Coyler Flashcards
What concentration is cAMP within a cell?
0.1-1uM
What are the physiological effects of cAMP?
Glucose metabolism from glycogen
Fight or flight response
Trigyceride breakdown
How many members of the adenlyl cyclase family are there?
6, with 50-92% identity
What is the structure of adenlyl cyclase?
2 NBD domains and 12TM helices
What is the turnover of AC?
1000/min
How does cAMP have localised effects?
APAK provides a platform to link targets and degradation close to AC
What is the main target of cAMP?
PKA
What are the targets of cAMP?
cAMP-dependant kinases,
Exchange Protein directly activated by cAMP,
ion channels
What are the sites of cAMP/ CaMK responses in muscle cells?
L-Calcium channels
RyR2 Calcium channels
Phospholambam
What enzyme does cAMP act on in muscle cells?
CamK11
How is cAMP degraded?
Phosphodiesterases
What structure is PDE?
58-125kDa, regulatory and targetting domains with catalytic core
How many forms of PDE are there?
11 classes, 30 isoforms
What is the structure of PKA?
Dimer of R/C
R is autoinhibitory by mimicking substrate, released by fast/slow cAMP sites
C has MgATP site and substrate binding site
What specificity does PKA have?
Low specificity for S/T in RRxSxxxx motif
What deterrents does PKA specificity have?
Y at 0
F at +4
Acidic residues at -1
K at -1
How is cAMP signalling restricted?
PDE degradation AKAP compartmentalisation Phosphatases counteracting downstream targets GPCR/GP inactive form Internalisation of GPCR
How is Adenlyl cyclase activated?
By GPCR stimulation, varying from which GP subunit
Is the [cAMP] uniform across a cell?
No-heterogeneous/localised
Which pathogens expliot cAMP signalling?
Cholera ADP-ribosylates Gas for permanent inactivation
Pertussis injects soluble AC
Which receptors are activated by cAMP in heart?
B1-AR and B2-AR
Which heart receptor have the most significant increase in contractility?
400% in B1-AR
Which heart receptor is cardioprotective?
B2-AR
What are the effects of overstimulation of PKA?
desensititation
How does EPac respond to high stimulation?
desensitised
Which cAMP target has a switch in activity when overactivated?
CaMK11 switches from contractility to apoptosis
What effect does GPCR phosphorylation have on a cell?
Cell growth/hypertrophy.
What is IP3?
inositol-1,4,5-triphosphate
What percentage of the membrane is PIs?
2-8%
Which 2 pathways can stimulate PLC activity?
PTK or GPCR
How are GPCRs activated for IP3 signalling?
Ach, 5HT, odour, light
Which PLC isoform do RTKs activate?
PLCy phosphorylation at Y771, Y783, Y1254
Which G proteins are involved in activating PLCs?
q, By, 11, 14, 16
Which PLC isoform does Gaq activate?
beta 1
How is PLCy inhibited?
PKA and PKC phosphorylation
What physiological effects does IP3 have directly?
fertilisation,
platelet aggregation
secretion from the adrenal cortex
smooth muscle contraction
What does DAG activate?
Ca-dependant kinases
What does PLC produce?
IP3 and DAG
How does IP3 mediate release of Ca?
Opens ligand gated Ca channels in ER
Where is the intracellular store of Ca?
In the ER, maintained by SERCA pump and buffer proteins calreticulin and calsequestrin
What is the structure of the IP3R?
tetrameric channel with 3 isoforms of 2701-2745 residues
How is IP3R regulated?
Cytosolic [Ca}
ATP
PKA phosphorylation
What is the Kd of IP3 for the IP3R?
10nM
What is the [intracellular Ca]?
0.1-1uM
What is the [Ca] in the blood?
1-2mM
How can [cytosolic Ca] be observed?
photoproteins based on aequorin
EGTA derived organic dyes that use acidic groups for chelation
How much Ca can be released in cytoplasm?
Quantal/fixed
How does Ca release vary with increasing [agonist]?
frequency of release varies
What is the capacitive Ca entry?
Quantal release of Ca from ER
Decline
Opening of blood channels
Increase in Ca
How does Ca enter cells from blood?
Low Ca causes Orai to release TRPC as open non-specific cation channel
Orai binds STIM in ER membrane
Clusters of complex lock TRPC in Ca form (Icrac form)
What direct effects does Ca have an a cell?
channels, proteases and lipases,
eg TCA, PKC, PDE, contractile proteins
Which regulatory proteins does Ca interact with?
calmodulin, annexins
What is the structure of CaM?
16.8kDa
4 EF hands
Where is CaM found?
Ubiquitous and abundant
What processes in CaM involved in?
Ca transport
Glycogen metabolism
Cyclic nucleotide metabolism
Replaces troponin in smooth muscle contraction
How is CaM activated?
conformational change buckles central helix
methionine puddle exposed
binding of substrate
What motif does CaM target?
positive residues-hydrophobes
How is CaM involved in neurotransmitter release?
CaMK11 releases synapsin on vesicles for interaction with actin cytoskeleton
