Section 2 Flashcards
calcium gradients
low cytosol
high extracellular and ER
maint chennels for Ca influx
IP3R
RyR
main channels for Ca efflux
PMCA
NCX
NCKX
SERCA
types of Ca signals
Blip/Quark (single channel - not important)
Puff/Spark
Waves
Ca changes during signalling
d
3 functions mediated by Ca signaling
T-Cells - increased Ca mediats funciton
Muscle contraction
Neurotransmitter release
tools to observe Ca changes
Imaging: fluoresecence
chemical indicators: 2 wavelenght and 1 wavelength
flura 2
genetic indicators like GFP
IP3R
on smooth er lets Ca out into cytosol activated by IP3 binding and low Ca most common result of second messenger cascade
Ryanodine Receptors
in excitable cells
opens for excitation contraction coupling so Ca gets out of sarcoplasmic reticulum
SERCA
cytosol to ER/SR
2 Ca for 1 ATP
PMCA
1 ca out of cell for 1 atp
high affinity slow activity
NCX and NCKX
1 Ca out for 3 Na in
1 Ca and 1 K out for 4 Na in
low afinnitiy but fast activity
CRAC
open to get more Ca when ER is empty
Puff/Spark
Cooperative activity in a microdomain of IP3R or RYR
Puffs- local, diffuse, super small
sparks - small and localized but bigger than puff?
waves
global elevations
domino effect
builds in a direction
intra and intercellular
calmodulin
finches
Ca plus calmodulin changes shape and binds to proteins to change them
cAMP
2nd messenger
ATP + AC = cAMP
cAMP production protein
Adenylyl cyclase AC
activated by G protein
cAMP signaling proteins
cAMP turns on PKA
to add P to stuff
cAMP degredation proteins
phosphodiesterases to AMP
G protein and cAMP interaction
G protein turns on AC to make cAMP
Camp and Ca interactions
Ca regulates cAMP
calmodulin upregulates cAMP
calcineurin downregulates
cAMP regulates Ca channels = can let more in for contraction
tools for cAMP
inhibit PDE
downstream with CREB
upstream depends on pathway
Cellular processes regulated by by G proteins
d
subfamilies of G proteins
d
effectors of G proteins
d
subtypes of Galpha
Gs - AC stim
Gi/o - AC inhibit
Gq - phospholipase activation
G12/13 - GPCR to Rho
PTM of G proteins
for membrane targeting
prenylation
myristoylation
palmitoylation
regulators of G proteins
GTPase off
GEFs on
techniques to study G proteins
toxins
inhibitors and probses
pathologies involving G proteins
Cancer endocrine disease psychiatric disorders cholera hypertension
groups of drugs that target G proteins and GPCRs
target: heterotrimeric G proteins and coupled receptors
Mechanisms that regulate GPCRs
removal of ligand GTPase activity desensitization degredation of GEF Second messenger elimination
major groups of G proteins
heterotrimeric - bind to GPCRs
Smal GTP binding proteins (monomeric)
conserved motifs of G proteins
N/TKXD - nucleotide base
GXXXXXGSK/T - phosphate binidng (P loop)
DXXG - guanine
activation/inactivation cycle of G protein regulators
d
G protein effectors
high affininty for GTP bound for and bind swithc domain
Ras
monomeric G protein
cell proliferation and differentiation
Rho
monomeric G protein
actin cytoskeleton, gene transcription, lipid metabolism
Rab
monomeric G protein
Vesicle trafficking and transport
Arf
monomeric G protein
formation of transport vesicle
Ran
monomeric G protein
chromosome dynamic
nuclear import/export
biological activation of monomeric G protein
PTM
efffectors of monomeric G protein
ser/thr kinass and Tyr kinases
switch domain
d
why signal transduction
to dynamically respond to changes in environment
different types of transductions
contact dependent
paracrine - local
endocrine - everywhere
synaptic
divergence
one ligand haveing many effects depending on cell type
convergence
multiple ligands acting via same effector pathway
why membrane receptor
membranes are not permeable to first messengers
definition of receptor
saturation, reversal, specificity, reconstitiutin
study of receptor fucntion
binding assay
biological effector assays
study of receptor structure
isolation from membrane
sequence analysis
hydropathy plot
different classes of receptors defined
Defined by second messenger - G protien or ionotropic
defined by function - death receptors
defined by activity - tyrosine kinase, notch signaling
structural features of receptor classes
dimerization - tyrosine kinase
multiple subunits - ionotropic
multiple membrane spanning domains - G proteins
Signaling complex= death receptors
receptor states for agonist/antagonist binding
Partial agonists and antagonists can bind ot both
Inverse agonists can bind to small r
Agonists can bind to R
Lig-R can transduce when agonist is around
Lig.r cannot transduce with inverse agonist binds to r
Partial agonist
Depends on which state it binds to
To R can transduce
To r cannot transduce
Preference to bind here with active agonist
Partial agonist binds to r
When antagonist is present
Partial agonist binds to R
Antagonist
Binds to either r or R
antagonist plus R can not transduce, no conformational change
hydropathy plots
X axis is aa by position Tells what each aa is/does Higher number more hydrophobic Charged > polar so charged more negative Polar and charged are negative So above x axis is in the membrane Doesnt show which orientatio
protein kinases
use ATP to add P to protein
Phosphorylases and pyrophosphorylases are different
purpose of kinases
mediate both TFs and non genomic signaling
types of kinases
based on aa they add P to
or substrate protein
structural features of kinases
regulatory and catalytic domains
some regulatory domains are separate polypeptides
catalytic domain binds to ATP, substrate and mediates PO4 transfer
Specificity for Ser/THr vs Tyr basesd on consensus sequence or catalytic cleft
PKA
cAMP regulated
separate catalytic and regulatory subunits hetero-tetramer
PKB
Akt
activated by phosphorylation
translocates to membrane and binds to PIP3
PKC
activated by Ca and diacylglycerol
translocates to membrane
exists in 4 states
regulated by phosphorylation
CaM kinase
regulated by Ca indirectly
varying active states
different tyrosine kinases
receptors
non receptors cytoplasmic
janus kinases non receptor TKs
regulation of Tyrsoinekinase
autophoshorylation RTKs
Phosphorylinaiton and de phosphorylation Src
SOCS/PIAS for JAK STAT
JAK-STAT
non tyr k
Regulatsion
SOCS - block JAKs
PIAS - block STATS from binding to DNA
MAP kinase regulation
activation by sequential phosphorylation
P on both Ser/THr and Tyr
Multisite p of RTKs for signal divergence
inositol head group phosphorylation site
4C and 5C
fate of Bis Pohsphate
catabolized by PLC
PLC
signaling intermediate IP3 and DAG
IP3 activateds intracellular calcium channels
DAG activates PKC
DAG gets P in T cell anergy
PLC activation
direct -phosphorylation
indirect - g proteins
another inosital head group P site
3C by PI-3 Kinase
PI (3,4,5) tris phosphate
docking site with PH domains
Facilitates signal transduction
PH domains
d
structure of PI-3 kinase
regulatorey and catalytic subunits
how many PI-3 kinases
differ by substrate T1,2,3
T1 PI3 kinase
d
phosphoinositides
2nd messengers docking sites membrane trafficking ion cahnnel activity cell structure
source of arachidonic acid
phospholipids
free arachidonic acid
becomes prostaglandind, leukotrienes, thromboxanes
phsyical and patholigic functions
signal via receptors
are fatty acids present only as part of phospholipids>
no some aer unesterified
they exist as part of teh membrane or are bound to proteins
free fatty acids and PGs
bind to PPARS (steroid hormone receptor family)
transcription factors
maine sphingolipid signaling molecules
ceramide
shingosine
ceramide and sphingosine functinos
apoptosis, enzyme regulaiton, cell motilitiy, gene transcription
ceramide source
de novo from sphingomyelin and sphiganine
shingosine source
ceramide
is shingosine the signalling molecule
no, it is converted to shingosine phosphate and secreted
extracellular S1P activates cognate GPCRs
Shingosiene and S1_ translocate to nuclease to be TF factors on histones
