BIOC DR. TISCHLER LECTURE 22 SIGNAL TRANSDUCTION: G-PROTEINS

OBJECTIVES 1.Mechanism by which glucagon/epinephrine produce cyclic AMP via hormone-receptor complex formation, G s protein, and adenylyl cyclase. 2.Mechanism by which cAMP activates protein kinase A and its role 3.Why caffeine prolongs the effects of epinephrine. 4.How cholera toxin causes cAMP accumulation 5.Mechanism for production of inositol trisphosphate, diacylglycerol and calcium via G q protein and phospholipase C. 6.How DAG/calcium activate PKC and calmodulin-protein kinase.

OVERVIEW OF HORMONES  classical definition: chemical substance secreted by a ductless gland into blood transported to a distant ` target organ whose activity it specifically affects  target tissues have specific protein receptors for each hormone o some receptors on plasma membrane (e.g., insulin, epinephrine, glucagon) o some receptors intracellular (e.g., steroid, vitamin D) o hormone + receptor = hormone-receptor complex  promote communication between tissues and coordinate activities of diverse organs in the complex organism

OVERVIEW OF HORMONES (cont) second messengers: hormone is the 1 st messenger; chemicals that transduce the signal in the cell are 2 nd messengers insulin: hormone used for fuel storage (anabolic) released from  -cells of pancreas in response to glucose glucagon: mobilizes fuel in starvation (catabolic) {glycogen; triacylglycerol} released from  -cells of pancreas when glucose low epinephrine: catecholamine produced by chromaffin cells of adrenal medulla “hormone of fight or flight response” – mobilizes fuels (catabolic)

Cyclic AMP is a Second Messenger cAMP phosphodiesterase AMP H2OH2O ATP 3’,5’ Cyclic AMP (cAMP) Pyrophosphate (PPi) Adenylate cyclase Adenosine

Table 1. Summary of the types of G-protein families. G proteinSignalEffected enzymeEffect GsGs epinephrine (  -receptors), glucagon adenylyl cyclasestimulatory GiGi catecholamines (  2 -receptor) adenylyl cyclaseinhibitory GqGq acetylcholine catecholamines (  1 -receptor) phospholipase Cstimulatory GtGt photonscGMP phosphodiesterasestimulatory Signal interacts with receptor to form hormone (signal)- receptor complex Complex interacts with G-protein

AC     [1] Hormone binds to receptor; hormone-R complex interacts with G-protein Hormone Receptor ss Figure 2. Signal transduction by G proteins [2] GTP displaces GDP on the  -subunit; hormone dissociates GTP GDP GTP GDP

AC     Figure 2. Signal transduction by G proteins  GTP ss ss  ss [3]  -subunit dissociates then interacts with Adenylyl cyclase (AC) GDP Receptor [4] GTPase activity of the  -subunit hydrolyzes GTP ss GDP PiPi PiPi PiPi

AC     Receptor GDP  [4] GTPase activity of the  -subunit hydrolyzes GTP PiPi  GDP [5] Without GTP bound to the  -subunit, it reassociates with the  and  subunits to restore the initial setup.  GDP  Figure 2. Signal transduction by G proteins

inactive catalytic subunits PROTEIN KINASE A regulatory subunits reassociation phosphorylates enzymes to regulate metabolism cyclic AMP adenylyl cyclase dissociation ATP 5’ AMP Phosphodiesterase active catalytic subunits Figure 3. Mechanism of activation of protein kinase A by cAMP. Binding of cAMP causes dissociation of regulatory subunits

Figure 4. The phosphatidylinositol system PIP 2 = phosphatidylinositol-bisP PLC = phospholipase C IP 3 = inositol-trisP DAG = diacylglycerol CaM = calmodulin PKC = protein kinase C CYTOPLASM Extracellular Space Receptor Endoplasmic Reticulum  inositol + 3 phosphates  Hormone-receptor complex inactive G q GDP active G q GTP GDP inactive PLC active PLC PIP 2 DAG  IP 3 Ca 2+  IP 3 CaM-dependent kinase Cellular responses   GTP displaces GDP (see step 2, Fig 2)  GTP Hormone   inactive PKC   activated PKC

   hormonal signal activating PIP 2 hydrolysis with release of DAG+ IP 3 (causes increased Ca 2+ ) Inactive protein kinase C free in the cytoplasm; active site blocked C1C1 C1C1 C2 Active site Activated protein kinase C bound to membrane with active site exposed C1C1 C1C1 C2 calcium ions  inner leaflet of membrane bilayer Figure 5. Mechanism for the activation of protein kinase C by DAG and Ca. Binding to the membrane exposes the active site where proteins are phosphorylated using phosphate from ATP. Substrates ATP+protein ATP  diacylglycerols