Second messenger systems: cAMP/cGMP Cyclic nucleotide production & regulation AGC family kinases Biological function

Cyclic Nucleotides Nucleotide cyclase Phosphodiesterase ATP GTP cAMP cGMP AMP GMP AC PDE GC Gs GoGiGt NO Ca2+

Cyclic nucleotide pathways Ligand  Receptor  Gs/Go  AC  cAMP Ligand  Receptor  Gi--|AC  cAMP Ligand  GC Receptor  cGMP NO  G-cyclase  cGMP Rhodopsin receptor  Gt  PDE--|cGMP Ca  CaM  PDE--|cAMP

Competitive control of AC (heart) Sympathetic NS (fight of flight) Parasympathetic NS (rest or relax) Wettschureck & Offermanns, 2005 cAMP AMP PDE3 Increased depolarization cAMP increases Ca 2+ influx Reduced action potential Reduced Ca 2+ efflux Increased Ca 2+ 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 /eLife.01319

AKAPs and cardiac contractility Adrenergic stimulation increases cardiac output – ↑ K V,I KS –Enhanced Ca signal – ↑ DHPR – ↑ RyR – ↓ Phospho- lamban-|SERCA Kritzer, et al., 2012 AKAP Target PKA

Refining PKA activity by AKAP modules PKA FRET reporter (Zhang 2001) Reporter + PKA anchor Reporter + PKA anchor + PDE anchor ReporterReporter + NLS Zhang et al 2001 Dodge-Kafka et al 2005 Add forskolin  AC 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 [Ca 2+ ] i Reduced calcium –GC activating protein –Restoration of cGMP