1. Signal Transduction BL 4010 12.07.06

4. Outline Extracellular signals Signal-Transducing Receptors –single membrane spanning receptors –7 TMS receptors Intracellular Second Messengers –cAMP –Calcium –G-proteins Enzyme cascades

5. Additional resources http://www.signaling-gateway.org/ http://web.indstate.edu/thcme/mwking/signal- transduction.html http://stke.sciencemag.org/

6. Extracellular Signals Light Small molecules –hormones –toxins –metabolites Large molecules –oligosaccharides –proteins

7. Transcriptional activation

8. Interferon

9. The phosphorelay system

10. Phosphorelay system

11. Figure 19-1aClassification of hormones. (a) Endocrine signals are directed at distant cells through the intermediacy of the bloodstream. Page 658

12. Figure 19-1bClassification of hormones. (b) Paracrine signals are directed at nearby cells. Page 658

13. Figure 19-1cClassification of hormones. (c) Autocrine signals are directed at the cell that produced them. Page 658

14. Classes of Hormones

15. Figure 19-3aBinding of ligand to receptor. (a) A hyperbolic plot. Page 660

16. Figure 19-3bBinding of ligand to receptor. (b) A Scatchard plot. Page 660

17. Classes of Hormones - Steroid Hormones Derived from cholesterol- (e.g. Glucocorticoids,vitamin D, sex hormones) –regulate metabolism, salt/water balances, inflammation, sexual function. May bind to PM receptor or enter nucleus directly May either act at nucleus or at plasma membrane Steroids are hydrophobic and cannot diffuse freely to nucleus Receptor proteins carry steroids to the nucleus Steroid receptor proteins are all apparently members of a gene superfamily and have evolved from a common ancestral precursor

18. Classes of Hormones - Steroid Hormones

19. Classes of Hormones - Nonsteroid hormones Amino Acid Derived Hormones (e.g. epinephrine) bind to PM receptors –regulate smooth muscle, blood pressure, cardiac rate, lipolysis, glycogenolysis

20. Effects of epinephrine ReceptorMoleculeEffect Alpha1Epinephrine, NorepinphrineIncreased Ca 2+ Alpha2Epinephrine, NorepinphrineDecreased cAMP Beta1Epinephrine, NorepinphrineIncreased cAMP Beta2EpinephrineIncreased cAMP Increased rate and force of contraction of heart muscle: predominantly an effect of epinephrine on beta receptors. Constriction of blood vessels: increased blood pressure. Dilation of bronchioles: assists in pulmonary ventilation. Stimulation of lipolysis in fat cells: provides fatty acids for energy production and conserves dwindling blood glucose. Increased metabolic rate Inhibition of certain "non-essential" processes: e.g. inhibition of gastrointestinal secretion and motor activity. Dilation of the pupils: particularly important in situations where you are surrounded by velociraptors under conditions of low ambient light

21. Peptide Hormones (e.g. insulin) - bind to PM receptors –regulate many processes in all tissues - including release of other hormones –All secreted polypeptide hormones are synthesized with a signal sequence (which directs them to secretory granules) –Usually synthesized as inactive preprohormones ("pre-pro" implies at least two precessing steps) –Proteolytic processing produces the prohormone and the hormone Classes of Hormones - Nonsteroid hormones

22. Insulin is a peptide hormone

23. Secretion of insulin

24. Single TMS Receptors What is a receptor? Three main classes Extracellular domain to interact with hormone Single transmembrane segment Intracellular domain with enzyme activity Activity is usually tyrosine kinase or guanylyl cyclase Each of these has a "nonreceptor" counterpart src gene kinase - pp60 v-src was first known Two posttranslational modifications

27. Receptor Tyrosine Kinases Membrane-associated allosteric enzymes How do single-TMS receptors transmit the signal from outside to inside?? Oligomeric association is the key! Extracellular ligand binding

29. Protein-Tyrosine Phosphatases The enzymes that dephosphorylate Tyr Some PTPases are integral membrane proteins But there are also lots of soluble PTPases Cytoplasmic PTPases have N-term. catalytic domains and C- terminal regulatory domains Membrane PTPases all have cytoplasmic catalytic domain, single transmembrane segment and an extracellular recognition site

31. Guanylyl Cyclases Soluble or Membrane-Bound Membrane-bound GCs are the other group of single- transmembrane-segment receptors (besides RTKs) Peptide hormones activate the membrane-forms Note speract and resact, from mammalian ova Activation may involve oligomerization of receptors, as for RTKs

33. Soluble Guanylyl Cyclases Receptors for Nitric Oxide NO is a reactive, free-radical that acts either as a neurotransmitter or as a second messenger NO relaxes vascular smooth muscle (and is thus involved in stimulation of penile erection) NO also stimulates macrophages to kill tumor cells and bacteria NO binds to heme of GC, stimulating GC activity 50-fold Read about NO synthesis and also see box on Alfred Nobel

37. Types of Receptors 7-TMS receptors (G protein receptors) –extracellular site for hormone (ligand) –intracellular site for GTP-binding protein Single-transmembrane segment receptors –extracellular site for hormone (ligand) –intracellular catalytic domain - e.g. kinase or guanylyl cyclase Oligomeric ion channels

38. Second Messengers Many and there may be more! The hormone is the "first messenger" The second messenger - Ca 2+, cAMP or other - is released when the hormone binds to its (extracellular) receptor The second messenger then activates (or inhibits) processes in the cytoplasm or nucleus Degradation and/or clearance of the second messenger is also (obviously) important

39. Steroid Receptor Proteins Hydrophobic domain near C-terminus that interacts with steroid itself Central, hydrophilic domain that binds to DNA Central DNA-binding domains are homologous to one another, with 9 conserved Cys residues Three pairs of Cys residues are in Cys-X-X-Cys sequences - as in Zinc-finger domains Steroid-receptor complex may bind to DNA or to transcription factors Thyroid hormone receptor proteins are similar

40. Steroid Receptor Proteins

43. cAMP and Glycogen Phosphorylase Earl Sutherland discovers the first second messenger In the early 1960s, Earl Sutherland showed that the stimulation of glycogen phosphorylase by epinephrine involved cyclic adenosine-3',5'-monophosphate He called cAMP a "second messenger" cAMP is synthesized by adenylyl cyclase and degraded by phosphodiesterase

45. How are the hormone receptor and AC coupled? Purified AC and purified receptor, when recombined, are not coupled. Rodbell showed that GTP is required for hormonal activation of AC In 1977, Elliott Ross and Alfred Gilman at Univ. of Virginia discovered a GTP-binding protein which restored hormone stimulation to AC Hormone stimulates receptor, which activates GTP-binding protein, which activates AC

46. Heterotrimeric G Proteins A model for their activity Binding of hormone, etc., to receptor protein in the membrane triggers dissociation of GDP and binding of GTP to -subunit of G protein G  -GTP complex dissociates from G  and migrates to effector sites, activating or inhibiting But it is now clear that G  also functions as a signalling device

47. Figure 19-13Activation/deactivation cycle for hormonally stimulated AC. Page 674

48. Signalling Roles for G(  ) A partial list Potassium channel proteins Phospholipase A 2 Yeast mating protein kinase Ste20 Adenylyl cyclase Phospholipase C Calcium channels Receptor kinases

49. Stimulatory and Inhibitory G G proteins may either stimulate or inhibit an effector. In the case of adenylyl cyclase, the stimulatory G protein is known as G s and the inhibitory G protein is known as G i G i may act either by the G i subunit binding to AC or by the G i complex complexing all the G i and preventing it from binding to AC

51. Figure 19-16Mechanism of receptor- mediated activation/ inhibition of AC. Page 676

53. The ras Gene and p21 ras An oncogene and its product a gene first found in rat sarcoma virus Normal cellular ras protein activates cellular processes when GTP is bound and is inactive when GTP has been hydrolyzed to GDP Mutant (oncogenic) forms of ras have severely impaired GTPase activity, so remain active for long periods, stimulating excessive growth and metabolic activity - causing tumors to form

55. G-protein coupled receptors Receptors that interact with G proteins Seven putative alpha-helical transmembrane segments Extracellular domain interacts with hormone Intracellular domain interacts with G proteins Adrenergic receptors are typical Note desensitization by phosphorylationby protein kinase A