Final: Ch 15 Signal Transduction & Assays Flashcards
pheremones (single-celled organisms)
secreted molecules that coordinate the grouping of cells for sexual mating or differentiation
in cells, a signal produces a specific response only in target cells with ______ for the signal
receptors
can the signal be a physical stimulus like light, touch, or heat OR a chemical molecule like gases, peptides, and proteins?
yes
how can extracellular signaling molecules that are too large and hydrophilic to penetrate the membrane affect intracellular processes?
bind cell-surface receptors (integral membrane proteins)
3 domains of cell-surface receptors
extracellular
transmembrane
intracellular
what happens when a ligand binds the cell-surface receptor
binding of ligand induces a conformational change in the receptor
transmitted through transmembrane domain to intracellular domain resulting in cytosolic activation or inhibition of proteins
proteins/second messengers carry signal to effector proteins
signal transduction
process of converting extracellular signals into intracellular responses
what is the most numerous class of receptor
G-protein coupled receptor (GPCR)
~900
structure of a GPCR
integral membrane receptor coupled to an intracellular G protein that transmits signals into the cell
subset of G-protein (7 transmembrane passes)
signal transduction through GPCRs usually induces short-term changes in ____ _____, such as a change in metabolism or movement
cell function
activation of cell-surface receptors that are not GPCR usually results in
alteration of a cell’s pattern of gene expression
2 cellular responses that occur when signaling molecules bind to their receptors
change in the activity or function of specific enzymes
change in the amount of specific proteins produced by a cell b/c of modification by transcription factors
what do transcription factors do
stimulate or repress gene expression in the nucleus
why is it called a signal transduction pathway
several intermediates convert the signal’s info from receptor to target
can signaling molecules act locally or at a distance?
yes
3 types of signaling from extracellular molecules
endocrine
paracrine
autocrine
endocrine signaling
signaling molecules synthesized and secreted by signaling cells
transported through circulatory system
act on distant targets
hormone is what type of signaling?
endocrine
ex. insulin (pancreas), epinephrine (adrenal glands)
paracrine signaling
the signaling molecules released only affect nearby cells
ex. nerve released nt, muscle cell
many protein ______ factors regulating development in multicellular organisms act at short range
growth factors
autocrine signaling
cells respond to substances that they themselves released
ex. tumor growth factors, cultured cells
binding specificity of a receptor refers to
its ability to bind or not bind closely related substrates
ligand binding depends on
weak noncovalent forces (van der waals, ionic, hydrophobic interactions)
molecular complementarity
examples of molecular complementarity
growth hormone
ACh
insulin (IGF-1 and IGF-2)
organisms have evolved to use a single ligand to…
stimulate different responses in cells
2 ways a single ligand can affect cells differently (effector specificity)
different cell types may have different receptors for the same ligand
the same receptor is found on multiple cell types, but binding of ligand induces a different response due to the types of proteins within the cell
2 examples of effector specificity
ACh released onto muscle induces contraction
ACh released in heart muscle slows the heart rate
what 2 enzymes are used in all signaling pathways?
protein kinases
phosphatases
act as switches (on/off)
what does a kinase do
covalently adds a phosphate
what does a phosphatase do
removes a phosphate
can a receptor posses intrinsic kinase activity?
yes, or it is rightly bound to a cytosolic kinase
if ligand is unbound, is the kinase active or inactive?
inactive
what does binding of ligand do to the kinase
changes conformation of the receptor, which activates the kinase
the kinase phosphorylates the monomeric inactive form of a TF
what happens when the monomeric inactive form of a TF is phosphorylated by the kinase
TF dimerizes and moves from cytosol to nucleus
TF activates gene transcription
what would a phosphatase in the nucleus do
dephosphorylate the TF –> 2 inactive monomers that move back into cytosol
do intracellular switch proteins turn upstream or downstream proteins on or off?
downstream
most important group of intracellular switch proteins
GTPase superfamily
bound GTP = on
bound GDP = off
conversion of inactive GTPase to active is triggered by…
a signal (hormone binding receptor)
mediated by guanine nucleotide exchange factor (GEF)
GEF causes…
release of GDP from the switch protein and replacement with GTP
GTPase-activating protein (GAP) and regulator of G protein signaling (RGS)
accelerate GTP hydrolysis –> GDP
active –> inactive
many regulators of G protein activity are controlled by …
extracellular signals
trimeric (large) G proteins
directly bind to and activated by cell-surface receptors
monomeric (small) G proteins
not bound to receptors
play roles in pathways of cell division/apoptosis
ex. Ras
what are second messengers
low MW signaling molecules that transmit and amplify signals from many receptors (bind to other proteins)
ex. Ca2+ released from ER stores or imported through channels
how to detect 2nd messenger Ca2+ changes
fluorescent dyes
ex. in muscle, rise in Ca = contraction
cAMP
2nd messenger that triggers activation of protein kinase A or opens/closes ion channels
protein kinase A (PKA)
phosphorylates target proteins to induce changes in cell metabolism / ion channels
how can GPC hormone receptors induce signal amplification?
a single receptor can activate MULTIPLE G proteins –> activate effector proteins
a single epinepherine-GPCR complex can activate up to 100 _____ cyclase molecules
adenylyl cyclase molecules –> catalyze synthesis of many cAMP molecules
2 cAMP –> activate 1 PKA –> phosphorylate multiple target molecules
2 functions of G proteins
signal transduction
signal amplification
______ activate G proteins
hormones
40% of medicines act…
biochemically by pituitary gland (GPCR pathway)
3 subunits of a trimeric G protein
alpha binds GTP
beta/gamma binds GDP
activation = release from receptor & beta/gamma subunits
adenylyl cyclase (enzyme)
catalyzes rxn of cycling ATP to cAMP (2nd messenger)
2 effects of 2nd messengers
amplification
integration - combining multiple signals (inhibitory or excitatory)
cAMP can bind to …
protein kinase C
protein kinase C (PKC)
releases catalytic subunits when activated
allosteric effect in binding cAMP
catalytic subunits
activate glycogen degrading enzymes to produce glucose
what is the dissociation constant a measure of
the affinity for a receptor for its ligand
measure of bound and unbound receptor
units = molarity
Kd =
[R] [L] / [RL]
concentration of ligand where 1/2 receptor is bound
the lower the Kd…
the lower the [L] required to bind 1/2 receptors
does a lower Kd = tighter binding?
yes
for a cellular response, usually less than ___ of the receptors need to be activated
1/2
~10^-10 M
what is TNF alpha (tumor necrosis factor alpha)
hormone secreted by immune cells to recruit more immune cells to site of inflammation
abnormal levels cause inflammation in autoimmune disease
treat excess TNF alpha disease with chimeric fusion protein
contains extracellular domain of TNF alpha receptor
fused to constant (Fc) region of human immunoglobin
binds tightly to TNF alpha to prevent it from binding cell-surface receptors
how to detect receptors and determine their affinity and specificity for ligand?
binding assay
see how much fluorescent or radioactive ligand is attached to receptors
what does a competition assay measure
measure weak ligand binding by comparing to a ligand that binds the same receptor with high affinity
how to perform a competition assay
add increasing amounts of unlabeled low-affinity ligand to a cell sample with a constant amount of labeled high-affinity ligand
calculate inhibitory constant (close to Kd)
what is competitive binding used to study usually
synthetic analogs of natural hormones
agonist
mimic function of natural hormone by binding receptor and inducing normal response
antagonist
binds to receptor but doesn’t induce a response
can block binding of natural hormone or agonist
beta-blocker
antagonist of B-adrenergic receptors in the heart (increase heart rate)
used to slow heart contractions
how is the sensitivity of a cell to signals determined?
by the number of surface receptors the cell has for that ligand
also by how high the affinity the receptors have for the ligand
desensitization
reduction in a cell’s sensitivity to a ligand
regulation of receptors
change affinity for ligand
change # of receptors present
________ of cell-surface receptors can lower the cellular response
endocytosis
how would you generate a cell with a large # of cell-surface receptor you want to purify?
recombinant DNA techniques
immunoprecipitation of kinases
Ab specific for kinase is reacted with small beads coated with protein A to bind via Fc region
beads mixed with cytosol or nucleus and washed with salt
only kinase and associated proteins are bound to Ab beads
how to determine kinase function unsing immunoprecipitation
add radiolabeled 32P ATP to mix, and see how quickly the phosphate is transferred to the product by the kinase
what use do monoclonal Ab have?
use one with selectivity for only phosphorlyated peptide to see if the kinase works with a certain signal
what is a pull-down assay used for
quantify the activation of a GTP-binding protein (western blotting)
what does a pull-down assay use
prepare beads with the binding domain of the target protein (of the GTP switch)
add to cell extract to pull-down (capture) GTP-bound form
centrifugation
western blotting with Ab
how to purify receptors that still bind ligand
like affinity chromatography but ligand is attached to beads instead of Ab
