Common Principles of Signal Transduction Flashcards
(47 cards)
signal transduction
process by which chemical or physical info from environment gets detected, transferred into cell, amplified, and results in biologically useful change in the cell
common features of signaling pathways
-detecting signal with surface molecules like proteins or they’re intracellular
-discriminating among the signals
-transferring info so that the extracellular signal can impact intracellular events
-form of energy might change in this process (Ex. if you’re a neuron, you might convert chemical signal into electrical signal)
-signals often need to be amplified since cells need to produce microscopic changes in response to vanishingly small quantities of extracellular signals
-cells need to adapt so that they don’t get saturated by a signal and lose their ability to respond to further changes in that signal
-cells need to take in info from multiple signals and coordinate response to that combo in way that’s advantageous
how can signal transduction processes be classified?
- type of signal
- type of signal transduction molecules involved
- site of detection
- origin and route of signal
type of signal
-most of the signals we think about are chemical in nature- ions, lipids, nucleic acids, carbohydrates, peptides, and proteins
-responding to a wide range of chemically diverse signals requires a broad repertoire of receptors
-changes in physical environment like EM forces, light, temp sensors, mechanical forces, distinguish potentially harmful stimuli
type of signal transduction molecules involved
-ligands- small molecules or large molecules that bind to sensor on surface (receptors) and there can be adaptor proteins that couple a ligand occupied receptor to downstream output molecules called effectors and these can act directly on the proteins in cell to carry out fundamental changes in cell behavior, catalyze the production of soluble secondary messenger molecules like cAMP that are in turn driving downstream activities
-sometimes all of these components exist as separate entities or you might have everything in one or receptors to interact directly without adaptor molecules or a given receptor might be able to interact with multiple adaptors to independently activate multiple downstream effectors
site of detection
used to thinking about signaling molecules being detected up at the plasma membrane but there are also receptors inside the cytoplasm and nucleus Ex. steroid hormones and viral RNA receptors
diffusible signals
-endocrine- diffusible signals produced by distant source
-paracrine- signal produced by nearby cells
-neuronal synapse- narrow distance separating two neurons and pre-synaptic neuron releases neurotransmitter into cleft and these are detected by receptors on the post synaptic membrane
-autocrine- cells respond to presence of molecules that they release as a way of gauging their own activity
anchored signals
-not all signals are soluble and are considered anchored
-some of these anchored signals are on other cells- 2 cells come together and molecules on their surface will interact with one another and results in changes in behavior of one or both cells
cell-cell interaction
-homotypic- same molecule on both cells binds to its counterpart on the other Ex. cell adhesion receptors
-heterotypic- one cell binds to another set of molecules on one cell binds to another set of molecules anchored on the surface of the other cell –> signaling pathway involving proteins called delta and notch where one is on one cell and one’s on the other
Ex. immunological synapse- another anatomically specialized structure in the immune system where antigen-presenting cells come into close contact with T lymphocytes and form highly organized structure that binds the 2 together and allows signaling to T cells
-cells can also respond to the surface they’re on like the bottom of a petri dish or ECM Ex. integrins that bind to ECM and lead to changes in integrins conformations
receptors
-molecules that bind the ligand or detect stimulus in the first place and they transduce signals across the plasma membrane or if it’s inside the cell it transduces it from one part of the cell to the other
-in the process of signaling, these receptors change in conformation or oligomerization state
catalytic activity
-some receptors have enzymatic activity in cytoplasmic domains that might lead to formation of new covalent bonds
-some receptors activate intracellular proteins like G protein signaling, kinases, txn factors
-ion channels- allow ions to flow in and out of cells
complex with other subunits
-polypeptides that form receptors to be part of multimers- constitutive multimeric complex or induced by ligand bonding or phosphorylation
-sometimes homomultimeric (multiple copies of same polypeptide to form this functional receptor) or heteromultimeric (multiple copies of different polypeptides)
–> multimeric complexes increases binding avidity with several ligand binding sites all in close proximity to each other –> increases effective sensitivity of the system b/c if one ligand molecule diffuses off of its ligand binding site, there’s another ligand binding site next door that has higher probability of it binding to
-they also increase specificity- binding pocket in multimeric cell surface receptor is formed right where 2 different subunits come together- only if you have this multimeric pocket do you have ability of ligand to bind
occupancy induced changes in activity
in the initial moments after a ligand binds a receptor, they might exhibit one level of signaling activity but over time they might either show an enhanced ability to signal or decreased ability to signal or affinity for ligand might change over time due to conformational changes
–> changes are often due to phosphorylation or PTMs in the cytoplasmic domains of the receptors
internalize ligand
-receptors can internalize ligand through receptor mediated endocytosis and that can serve through miltiple purposes: allows system to desensitize so the cell stops responding over time to continued presence of a stimulus, might be a way of bringing that ligand molecule into the cell where it can serve other purposes, or means of bringing receptor-ligand complex into the cell where it can signal another compartment
experimental design of a binding assay
-receptor needs to recognize ligands and bind them
-need affinity for ligands that’s appropriate for whatever physiological [] of ligand is
-set up binding assay to measure ligand binding affinity
-use labeled ligand mixed with source of receptor like intact cells or membrane prep from those cells or even purified protein and allow binding rxn to come to equilibrium and somehow remove the unlabeled ligand
-count how much ligand you have bound
potential issue: small number of receptors/cells
you can use radioactive or fluorescent ligand molecule, you can overexpress receptor heterogeneously for more of the receptor, or you can purify the receptor
potential issue: on/off rate of ligand can be very fast
-allow it to come to equilibrium and you go to remove the unbound with washes and over time if you have low affinity, the ligand is going to continue to leech off the cell
-you can do rapid filtration of receptor where the ligand complexes through membrane that retains complex but lets ligand through, centrifuge cells through cushion of mineral oil so the ligand molecules stay on top and cells with their ligand bound go to bottom or use size exclusion chromatography
potential issue: ligand binding to non-receptor (nonsaturable) sites- cell membrane, trapped between cells
-sometimes labelled ligand are stuck non-specfically to the PM or trapped between the cells or bound to other receptors
-separate set of binding rxns where you have excess of unlabeled ligand and unlabeled ligand displaces ligand from receptors but not able to displace the ligand trapped between cells
potential issue: ligand binding to other receptors (nonspecific)
you can add excess of site-specific ligand
experimental design of a binding assay
-cells expressing receptor of interest and radio-labelled ligand like a hormone molecule
-set up series of tubes with fixed number of cells and increasing [] of labelled ligand and no unlabelled ligand
-set up parallel series of tubes with same contents but you also include in every tube a vast excess of unlabelled ligand
typical binding assay results
-in the presence of low [] of labelled ligand and no unlabelled ligand, some of the labelled ligand molecules will bind to receptor and others get trapped
-if you have high [] of labeled ligand, you get to the point where all of the receptor sites are occupied and there’s even more of this nonsaturated binding in between cells
-in the presence of an excess of unlabelled ligand at low []s of labelled ligand, the unlabelled ligand is able to displace the labelled ligand from all of the receptor sites –> none of your receptor sites have your labelled ligand bound to them but you still have these molecules trapped between the cells
-at high []s of labelled ligand with excess of unlabeled ligand, you have displaced your labelled ligand from all of the receptor sites but now you have even more trapped labelled ligand between the cells
-set this up over big range of []s and plot it –> total binding is binding you measure at high []s in the absence of any unlabelled ligand and as you increase the [] of labelled ligand it goes up then continues to slowly climb
-low []s of labelled ligand with a lot of excess unlabelled ligand- displace all of the labelled ligand from the receptors and as time goes on shallow increase in the amount of labeled ligand that gets trapped between the cells
-curve plateaus since you only have so many receptors/cell –> peak it reaches is the Bmax (binding max), which tells you how many receptors you have on the surface of cell
-if you go to the ligand [] at which you have occupied half of the receptors, it gives you the dissociation constant to tell you the affinity of the receptor for the ligand
saturable binding
total binding minus nonsaturable binding
biochemical purification
have a ligand binding site in cells and you fractionate the cells and isolate the protein component that’s capable of binding the ligand and once you have purified that protein, you can sequence it to deduce the gene encoding it
cDNA cloning
-from partial protein sequence you are able to ID biochemically
-there’s also functional expression cloning or homology cloning that allow you to ID the cDNAs encoding a receptor
-once you have cDNA, you can tell its membrane topology like how many transmembrane proteins it has, recognizable functional domains like kinases, and you can use computational methods to predict its 3D structure
