Theresa Filtz, hd har 735, Winter 2006 G protein-coupled Signal Transduction Main Objectives (the big chunks) Describe in molecular detail the cascades of events in a generalized G protein-coupled signaling pathway Describe the structure of a and how it relates to function List the four major classes of Gα subunits and the effects of toxins on their activity List the major, proximal second messengers produced by adenylyl cyclase and LC-β Compare the effects of changing cyclic AM and calcium levels on contraction and relaxation in smooth muscle cells G protein coupled receptors () Function The receptor subtype mediating the actions of hundreds to thousands of endogenous and exogenous substances Couple to a guanine nucleotide binding protein (G protein) G proteins activate effectors to initiate signaling cascades Examples of drug acting through s Bronchodilators-albuterol (Ventolin, etc) Stimulants- Ritalin, ephedra Decongestants-Sudafed Antipsychotics-haloperidol, Zyprexa, Geodon Antihistamines-Benadryl, Claritin, Allegra ain medication-morphine and opioid analgesics Labor inducing agents-pitocin Anti-hypertensives-β-blockers Cardiac stimulants -atropine, dopamine, epinephrine, adenosine Epinephrine for severe allergic reaction Structure One of the most abundant protein families At least a thousand different genes for in the human genome otentially a unique for every dectectable odor age 1
Seven transmembrane α helices External NH 2 terminus and internal C tail Binding pocket Inside the α helices for small molecules and at the NH 2 terminus for larger peptides Intracellular loops Binding of activators to α helices affects configuration of the intracellular loops 2 nd and 3 rd loops and C tail interact with G proteins β-adrenergic receptor Guanine nucleotide binding proteins (G roteins) Structure and Function Turn on effector enzymes Heterotrimer--composed of three protein subunits Gα, Gβ, and Gγ Other families of G proteins exist which serve other intracellular functions Bound by lipid chains to the intracellular surface of the plasma membrane Gα subunit Binds GD and associates with Gβγ in the resting state Binds GT and dissociates from Gβγ in the active state Upon activation by receptor, releases GD and picks up GT Dissociates from Gβγ upon binding GT Activates effector enzymes when bound to GT Intrinsic GTase activity Self-Hydrolyzes GT back to GD Rejoins with Gβγ when bound to GD to turn itself off Gβγ subunit Exists mainly as a non-dissociable dimer Active when dissociated from Gα subunit Activates different effector enzymes when separated from Gα Inactivated by binding Gα age 2
Subtypes Gα subtypes--four major families, multiple subtypes Gαs Activates adenylyl cyclase Cholera toxin permanently turns it on G olf olfactory Gα subunit, activates ion channels in nasal epithelia Gαi Inhibits adenylyl cyclase ertussis toxin permanently turns it off Gαt, tranducin-the retinal light transduction subunit activates cyclic GM phosphodiesterase in response to light Gαo inhibits some types of Ca channels Gα12,13 Acivates p115rhogef Involvement with growth regulatory pathways Gαq Activates phospholipase C β enzymes Gβγ subtypes Multiple subtypes of each subunit Activate many enzymes including phospholipase C-β, cation channels, kinases, subtypes of adenylyl cyclase, among other signaling proteins Which combination of subtypes activate which effectors is still being investigated age 3
Effector enzymes hospholipase C-β Activated by Gα q subunits OR Gβγ subunits (depending on isoform) Hydrolyzes a low abundance membrane phospholipid, I 2 (phosphatidylinositol 4,5- bisphosphate) into two products, I 3 (inositol trisphosphate) and DAG (diacylglycerol) I 3 I 3 is water soluble and diffuses through the cytoplasm to bind to receptors on the endoplasmic reticulum (ER) I 3 receptor activation releases Ca from the ER into cell cytosol Cells are exquisitely sensitive to changes in cytosolic Ca concentration Calcium activates mainly pathways including kinases associated with Ca-dependent calmodulin Lithium carbonate inhibits inositol recycling back into I 2, causing a build-up of I 3 and depletion of I 2 DAG Membrane bound lipid Activates protein kinase C (KC) KC initiates multiple phosphorylation cascades horbol esters are tumor promoters that mimic DAG and activate KC Activation of LC-β leads to many physiologic responses including smooth muscle contraction, learning and memory, and cell growth and differentiation Hormone G!q G"# I 2 LC-" OH DAG I 3 KC inositol I 3 receptor Ca 2 Ca 2 Endoplasmic Reticulum Ca 2 /CaM Kinases = G protein coupled receptor KC = protein kinase C LC-" = phospholipase C-" CaM = calmodulin age 4
Adenylyl Cyclase Activated by Gαs subunits Inhibited by Gαi subunits Transforms AT into cyclic AM cyclic AM Increases and decreases in cyclic AM levels lead to different effects Activates cyclic AM-dependent protein kinase (KA) Hydrolyzed by phosphodiesterases (DE) DE is inhibited by theophylline (bronchodilator), caffeine, sildenafil (Viagra ), etc cyclic AM also activates transcription factors cyclic AM response element binding protein (CREB) Increased cyclic AM is associated with relaxation of smooth muscle, contraction of cardiac muscle, release of insulin, glycogenolysis in the liver and skeletal muscle, and regulation of circadian rhythm among many other functions Stimulatory Hormone Inhibitory Hormone G!s G"# Adenylyl Cyclase G!i G"# - AT cyclic AM DE AM KA = G protein coupled receptor KA = cyclic AM regulated protein kinase DE = phosphodiesterase age 5
Integrating G protein coupled signaling pathways Smooth muscle (e.g. blood vessel) contraction and relaxation I3 Increases lead to increased cytosolic Ca Increased Ca leads to activation of Ca /CaM dependent myosin light chain kinase (MLCK) hosphorylation of MLCK phosphorylates myosin -myosin stimulates contraction Cyclic AM Activation of KA leads to activation of membrane and sarcoplasmic Ca pumps Activated pumps increase storage of Ca Decreased cytosolic calcium inhibits contraction M1 Muscarinic receptor Angiotensin receptor Histamine H1 receptor Cysteinyl leukotriene receptor Oxytocin receptor 2Y purinergic receptors "2-adrenergic receptor Endothelin receptors G!q I 2 LC-" DAG I 3 KC G!s Adenylyl Cyclase AT cyclic AM Relaxation myosin myosin Ca 2 /calmodulin MLCK - Ca 2 Ca 2 ER/SR Ca 2 pump Ca 2 KA Contraction Cyclic GM Soluble guanylyl cyclase is stimulated by diffusible NO gas Cyclic GM levels increase Smooth muscle relaxes Mechanisms unclear Ca levels decrease Cyclic GM phosphodiesterase breaks down cyclic GM Inhibitors (sildenafil) increase smooth muscle relaxation age 6