1. Lecture 22 Signal Transduction 1

2. Important Concepts in Signal Transduction Primary messengers Membrane receptors Second messengers Amplification Signal termination 7TM receptors G proteins Adenylate cyclase - Protein Kinase A Phospholipase C- Protein Kinase C, Ca 2+ Channels

3. Cells respond to the environment: Signal Transduction

4. The term signal transduction refers to the biochemical mechanism responsible for “transmitting” extracellular signals inside the cell, which ultimately lead to the activation of target proteins that control metabolic pathways or regulate gene expression. Courtesy: Roger Miesfeld

5. 1.Signal molecule (primary messenger, first messenger, ligand) travels to the cell. 2.Primary messenger binds to the extracellular domain of a receptor protein and initiates a structural change in the receptor which is propagated across the membrane. –Membrane receptors sense the stimulus and transfer info across the membrane. –Exception: some molecules, for example steroid hormones, move across membranes, bind to proteins and act, generally at the nucleus. –Most molecules are too polar or too large to cross the membrane, so the stimulus does not enter without membrane receptors. –Generally, receptors are intrinsic (integral) membrane proteins with extra- and intracellular domains. Steps of Signal Transduction

6. 3.Receptor protein stimulates signaling proteins 4.Second messengers amplify the signal –Free to diffuse –Cross talk between pathways exists 5. Second messengers bind to additional signaling proteins 6.Signal is propagated, often by a protein kinase cascade Steps of Signal Transduction

7. 7. Target proteins are affected (activated, inhibited) –Transcription factors –Metabolic enzymes –Cytoskeletal proteins –Transport proteins –Etc. 8. Signal is terminated –Phosphatases Steps of Signal Transduction

8. The biochemical basis for signal transduction involves three primary mechanisms: 1) protein conformational changes 2) covalent protein modifications 3) altered rates of gene expression

9. First/primary messengers are small diffusible biomolecules. These can be produced through endocrine mechanisms and act at a distance. Or they can function locally as paracrine or autocrine signals. Courtesy: Roger Miesfeld

10. Small molecules act as diffusible signals First/Primary Messengers Human growth hormone and insulin are peptide hormones Cortisol is a steroid that is derived from cholesterol Epinephrine, also known as adrenaline, is a derived from the amino acid tyrosine Acetylcholine is a neurotransmitter that binds the acetylcholine receptor NO is produced by deamination of L- arginine

11. Small molecules act as diffusible signals Second Messengers Second messengers amplify the receptor-generated signal Fine tuning Rapid production of maximum response

12. Produced by the enzyme adenylate cyclase from ATP. Receptor activation of adenylate (adenylyl) cyclase generates large amounts of cAMP, which in turn, binds to and activates downstream signaling proteins such as cAMP dependent protein kinase A (PKA). Importantly, the intracellular concentration of cAMP is carefully controlled by the relative levels of receptor-activated adenylate cyclase and soluble forms of cAMP phosphodiesterase (PDE) which converts cAMP to AMP. Courtesy: Roger Miesfeld One of the best characterized second messengers is cyclic AMP (cAMP).

13. The intracellular concentration of cAMP is carefully controlled Relative levels of receptor-activated adenylate cyclase and soluble forms of cAMP phosphodiesterase (PDE) which converts cAMP to AMP. Courtesy: Roger Miesfeld

14. Produced from GTP by the enzyme guanylyl cyclase. The cGMP analog Sildenafil, also know as Viagra, is used to treat sexual dysfunction by inhibiting the activity of cGMP phosphodiesterase (PDE). The molecular structure of sildenafil is similar to cGMP and binds tightly to cGMP PDE. Courtesy: Roger Miesfeld Another second messenger important in signal transduction is cyclic GMP (cGMP)

15. Courtesy: Roger Miesfeld The Kissing Bug, Rhodnius prolixus, delivers NO to their victims by injecting heme-containing proteins called nitrophorins that carry NO into the wound along with their saliva.

16. Intracellular levels of DAG, IP3 and Ca 2+ are controlled by the activity of a membrane associated enzyme called phospholipase C (PLC). Receptor-mediated activation of phospholipase C leads to cleavage of the membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2) to form DAG and IP3. Courtesy: Roger Miesfeld Some Other Second Messengers: diacylglycerol (DAG), inositol 1,4,5-trisphosphate (IP3) and calcium ion (Ca2+).

17. The term signal transduction refers to the biochemical mechanism responsible for “transmitting” extracellular signals inside the cell, which ultimately lead to the activation of target proteins that control metabolic pathways or regulate gene expression. Courtesy: Roger Miesfeld

18. 5 Major classes of receptor proteins: the gatekeepers of the cell Courtesy: Roger Miesfeld

19. Seven-Transmembrane-Helix Receptors (7TM receptors) Fig. 14-4 All seven-transmembrane-helix receptors are coupled to G Proteins : G Protein Coupled Receptors or GPCRs

21. β-Adrenergic receptor signal transduction pathway Binding of ligand on the extracellular domain of the β- adrenergic receptor causes a structural change on the cytoplasmic side This structural change causes a change in an associated signal-coupling protein called a G- protein (guanyl nucleotide binding protein) The change in the G-protein involves GDP vs. GTP binding affinity. The G-protein activates adenylate cyclase, increasing cAMP levels

22. Inactive form of G protein heterotrimer binds GDP Notice the three different subunits here

23. G Protein Activation Receptor conformational change causes structural change in the G protein – GDP leaves, GTP binds GTP binding cause a structural change in the G protein—molecular switch βγ dimer then dissociates and activated α subunit goes off to affect other proteins

24. The human genome contains 15 α, 5 β and 10 γ subunits, leading to many different possible combinations (~1000) and functions

25. The activated Gα subunit binds to other proteins to activate them G αs binds to adenylate cyclase, activating it, leading to increased levels of cAMP, which turns on Protein Kinase A. Fig. 14-7

26. Examples from the adrenergic systems

27. Glycogen is a storage form of glucose. Glycogen is broken down to glucose when the body needs energy. Glucose levels in the blood are tightly controlled. Glycogen synthesis (anabolism) and degradation (catabolism) are highly regulated by hormone signaling. The liver is one of two major storage depots for glycogen, which in response to epinephrine or glucagon signaling, provides an important source of glucose for export throughout the body when dietary glucose is limiting. Epinephrine is the fight or flight hormone. Glucagon is released by the pancreas and has been called the hunger hormone because it signals low blood glucose levels.

28. Examples from the adrenergic systems

29. Switch II, undergoes a conformational change in the presence of GTP and is critical for stimulation of adenylate cyclase activity

30. How does cAMP binding activate the phosphorylating function of protein kinase A?

31. Stimulation of PKA signaling events in liver cells by epinephrine

32. Stimulation of PKA in liver cells by epinephrine

33. Stimulation of PKA signaling events in liver cells by epinephrine

34. Activation of phospholipase C Human liver cells contain α1 adrenergic receptors bind epinephrine and signal glycogen degradation through a second messenger pathway linked to a phosphorylation cascade. α1 adrenergic receptors are coupled to a heterotrimeric G protein containing Gαq activates the enzyme phospholipase C (PLC) through a mechanism very similar to Gαs stimulation of adenylate cyclase activity Phospholipase C is a membrane associated protein catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) to form the second messengers DAG and IP3.

35. PLC produces second messengers DAG and IP3

36. Function of second messengers IP3 & DAG IP3 binds to calcium channels on the endoplasmic reticulum causes a rapid increase in intracellular Ca 2+ levels. Released Ca 2+ binds to protein kinase C (PKC) stimulates its association with DAG at the plasma membrane resulting in activation of the PKC kinase function Ca 2+ binds to calmodulin, activating: phosphorylase kinase calmodulin dependent kinase PKC and calmodulin dependent kinase phosphorylate and inactivate glycogen synthase Calmodulin-activated phosphorylase kinase stimulates glycogen degradation by activating glycogen phosphorylase

37. Stimulation of PKC signaling events in liver cells by epinephrine

38. G proteins reset themselves Possesses GTPase activityThe βγ dimer then re-associates with the α subunit, preventing it from further propagating the signal Ready to start all over again. Fig. 14-9

39. Don’t forget the 7TM receptor, it must be reset as well, or it activates more G protein Fig. 14-10

40. 1. Receptor-mediated activation of GDP-GTP exchange in Gα subunits. 2. Gα stimulation of an effector enzyme that generates 2nd messengers. 3. Activation of a phosphorylation cascade by 2nd messenger signaling. 4. Inactivation of Gα by effector stimulation of the intrinsic GTPase activity. 5. Signal duration is controlled by loss of 2nd messengers and receptor desensitization. Summary of G protein coupled receptor signaling

41. What happens when receptors aren’t reset? Dopamine receptor and cocaine / amphetamines Opiate receptor and heroin Serotonin 5-HT1A receptor and MDMA (Ecstasy) These are all G protein coupled receptors (GPCRs)! First you get high, then you become addicted. Why? Because the dopamine D2 receptor and the opiate and the 5- HT1A receptors bind these drugs much more tightly than they do their natural ligands. They don’t get reset properly, the whole signaling cascade downstream is “messed up”. After exposure, more receptor is needed to get the “normal” physiological response. The body responds by altering gene expression and receptor levels in the brain. One exposure to MDMA or Meth permanently alters brain function! There is no going back. This is a huge social problem right now.