12: Biosignaling Flashcards
4 features of signal transduction systems
SADI
Specificity: signal molecule fits binding site on complementary receptor, other signals don’t fit
Amplification: the number of affected molecules increases geometrically in a cascade
Desensitization/Adaptation: recepter activation triggers a feedback circuit that shuts off the receptor or removes it from cell surface
Integration: when two signals have opposite effects on a metabolic characteristic the regulatory outcome results from the integrated input from both receptors
affinity vs specificity
specificity: how well it fits
affinity: how tight it binds
how can receptor-ligand binding be quantified?
similar to enzyme substrate concepts. binding of L to R depends on concentration of L and R. Kd is the dissociation constant and is equivalent to 50% occupancy of R with L. Kd=[R][L]/[RL]
describe Scatchard analysis
Scatchard can be used if investigating how many binding sites there are or determining purity. Uses the equation:
[bound]/[free] = [RL]/[L] = 1/Kd(Bmax - [RL])
what we can measure is [bound] and [free]
Bmax is the total number of sites. Bmax = [R] + [RL]
12 pt1 slide 6
6 basic signaling mechanisms
gated ion channel: opens/closes in response to signal
serpentine receptor: ligand binding receptor activates an intracellular GTP binding protein which sets of cascade
steroid receptor: steroid binding to a nuclear receptor protein allows regulation of gene expression (only receptor NOT in PM)
receptor enzyme: ligand binding extracellularly stimulates enzyme activity intracellularly
-receptor with no intrinsic enzyme activity: activates gene regulating cascade
-adhesion receptor: alters interaction with cytoskeleton
describe ligand gated ion channel receptors. specific example is nicotinic acetylcholine receptor
the receptor channel is closed when no acetylcholine (Ach) is present. cooperative binding (2nd molecule is easier to bind than 1st) occurs when 2 molecules of Ach bind and the gate opens allowing Na+ and Ca2+ to flow into cell, causing depolarization. desensitization occurs after continued excitation and the gate closes with Ach still bound. Ach dissociates and the receptor is resting again ready to be excited.
describe voltage gated channels. specific example is Na+, K+, and Ca2+ channels in action potential
when the membrane is at rest, all voltage gated channels are closed. the Na+ and K+ channels open when membrane is depolarized (caused by Ach gated receptors that allow Na+ to flow in). As depolarization reaches the tips (synaptic cleft) the Ca2+ channels open and Ca2+ flows in which causes release of Ach and signal is passed on to next cell.
review membrane potential and the passive flow of ions, Na+, K+, Ca2+, and Cl-
the membrane potential is -60 to -70 mV as established by the Na+K+ ATPase which actively transports 2 K+ inside and 3 NA+ outside cell, giving cell negative charge inside. this pump action and voltage dictate passive ion flow direction
Na+ flows high to low (out to in)
K+ flows high to low (in to out)
Ca2+ flows high to low (out to in)
Cl- flows LOW to HIGH (in to out)
Cl- passive flow is explained by the negative charge and membrane voltage. according to equation, passive transport is a function of concentration in/out, ∆Vm, and the ion charge
how do neurons show integration?
they have both excitatory and inhibitory signals that are summed to determine the overall response. action potential occurs depending on whether summation of EPSPs and IPSPs results in reaching threshold or not.
describe receptor enzymes
they have an extracellular domain which binds ligand and intracellular domain which contains enzyme domain, often a kinase. ligand binding causes a conformational change which leads to change of activity of enzyme domain. cascades occur
describe insulin receptor try-specific protein kinase pathway
insulin bindes the insulin receptor which undergoes autophosphorylation on Tyr residues. the insulin receptor phosphorylates IRS-1 on its Tyr residues. SH2 domain of Grb2 binds to the IRS-1 phosphoTyr, Sos binds Grb2 SH3 domain, Sos binds to Ras and causes release of GDP and binding of GTP. Activated Ras binds and activates Raf-1. Raf-1 phosphorylates MEK, activating. MEK activates ERK. ERK activates transcription factors such as Elk1 which stimulate transcription of genes needed.
12 pt2 slide3
explain how insulin receptor is activated
autophosphorylation occurs as the activation loop, which blocks substrate binding site, is moved 30 angstroms after insulin binds. when inactive, the loop position is stabilized by Try and Asp residues, but phosphorylation of the Tyr residues causes the loop to move. this opens up the binding site for a target protein.
explain the domains on Grb2
SH2 has affinity for phosphotyrosine and SH3 has affinity for proline residues. This means that SH2 likes to bind to the activated IRS-1 when it has phosphotyrosine residues. SH2 likes to bind to Sos.
These domains are protein linkers/adapters which are discussed more later (?)
describe MAPK cascades in insulin signaling
MAPK = mitogen activated protein kinases. includes ERK, MEK, Raf-1 in insulin signaling
MAP kinases work in a variety of signal cascades
describe the other pathway activated by IRS-1 that controls glycogen synthesis
IRS-1 is phosphorylated by insulin receptor then it activates PI-3K by binding to its SH2 domain. PI-3K converts PIP2 to PIP3. PIP3 binds to PKB and it is phosphorylated by PDK1, activating, so it can then phosphorylate GSK3 and inactivate it. Inactivated GSK3 cannot convert glycogen synthase to its inactive form, so glycogen synthase is active and creates glycogen from glucose. PKB also stimulates movement of GLUT4 to the PM to increase glucose uptake
overall: blood glucose increases, insulin increases, more glycogen is synthesized
what are phosphoinositides and their structure?
important second messengers in signaling. there are many types depending on where they are phosphorylated. the general structure is the same: a glycerol backbone with two FA groups and a phosphate group linking to a hexose sugar. the sugar can be phosphorylated in many different ways
how is the insulin signal integrated?
The insulin signal causes two results: altered gene expression via Grb2-Sos-Ras and MAPK, and altered glycogen metabolism via PI3-K and PKB. the net result of the signal is determined by integration of these two processes
describe guanylyl cyclase (2 forms)
a receptor enzyme that is transmembrane, the structure is generally similar to other receptor enzymes but instead of kinase domain it has guaylate cyclase domain.
a receptor enzyme that is entirely cytosolic, the structure has a bound Heme group and is activated by nitric oxide NO binding to heme
it catalyzes conversion of GTP to cGMP, a second messenger that carries different messages throughout the cell.
where does NO come from and how does it effect vasodilation?
NO is a product of NO synthase converting arginine to citrulline. soluble guanylyl cyclase is activated by NO and it increases cGMP and out flow of Ca2+, which causes vasodilation
how is cGMP concentration regulated?
phosphodiesterase will convert cGMP to 5’GMP, which will decrease cGMP signaling such as decreasing vasodilation. (basis of viagra, which inhibits phosphodiesterase to prolong cGMP signaling and vasodilation)
components of serpentine receptors (G protein coupled receptors)
Plasma membrane receptor with 7 transmembrane segments
An enzyme to generate a second messenger (like AC or PLC)
G protein (Gs or Gi) that has three subunits
describe B-adrenergic receptor signal pathway
epinephrine signal binds to the extracellular domain of the serpentine receptor which causes the Gs protein to release GDP and bind GTP, activating. Gs alpha subunit moves to adenylyl cyclase and activates. AC catalyzes formation of cAMP which then activates PKA which phosphorylates proteins and causes cellular response of glucose mobilization. cAMP is degraded by phosphodiesterase to dampen PKA activation
what does adenylyl cyclase do? what activates it?
catalyzes formation of cAMP, a secondary messenger, from ATP. it is a counterpart to guanylyl cyclase. AC is activated by Gs-GTP (only GTP bound, GDP does not activate)
what does Gs do and how?
Gs is a self-limiting molecular switch that activates downstream proteins like AC. When GDP bound, Gs is off and cannot activate AC. contact with the hormone-receptor complex causes displacement of GDP and GTP binds. When GTP bound, Gs dissociates into a and By subunits. Gs alpha can activate AC. The protein has intrinsic GTPase activity that will eventually hydrolyze GTP to GDP and turn itself off (self-limiting) and subunits reassociate
how is PKA activated?
PKA is a tetramer with two regulatory and two catalytic domains. when no cAMP is present, the regulatory domains bind and block the catalytic active site. cAMP binding (4x) causes dissociation and opens the catalytic domain active site for substrate binding.
