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