Second messenger systems: cAMP/cGMP
• Cyclic nucleotide production & regulation
• AGC family kinases
• Biological function
Cyclic Nucleotides
• Nucleotide cyclase
• Phosphodiesterase
ATPGTP
cAMPcGMP
AMPGMP
ACPDE
GC
GsGo Gi Gt
NO
Ca2+
Cyclic nucleotide pathways
• LigandReceptorGs/GoACcAMP
• LigandReceptorGi--|ACcAMP
• LigandGC ReceptorcGMP
• NOG-cyclasecGMP
• Rhodopsin receptorGtPDE--|cGMP
• CaCaMPDE--|cAMP
Competitive control of AC (heart)Sympathetic NS(fight of flight)
Parasympathetic NS(rest or relax)
Wettschureck & Offermanns, 2005
cAMPAMP
PDE3
Increaseddepolarization cAMP increases
Ca2+ influx
Reduced action potential
Reduced Ca2+ effluxIncreased Ca2+ influx
Increased force
Nucleotide cyclases
• Dimeric cyclase homology domain
• Regulation by accessory proteins-G, CaM,…
Sunahara & Taussig., 2002
Cyclic Nucleotide Effectors
• Cyclic nucleotide kinases (ser/thr)– PKA– PKG
• cGMP binding displaces autoinhibitory domain
• BKCa, IP3R, CFTR
• cGMP ion channel
• cAMP sodium channel
AGC Kinases
• Allosteric regulation• Cyclic nucleotides, PIP3, DAG
• PKA– cAMP causes release of regulatory subunit
• PKG– cGMP binding exposes active site
• PKC– DAG (& calcium) cause membrane association
• PDK– PIP3 causes membrane association
PKA
• Ubiquitous, multifunctional– Two of 3 catalytic subunits– Two of 2 regulatory subunits– cAMP binding displaces regulatory subunit
• Transcription factors– CREB, TTF-1, GATA-4
• Metabolic processes– Glycogen phosphorylase kinase (GPK), GS
• Ion channels– AMPA, CFTR
PKA Regulatory subunit
AKAP scaffold
• Subcellular targeting of PKA– 250+ isoforms– Membrane bound
• Scaffold– PKA Regulatory subunit– Acylation– Anchor (receptor,etc)– PDE
• Spatially specific activity
Smith & al 2013 DOI 10.7554/eLife.01319
AKAPs and cardiac contractility
• Adrenergic stimulation increases cardiac output– ↑ KV ,IKS
– EnhancedCa signal
– ↑DHPR– ↑ RyR– ↓Phospho-
lamban-|SERCA
Kritzer, et al., 2012
AKAPTargetPKA
Refining PKA activity by AKAP modules
PKA FRET reporter(Zhang 2001)
Reporter + PKA anchor
Reporter + PKA anchor + PDE anchor
Reporter Reporter + NLS
Zhang et al 2001Dodge-Kafka et al 2005
Add forskolinAC at t=0
Add cAMP at t=0
PKG/ G Kinase
• cGMP production– Guanylate cyclase receptor– Nitric oxide
• PKG– Ser/Thr– Type I: soluble, neural & smooth muscle– Type II: membrane bound, gut
• Inhibits cell proliferation– MAPK stimulates differentiation– VASP actin binding protein
PKG reduces contractility
• Reduced Ca2+ influx
• Reduced contractile function
Gt/Transducin
• Rhodopsin receptor• cGMP phosphodiesterase
– Reduces cGMP– Closes cGMP Na+ channels– Hyperpolarizes rod– Decreases firing
– Decreases [Ca2+]i
• Reduced calcium– GC activating protein– Restoration of cGMP