lecture 9 Flashcards
signal transduction
- conversion of one signal to another
types of signals
growth factors, cytokines, hormones, ECM, neurotransmitters, light sound
small signalling molecules
epinephrine, acetylcholine, steroids, peptides
large signalling moleucles
growth factors, cytokines, proteins
receptors
- cell surface receptors and intracellular receptors
Intracellular signaling proteins:
G proteins, protein kinases/phosphatases, etc.
Different forms of cell signalling: endocrine, paracrine, autocrine, cell-cell.
endocrine, paracrine, autocrine, cell-cell
signalling molecules act locally or at a distance
endocrine
tissue produces the ligand that enters the blood stream to act at a distance
paracrine
once cell secretes a ligand that acts on an adjacent target cell and moves only a short distance
autocrine
cell produces a ligand and expresses a receptor itself (self-stimulation)
signal by plasma membrane attached proteins
cell produces transmembrane protein that can act on adjacent target cell that has cell surface receptor
epinephrine
both endocrine and paracrine singlaing
EGF
- autocrine, cell-cell or paracrine
types of nuclear receptors in nuclear-receptor superfamily
- thyroxine receptor
- rteinoic acid receptor
** involves phobic signalling molecules
cytoplasmic receptors in nuclear receptor superfamily
- estrogen receptor
- progesterone receptor
- glucoroctioic recepotr
Signal Transduction overview of process
Primary messenger or molecules.
Signaling of cell-surface receptors.
Short-term and long-term intracellular responses.
effector proteins (metabolic enzymes to alter metabolism, alter gene expressionn or alter cell shape or movement)
Termination of cellular response.
slow behaviour changes in target cell
- gene regulated changes in cell growth and division
faster behvaviour hangs in target cell
- changes in ion transport, cell movement, secretion or metabolism
what do cell signals tell it
- survive
- grow and divide (mytogenic factors)
- differentiate (response to growth factors)
- die
- Ligand-Receptor Interactions:
Binding specificity based on molecular complementarity.
Triggers receptor conformational change.
Often induces receptor dimerization (which is when interacting with another receptor which is important for signal transaction events to inside of cell)
- Dissociation Constant (Kd):
Measure of receptor-ligand affinity.
Smaller Kd indicates higher affinity.
Determines ligand concentration to occupy 50% of receptors.
Functional expression Assay
- use cells that do not have receptor for that ligand
- transfer the cells with cDNA from cell that expresses the receptor for growth factor
- pick the colonies that express that repceotr and that are associated with that phenotype
- deduce the receptor protein sequence from cDNA sequence
- or mutating specific amino acids to determine essential ligand binding domain
. Regulation by Kinase/Phosphatase Switch:
.
Tyrosine/Serine/Threonine Kinases.
Tyrosine/Serine/Threonine Phosphatases.
Phospho-specific antibodies reveal pathway activation
- phosphoryalation leads to activation
- not phosprylated = inactive
Switching Mechanism of G Proteins:
- proteins that responds to GTP instead of phosphate group
- Existence in active (GTP-bound) and inactive (GDP-bound) forms.
Activation triggered by signals, assisted by GEF.
Inactivation through GTP hydrolysis, mediated by GAP or other GTPase (intrinsic)
G-protein coupled receptors
- 7 membrane spanning domains
- respond to stimuli such as vision, pathogens, smooth muscle relaxation in a arteries, drugs
G protein coupled receptor structure
- 7 transmembrane domains that respond to different ligands
- used because it can be activated by chemical agonists and inhibited by antagonists
heterotimeric G protein comlex
- G alpha and G gamma subunit are lips anchored proteins at the cytoplasmic face
- G alpha controls activity of teh complex, is bound to GTP when active
- function in single transduction by coupling ligand-bound receptors to specific effector molecules
what happens when ligand binds to G protein coupled recptor
- recuritemtnof the g complex
- G alpha can inhibit tffector and g beta gamma will activate the effector
FRET showing dissociation of G alpha and G beta gamma
- g alpha linked to cFP
- g beta gama linked to YFP
- excite CGP with 440 nm
- emit light to excite YFP indicating that the proteins are interacting
- however, when ligand is added, loss of fret signal tdue to dissociation for he complex
G alpha s
activates adenylyl cuclase, increase cAMP
G alpha I
inhibits adenyl cyclase, decrease cAMP
activation of adenylyl cyclase
- stimulatory hormone (ACTH, glucagon, epi)
- alpha subunit moves along to activate effector protein (e..g adenylyl cyclase)
- this leads to production of secondary molecule cAMP
inhibtion of adenelyl cyclase
- inhibitory hormone (PGE, adenosine)
- binds to receptor for inhibitory homrone
- Galphai subunit to inhibit adenelyl cyclase
- no production of cAMP
cholera
- bacteria produce toxin in intestine that enters eptithelial cells using GM1 ganglisodie receptor
- it maintains the Galphas in activate state, lets to activation of adenylyl cyclase
- increase in cAMP
- activates PKA
- activates CFTR potassium channel
- increase in activity of CFTR ion channel
- it goes on to activate protein kinase
- flux of chloride ions into small intestine lumen and sodium
- water enters lumen of small intestine due to salt, diarrhea and dehydration
whooping cough
- infectes lung tissue
- toxin leads to activation of g alpha I subunit
- inhibitn of inhibitory actibity
- increase in g alpha s activity, more adenyl cyclase activation, more cAMP,
more activation of CFTR - increase cl ion efflux , watery mucous in lung
how does cAMP activate PKA
- epinephrine results in cAMP production
- PKA has two regulatory and two catalytic subints
- cAMP binds to regulatory components,
- regulatory are then released from catalytic domains so they can go and phosyrlate other proteins
cooperative binding of cAMP to PKA a and b domains on regulatory subunnits
- cAMP binds first to B to lower Kd for binding to A
cAMP
- activates PKA
cyclic GMP
- activates PKG, and opens cation channels in rod cells
DAG
- activates PKCE
inositol
opens Ca channels in ER
four intracellular secondary messengers
- cAMP
- cGMP
- diacylglycerol
- inositol
cAMP and PKA- short term or long term effects
short term, e.g. activity of enzymes modulation
and long term (gene activity)
gene regulation by pKA
- they must have specific cnuceltoide sequence (TGACGTCA) (this is the cRE)
Activation of cREB transcription factor
- by phosphytlation by pKA in nucleus
phospholipase C
- cleaves pip2 into DAG and IP3
