Introduction to Receptors & Signal Transduction in Cell Membranes

1. Membrane Function Signal Transduction

2. I. Introduction to Receptors & Signal Transduction

3. The Players • Signaling molecules • Receptors • G-proteins • Second messenger systems • Effector proteins

4. Signaling Molecules • Neurotransmitters • Hormones • Growth factors • Drugs • Other nomenclature • Ligand • Agonist / Antagonist

5. Receptors • Receptors are proteins associated with cell membranes • Receptors “recognize” signaling molecules by binding to them. • Binding of receptors by signaling molecules ---> Cell behavior change

6. Transmitters Hormones Transmitters Second Messangers Ion Channels Growth Factors Tyrosine Kinase Protein Kinases Hormones: Steroids Thyroid mRNA Synthesis Protein Synthesis Figure 1: Overview of Signaling

7. Neurotansmitters: Biogenic Amines. • Catecholamines • Epinephrine • Norepinephrine • Dopamine • Esters: Acetylcholine • Indolamines • Histamine • 5-HT

8. Neurotransmitters: Peptides • Substance P • Neuropeptide Y (NPY) • Enkephalins • Somatostatin • VIP

9. Neurotransmitters: Amino Acids • Excitatory • Glutamate • Aspartate • Inhibitory • g-aminobutyric acid (GABA) • Glycine

10. Neurotranmitters: Other • Nitric Oxide • Arachadonic acid • Carbon Monoxide • PAF • Zinc

11. The G-Proteins • Involved in most signaling processes • Link receptor proteins to effector proteins. • Trimeric proteins composed of a, b, and g-subunits.

12. A R b a g A GDP A R R b b a a g g GTP A GTP (GTPase) GDP -Pi R b a g GTP Adenylate Cyclase Phospholipase C Ion Channels Phospholipase A2 Phosphodiesterase Figure 2: G-Protein Cycling

13. Functional G-Protein Units • GTP-activated a-subunit • produce second messenger • and/or opens ion channels. • bg-complexes • Initially thought to be inert. • Probably not inert • Exact role currently ill-defined.

14. Second messengers produced by G-protein activation. • Adenylate Cyclase • cAMP • Phospholipase C (PLC) • Inositol triphosphate (IP3) • Diacylglycerol (DAG) • Ion Channel Activity

15. Families of G-proteins • Unique structure of their a-subunits. • bg subunits appear to be similar across families. • Main families: • Gas • Gai • Gaq

16. II. cAMP Second Messenger System

17. Ai As Adenylate Cyclase R2 R1 Gs Gi GTP GTP GDP GDP ATP-Mg++ cAMP AMP PDE Protein C C Reg Reg C C Protein Kinase A (PKA) PKA Protein-P Figure 3: Adenylate Cyclase

18. Summary of Adenylate Cyclase Activation • Receptors which associate with Gs -type G-protein • Stimulates adenylate cyclase. • Increases cAMP • Receptors which associate with Gi -type G-protein • Inhibit adenylate cyclase. • Decreases cAMP

19. Summary of cAMP action • cAMP exerts its effect by activating protein kinase A (PKA) • PKA phosphorylates proteins • Enzymes, pumps, and channels • Phosphorylation can either increase or decrease activity depending on the protein.

20. Adenylate Cyclase • Family of membrane spanning enzymes. • Types I through IV have been well characterized. • Additional types probably exist. • Types differ with respect to activity modulation by other second messenger systems

21. Adenylate Cyclase Activity and Other Messenger Systems • Kinases (PKA, PKC, other) can phosphorylate adenylate cyclase in some cells. • Binding of adenylate cyclase by: • bg-subunits of other G-proteins • Ca++/calmodulin complexes • Allows other second messenger systems to interact with cAMP system

22. III. The Phospholipase C Second Messenger System: IP3 and DAG

23. Ca++ A R PLC Gq DAG PKC PIP2 Protein Protein-P IP3 Endoplasmic Reticulum Ca++ Figure 4: Phospholipase C System

24. Summary of the Phospholipase C Messengers • Agonist binds receptor • Occupied Receptor ---> activation of PLC (Gq -mediated) • PLC Produces second messengers: IP3 and DAG • PLC activation associated with Ca++-channel activation

25. Action of IP3 • IP3 binds to IP3-receptors on the endoplasmic reticulum • Releases intracellular Ca++ stores.

26. Action of DAG • Remains membrane associated. • Activates Protein kinase C (PKC) which translocates from the cytosol to the membrane • Activated PKC phosphorylates other proteins and alters their function state.

27. PLC System and Calcium • PLC causes the IP3-mediated Calcium • PLC also causes the influx of Ca++. • Ca++ binds one of a family of Ca++ binding proteins (calmodulin). • Ca++/calmodulin complex • binds to yet other proteins and changes their functional activity.

28. IV. Guanylate Cyclase: cGMP and Nitric Oxide As Second Messengers

29. Intracellular Ca++ Stores Ca++ Membrane Bound Guanylate Cyclase C.M. Ca++ NO GTP Ca++ Soluble Guanylate Cyclase NO NO Synthetase + Citrulline GTP PDE GMP cGMP Arginine Ion Channels cGMP-Dependent PK PDEase Activity Figure 5: Nitric Oxide and cGMP

30. NO is Membrane Soluble. • Diffusion to nearby cells • Increase cGMP levels in nearby cells • Vascular endothelial cells and nearby smooth muscle cells.

31. V. SIGNALING BY ACETYLCHOLINE

32. Acetylcholine As a Neurotransmitter • Both the central and peripheral nervous systems. • Binds two broad classes of receptors: • Nicotinic receptors • Muscarinic receptors.

33. Nicotinic Receptor Features • Composed of 5 subunits: • 2 a, b, g and d. • Subunits are arranged to form a central cavity that extends across the membrane. • Nicotinic receptors are also channels • ACh-binding opens gates and allows ion fluxes across the channel

34. Channel Agonist Binding Site Gate Figure 6: Nicotinic Receptor

35. Subclasses of Nicotinic Receptors • Skeletal muscle (N1 or Nm) • Unique a and b subunits • Autonomic ganglia (N2 or Ng). • Both N1 and N2 are gene-product families not single receptor types.

36. Other Ligand-Gated Channels • Structural and sequence similarity to nicotinic receptors. • Example agonists for these channels include: • Serotonin (5-HT) • Glutamate • GABA • Glycine

37. Muscarinic receptors • Muscarinic receptors are not channels. • Operate through G-proteins to alter second messenger systems. • 5 muscarinic subtypes have been cloned and sequenced (M1, M2, M3, M4, M5).

38. Grouping Muscarinic Receptors • M1, M3, and M5 receptors: Activate Phospholipase C through Gq. • PLC activation ---> increased IP3 --> increased intracellular Ca++ • Increased intracellular Ca++ --->Activation of Ca++-sensitive K+ & Cl- channels.

39. Grouping Muscarinic Receptors • M2 and M4 receptors • Gi -coupled inhibition of adenylate cyclase • Go or Gi -coupled regulation of certain Ca++ & K+ channels.

40. VI. Signaling by Epinephrine and Norepinephrine and Coupling Through Adrenergic Receptors

41. Three Families of Adrenergic Receptors: • b -receptors: Three subtypes b1, b2, and b3. • a1 -receptors: Three subtypes a1A, a1B , and a1C • a2 -receptors: Three subtypes a2A ,a2B ,and a2C

42. . All adrenergic receptors appear to be coupled to cellular processes through G-proteins

43. Occupation of b - Adrenergic Receptors • Gs-mediated stimulation of adenylate cyclase • Increased cAMP • Increased PKA activity.

44. Occupation of a1 -Adrenergic Receptors • Mechanistic details sketchy • Possibly Gq-mediated PLC activation • Increases IP3 and DAG for some subtypes (1B)? • Activates Ca++-channels for other subtypes (1A)?

45. Occupation of a2 -Adrenergic Receptors • Gi -mediated inhibition of adenylate cyclase. • Decreased cAMP • Decreased PKA activity.