13 | cell signaling Flashcards
what can initiate signal transduction?
involves / can be initiated by growth factors, cytokines, hormones, ECM, neurotransmitters, light, sound, etc.
what are the basic elements of cell signaling?
signal/signaling molecule (ligand, primary messenger)
receptors
intracellular signaling and effector proteins
secondary messengers
what are some small signaling molecules?
epinephrine, acetylcholine, steroids, peptides
what are some large signaling molecules?
growth factors, cytokines (proteins)
what are some secondary messengers?
Ca2+
cAMP
cGMP
IP3 / DAG
NO
how do hydrophobic molecules initiate signal transduction?
can cross the plasma membrane, entering the cell where it can then bind to a receptor
MORE DIRECT
how do hydrophilic molecules initiate signal transduction?
can’t cross the plasma membrane, so it binds to a receptor on the surface
can recruit signal transduction proteins and secondary messengers which will then eventually lead to an effector protein, a protein that causes changes in the cell (modification of cellular metabolism, function, movement)
MORE INDIRECT
what are the 4 forms of intercellular signaling?
endocrine signaling
—- hormone secretion into blood by endocrine gland (e.g. from pancreas)
paracrine signaling
—- ligand moves short distance to adjacent cell
autocrine signaling
—- ligand comes from same cell
signaling by plasma membrane-attached proteins
—- ligand moves to cell physically attached to it
give an example of a signaling molecule that can act in both endocrine and paracrine signaling
epinephrine
describe the nuclear receptor superfamily
ligands are hydrophobic (e.g. steroids) as they must be able to cross the plasma membrane
because hormones bind to the receptor inside a cell – cell immediately elicits an effect (forgo the lengthy signal transduction process)
nuclear receptor superfamily have DNA binding domain and a ligand binding domain that can accept the hydrophobic ligands
describe the activation of glucocorticoid, which is part of the nuclear receptor superfamily
the glucocorticoid receptor is generally held in check by an inhibitor protein called HSP90 —- this protein can later be displaced by a ligand
this causes the glucocorticoid receptor to bind to another glucocorticoid receptor and dimerize — the dimer then enters into the nucleus
the DNA binding domains of the individual receptors come close together and bind to response elements, sequences generally found in the promotor sequence of genes, exposing activation domains
these activation domains help bind and recruit transcription factors to increase gene expression (some times decrease)
describe the signal transduction of a hydrophilic ligand
for a hydrophilic ligand, it’ll have to bind to a receptor with an extracellular domain, activating the receptor…
usually causing a change in the structure’s cytoplasmic domain, recruiting intracellular signaling proteins/molecules
which will then activate another (series of events)…. eventually gets to an effector protein, which will lead to a change in the cell
what type of changes could signal transduction elicit in a cell?
a cytoskeletal protein may alter the shape of the cell or its movement
a gene regulatory protein may alter gene expression
metabolic enzyme may alter metabolism
describe the types of responses a cell-surface receptor could cause
can be an immediate response (short-term response) or a chain of events (long-term cellular response)
e.g. short-term, opening of an ion channel
e.g. long-term, activation of a transcription factor and then synthesis of RNA, DNA, protein, etc.
describe the GENERAL signal transduction pathway
synthesis and release the signaling molecule by the signaling cell
transport and bind the signal to a specific receptor of the target cell (change in the conformation)
initiation one or more intracellular signal-transduction pathways
short term / long term responses
termination of cellular response
describe the ligand-receptor interaction
binding specificity is based on the molecular complementarity between the interacting surfaces of a receptor (binding interface) and ligand (noncovalent forces)
—— physical compatibility, right types of charges (charge interaction), etc.
it triggers a conformational change in the receptor
very often signaling molecules (ligands) induce receptor dimerization —> recruitment of another receptor… a common way of eliciting signal transduction
what is the formula for dissociation constant. describe the formula
Kd = [R][L] / [RL]
Kd is the measure of affinity of a receptor to its ligand
the smaller the Kd, the lower the ligand concentration required to occupy 50% of the cell surface receptors = higher affinity and specificity between receptor and ligand (ideal!) as it will unlikely leave to inappropriate activation of other receptors
what is a functional assay?
a functional expression assay is used to identify the cDNA that encodes a cell surface receptor, especially when the receptor is membrane-bound and could be damaged by cell lysis.
how to perform a functional assay?
Start with cells that don’t respond to ligand X (they don’t have the receptor).
Get RNA from cells that do respond to ligand X (they express the receptor).
Convert the RNA into cDNA to make a cDNA library (a collection of all expressed genes, including the receptor).
Introduce (transfect) the cDNA library into the non-responsive cells.
Screen for cells that now respond to ligand X (e.g. show proliferation or signaling), meaning they likely took up the receptor cDNA.
Identify the cDNA responsible using PCR and sequencing.
Use the sequence to determine the receptor protein, and optionally, mutate parts of it to find out which regions are critical for ligand binding.
This is a genetic approach to finding receptors without damaging them, by observing function in cells
what are the 2 main categories of kinases?
tyrosine kinase
serine/threonine kinase
how is protein activity regulated?
kinases activate proteins by adding phosphate groups
phosphatases inactivate proteins by removing the phosphate group from the amino acid
how do we observe the activation of signal transduction pathways?
via phospho-specific antibodies
these antibodies will bind to the protein only when it’s phosphorylated on a particular amino acid
mouse is injected with phospho-peptide to generate a monoclonal antibody via immune response
what are G-proteins
called G-protein because interacts with guanine molecules GTP/GDP
what are the 2 forms in which G-proteins exist?
active –> bound to GTP, causes a physical change in the protein… the “arms” fold inward
inactive –> bound to GDP… “arms” swing back
how are G-proteins regulated?
activation is triggered by a signal (a hormone binding to a receptor) and is helped by guanine nucleotide exchange factor (GEF) which removes the GDP. subsequent binding of GTP leads to activation
conversion back to inactivate state is mediated by GTPase (often part of the GTP binding protein itself)
GTPase-activating proteins (GAP) can accelerate GTP hydrolysis (GTP -> GDP, helps turning it off)
what are GPCRs?
G-protein coupled receptor system
a receptor with 7 membrane-spanning domains
the receptor itself is not a G-protein but it instead causes the recruitment of G protein complexes (that are trimeric)
it is a membrane-bound effector protein
a second messenger in many GPCR pathways
differentiate between agonists and antagonists
agonists = turn receptor on
antagonists = turn receptor off
describe the G-protein complex
heterotrimeric (3 components)
—- G-alpha, G-beta, G-gamma
G-alpha and G-gamma are lipid-anchored proteins at the cytoplasmic face of the plasma membrane
G-alpha links to the receptor (GPCRs)
G-alpha-GDP is inactive … G-alpha-GTP is active
what is the active and inactive form of G-alpha?
active = G-alpha-GTP
inactive = G-alpha-GDP
describe the activation pathway of GPCRs
after ligand binds to receptor, a conformational change occurs… the tail sticking out in the cytoplasm will now have high affinity for the heterotrimeric G-protein complex (specifically the alpha subunit)
when binding to the receptor, the alpha subunit undergoes a conformational change. GDP is removed and replaced with GTP.
the alpha subunit detaches from the beta and gamma subunit… the alpha subunit slides along the plasma membrane to an effector enzyme protein, activating it… while this occurs, its GTP is hydrolyzed to a GDP
the alpha subunit reattaches to beta-gamma
note: in some cases, G-alpha inhibits effector proteins and G-beta-gamma activates effector proteins – usually an ion channel, generally causing an efflux of ions out of the cytosol
what is a common effector protein for the G-alpha subunit of the G-protein complex?
adenylyl cyclase
