CAMP produced directly from ATP Catalysed by adenylate cyclases Stable 2nd messenger (1 out of 4 2nd messenger) NH2 O N N. N NH 5 N O-CH2 5 0-CH2 1 N N NH2 CH3 -(CH2), -C-O-CH, PO.2 OPO, 5 0 O 1 4 1 0 2 CH3 -(CH2),- C-O-CH 0 6 OH 1 OH HO 3 2 0-P O OH 3 2 0-P O OH 2 3 0 3',5'-Cyclic AMP (CAMP) 0 3',5'-Cyclic GMP (cGMP) O Fatty acyl groups 3 CH2OH Glycerol 1,2-Diacylglycerol (DAG) 4 OPO,2- Inositol 1,4,5-trisphosphate (IP2) Activates protein kinase A (PKA) Activates protein kinase G (PKG) and opens cation channels in rod cells Activates protein kinase C (PKC) Opens Ca2+ channels in the endoplasmic reticulum Figure 15.8 Molecular Cell Biology, Seventh Edition @ 2013 W.H. Freeman and Company Synthesised by: · adenylate cyclase-large complex with 12TM domains and and two conserved cytosolic loops (between TM 6-7 and the C-terminal sequence) which form the active site. · Low abundance, plasma membrane associated enzyme, with an active site on the cytoplasmic face of the membrane . Large multigene family which may be activated by Gas or inhibited by Gai . Catalyses the conversion of ATP to CAMP and PPi · Adenylate cyclase activity is controlled by heterotrimeric G proteins · Activation or inhibition of AC activity is dependent upon the type of heterotrimeric G protein binding Forskolin Function as an activator of adenylate cyclase. Natural plant product Increases CAMP in cells In-Vivo-vasodilators. Dosage-10mg=£61 Activated and Inactivated · Different signals, functioning through different receptors, regulate release of different heterotrimeric G proteins - Gas activate AC - Gai inactivates AC · CAMP acts to regulate protein kinase A activity (PKA) and CAMP-dependent Rap GEFS · PKA comprises regulatory and catalytic subunits
• Four different regulator subunits and three different catalytic subunits exist in humans Norepinephrine Dopamine B- adrenergic BOOK Dopamine D2 Gs + Adenylyl cyclase Gi O Adenylyl cyclase + CAMP CAMP + Protein kinase A Protein kinase A + Increase protein phosphorylation Decrease protein phosphorylation Inactive PKA · is an holoenzyme · comprises of tetramer (2 regulatory and catalytic subunits) · Dimer of regulatory subunits bound to catalytic subunits in an inactive state . Catalytic cleft of C-subunit of PKA is occluded by inhibitory domain of R-subunit so substrates cannot bind . Elevated levels of CAMP leads to binding of 2 molecules of CAMP to each PKA regulatory subunit · Causes structural change releasing 2 x active catalytic subunits · Catalytic subunits are Serine/Threonine protein kinase activity
Pseudosubstrate sequence CAMP R C R + 4 cAMP C C + R + R C Active Active CAMP-binding domains Figure 10-17 Biochemistry, Sixth Edition @ 2007 W. H.Freeman and Company PKA Activity- · PKA phosphorylation motif · Consists of 2 basic amino acids that can be separated by unspecified amino acids from serine and threonine residue to be phosphorylated · target proteins including receptors and cytosolic proteins · Phosphorylation directly alters target activity or binding to other proteins · Dependent upon cell