Page%9%of%1%% Lecture 3 GPCR signaling cont. 3 main families of α subunits: • Gαs -> Increase Adenylyl cyclase; turns ATP to cAMP->Stimulates/phosphorylates Protein Kinase A(PKA) at a Ser or Thr-> Phosphorylates other cytosolic proteins and catalytic subunits. Can also enter the nucleus and affect gene transcription by phosphorylating CREB. Gαs is seen in all of the β adrenergic receptors o PKA is a tetramer w/ 4 subunits; two catalytic and 2 regulatory. cAMP binding changes regulatory subunits conformation, causing the m to dissociate exposing catalytic subunits. o Reaction performed by kinases: ATP + Protein -> Protein-Phosphate + ADP • Gαi (inhibitory)-> opposite effects of Gas. Decrease AC-> Decrease cAMP-> Decrease PKA. Seen in α2 receptors • Gαq-> Activates Phospholipase C->Cleaves PIP2 into IP3 and DAG. Seen in α1 receptors o IP3 binds to gated Ca channel receptors in the ER to increase [Ca] ->Signals smooth muscle contraction and exocytosis of neurotransmitters. Ca binds to Calmodulin (component of troponin)->Activation of CaM Kinase phosphorylates proteins and enters nucleus to phosphorylate CREB and increase transcription. o DAG activates Protein Kinase C (PKC) which phosphorylates proteins. PKC also needs Ca to be activated Summary: β adrenergic receptors -> Gs α2 adrenergic receptors-> Gi α1 adrenergic receptors-> Gq How to terminate signaling: • Dissociating the hormone from the receptor BICD 150 Sp’14 Fortes 2 4/8/14 1 of 9Page%9%of%2%%• GTPase hydrolyzes GTP bound to the α subunit to GDP, turning it "off" after it rebinds βγ subunits o Bacterium- Cholera bacteria has an enzyme toxin it secretes which is taken up into cells . Catalyzes the reaction Gα + ADPribose -> Gα-ADPribose, inhibiting the GTPase activity causing continual stimulation of the cAMP/PKA pathway. A gated Cl channel gated by cAMP in the intestinal cells is continuously open releasing Cl. Cl attracts Na ions which in turn attract H20-> Diarrhea. High mortality from dehydration o Pertussis bacteria also has a toxin with similar effects • Second messengers can also be dephosphorylated by phosphodiesterases to stop signal pathway (cAMP-> AMP) • Protein phosphatases dephosphorylate proteins to stop signaling *Synthesis and expression of receptors can increase or decrease. Receptors can also be removed from the membrane by endocytosis (down-regulation) When there is overstimulation of specifically β adrenergic receptor over a period of time: • PKA will phosphorylate and activate β-ARK-> B-ARK Phosphorylates the β receptor-> phosphorylated receptor binds the soluble protein Arrestin, which stops the receptor from functioning. • Later, a phosphatase will remove the phosphate-> removes Arrestin-> reactivates receptor Growth Factor Receptors: • Insulin and IGFs bind these receptors • Single transmembrane segment (how does the info of the hormone binding on the outside reach the inside?) • These receptors frequently dimerize after binding their ligand or already exist as dimers • On the intracellular portion of these receptors there is at least one Tyrosine residue • The receptors have intrinsic tyrosine kinase activity on their intracellular domain • After binding the ligand the dimerized receptors will phosphorylate each other • The phosphorylated tyrosine will bind proteins w/ SH2 domains which will attract proteins w/ SH3 domains that also contain tyrosines. • This results in the recruitment of other proteins that get phosphorylated that result in cell division and growth • Remove ligand/tyrosine phosphatases turn off signaling Cytokine receptors: • Growth hormone, EPO, Prolactin bind these receptors • Used GH receptor specifically as an example • Receptors are dimers in the membrane and bind a single GH molecule at different spots • Receptors have intracellular tyrosines. No intrinsic enzymatic activity! • Binding GH will cause a change that allows a soluble kinase called JAK to bind and phosphorylate the tyrosines and itself. • Phosphotyrosines on the receptor can bind another protein called STAT • JAK phosphorylates STAT.Page%9%of%3%%• STAT enters the nucleus and stimulates transcription • Remove hormone/phosphatases end signaling TGF receptors not covered in this class Guanylyl cyclase receptors: • Natriuretic peptides bind these receptors; inhibit Na reabsorption in kidneys, regulate pressure in CNS to decrease secretion of ADH -> Decrease Na and BP • They have Guanylyl cyclase activity • Synthesizes cGMP Soluble guanylyl cyclase situations Smooth muscle: • Receptor for cytokines • Receptor stimulates NOS to make NO • NO will increase synthesis of cGMP • cGMP will activate PKG • PKG causes Vasodilation by inhibiting contraction Endothelium: • Receptor for Acetylcholine ( remember parasympathetic system is responsible for arousal in the genitals) or Bradykinin, which will increase [Ca] by binding to specific Gaq receptors • Ca stimulates NOS to make NO • NO diffuses out of endothelium into the smooth muscle to increase cGMP End signaling: • Remove hormones • NO has a very short half life • Phosphodiesterases cleave cGMP • Phosphatases remove phosphates from PKG NO derivatives are used as vasodilators to treat angina pectoris (chest pain from heart disease) • Nitroglycerin pills release NO Years ago a drug company made a compound as a dilator to treat angina pectoris. It didn't work that well but it was found that patients were frequently getting erections • The drug Viagra is a phosphodiesterase inhibitor • Increase cGMP -> Increase PKG -> Increase vasodilation • Erection! • The signaling pathway upstream of cGMP must still be working for the drug to be effective • Photoreceptors in the retina function by activating a Guanylyl cyclase. ED drugs can cause visual changes • Don’t take ED drugs with nitrates. Can cause such vasodilation that the BP drops tremendously and fatallyPage%9%of%4%%Intracellular receptors: • Hormones must be able to enter the cell; steroid hormones, Eicosanoids and vitamin D Cytoplasmic receptors: • Exist in an inactive state with hydrophobic binding sites. They would tend to clump together to hide these sites and precipitate • Instead the sites are covered by what are known as Heat Shock Proteins, HSPs. • In the rough ER proteins that are destined to be secreted like milk, digestive enzymes, protein