosphotyrosine antiserum (lane 2); this protein co-migrates upon gel electrophoresis with the EGF-R and is not detectable in the absence of prior EGF treatment (lane 1).
(B) The 32PO4-labeled phosphoamino acids borne by the proteins in such cells in the absence of EGF (as resolved by electrophoresis; are seen here.
(C) Following the addition of EGF to A431 cells, a spot in the lower right becomes darker; internal markers indicate that this is phosphotyrosine.
What is the Activation Loop?
Activation loop (A-loop, phosphorylation lip; pseudosubstrate)
How is Autophosphorylation not Limited to the A-loop
- In the absence of extracellular signals, most RTKs exist as monomers in which the internal kinase domain is inactive. Binding of ligand brings two monomers together to form a dimer.
- In most cases, the close proximity in the dimer leads the two kinase domains to phosphorylate each other, which has two effects.
- First, phosphorylation at some tyrosines in the kinase domains promotes the complete activation of the domains. Second, phosphorylation at tyrosines in other parts of the receptors generates docking sites for intracellular signaling proteins, resulting in the formation of large signaling complexes that can then broadcast signals along multiple signaling pathways. Mechanisms of dimerization vary widely among different RTK family members.
- In some cases, as shown here, the ligand itself is a dimer and brings two receptors together by binding them simultaneously. In other cases, a monomeric ligand can interact with two receptors simultaneously to bring them together, or two ligands can bind independently on two receptors to promote dimerization.
- In some RTKs—notably those in the insulin receptor family—the receptor is always a dimer, and ligand binding causes a conformational change that brings the two internal kinase domains closer together.
- Although many RTKs are activated by transautophosphorylation as shown here, there are some important exceptions, including the EGF receptor
What allows visualization of receptor activation?
- The use of a fluorescent reagent that binds specifically to a phosphotyrosine residue on the EGF-R enables the visualization of receptor activation following ligand binding. Here, receptor activation is measured on a monkey kidney cell at a basal level (0 second), as well as 30 and 60 seconds after EGF addition.
- In addition, following a 2-minute stimulation by EGF, the effects of a 60-second treatment by a chemical inhibitor of the EGF-R kinase (AG1478) are shown (right), indicating that the EGF-induced activation of the EGF-R can be rapidly reversed.
- Receptor activity above the basal level is indicated in blue, while activity below the basal level is indicated in red. The response to AG1478 treatment indicates that a significant basal level of EGF-R activity was present (at 0 second) even before EGF addition
Activating Mutations in the Tyrosine Activating Mutations in the Tyrosine Kinase Domain Occur Throughout the Kinase Domain
(RET=fuchsia)
Mutated RTK in Cancer
Inactivating mutations can create dominant negatives
