1. 1 Eric P. Widmaier Boston University Hershel Raff Medical College of Wisconsin Kevin T. Strang University of Wisconsin - Madison *See PowerPoint Image Slides for all figures and tables pre-inserted into PowerPoint without notes. Chapter 05 Lecture Outline * Control of Cells by Chemical Messengers Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

2. 2 Receptors Chemical messengers bind to proteins called receptors. Most chemical messengers are water-soluble and bind to receptors located at the plasma membrane. Some messengers like steroids are lipid- soluble and bind to an intracellular receptor.

3. 3 Transmembrane Receptor Protein Fig. 5-1

4. 4 Receptor Specificity Fig. 5-2 Remember that not all cells express the same receptors. This selective expression leads to specificity in the systems. The response of individual cells with the same receptor also vary based on the cell type, intracellular signaling cascade coupling, and other simultaneous signals being received.

5. 5 Receptor Affinity Fig. 5-3

6. 6

7. 7 Table 5.1 continued

8. 8 Signal Transduction Pathways

9. 9 Pathways Initiated by Lipid-Soluble Messengers Fig. 5-4

10. 10 Pathways Initiated by Lipid-Soluble Messengers Critical Points to remember: 1. Lipid messengers can diffuse through the plasma membrane. 2. They have intracellular receptors. 3. The receptors bind directly to recognized sequences in the DNA and alter gene transcription. 4. This is a slower response compared to membrane receptors, but it is a sustained response.

11. 11 Pathways Initiated by Water-Soluble Messengers Fig. 5-5

12. 12 Pathways Initiated by Membrane-Bound Receptors Critical Points to remember: 1.This is a broad range of receptors: ion channels, G-protein coupled receptors, receptors with intrinsic kinase activity, etc. 2. These receptors activate intracellular signaling cascades that affect cell function. 3. These receptors can activate downstream mediators which affect DNA transcription but also have many other effects in the cell. 4. This is a faster response compared to lipid/steroid receptors, but it is a less sustained response.

13. 13 Receptors that are Ligand-Gated Ion Channels Activation of the receptor by a first messenger (ligand) results in a conformational change of the receptor so it forms an open channel through the plasma membrane. Because the opening of ion channels has been compared to the opening of a gate in a fence, these types of channels are known as ligand-gated ion channels. The opening of ligand-gated ion channels in response to binding of a ligand results in an increase in the net diffusion across the plasma membrane of one or more types of ions specific to that channel. This often results in a change in the membrane potential. –Examples: Na +, K +, Ca 2+ channels

14. 14 Receptors that Function as Enzymes Some receptors ( like the insulin receptor) have intrinsic enzyme activity. Most of these receptors that possess intrinsic enzyme activity are all protein kinases that specifically phosphorylate the amino acid tyrosine (receptor tyrosine kinases). The typical sequence of events for receptors with intrinsic tyrosine kinase activity is: 1.The binding of a specific messenger to the receptor changes the conformation of the receptor so that its enzymatic portion, located on the cytoplasmic side of the plasma membrane, is activated.

15. 15 2.This results in autophosphorylation of the receptor (the receptor phosphorylates its own tyrosine groups). 3.The newly created phosphotyrosines on the cytoplasmic portion of the receptor then serve as docking sites for cytoplasmic proteins. 4.The bound docking proteins then bind and activate other proteins, which in turn activate one or more signaling pathways within the cell. The common denominator of these pathways is that they all involve activation of cytoplasmic proteins by phosphorylation. Receptors that Function as Enzymes

16. 16 Signaling The number of kinases that mediate these phosphorylations can be very large, and their names constitute a veritable alphabet soup— RAF, MEK, MAPKK, and many others. Most of the receptors with intrinsic tyrosine kinase activity bind ligands that typically influence cell proliferation and differentiation, and are often called growth factors.

17. 17 cGMP The one major exception to the generalization is Guanylyl Cyclase. Guanylyl cyclase is a receptor that acts to catalyse the formation, in the cytoplasm, of a molecule known as cyclic GMP (cGMP). In turn, cGMP functions as a second messenger to activate a protein kinase called cGMP-dependent protein kinase. This kinase phosphorylates specific proteins that then mediate the cell’s response to the original messenger.

18. 18 cGMP cont’d Receptors that function both as ligand-binding molecules and as guanylyl cyclases are present in high amounts in the retina of the vertebrate eye, where they are important for processing visual inputs. This signal transduction pathway is used by only a small number of messengers and should not be confused with the much more prevalent cAMP system. Also, in certain cells, guanylyl cyclase enzymes are present in the cytoplasm. In these cases, a first messenger—nitric oxide—diffuses into the cell and combines with the guanylyl cyclase there to trigger the formation of cGMP.

19. 19 Receptors that Interact with Cytoplasmic Kinases There are several families of cytoplasmic protein kinases: src, JAKs, etc. These receptors do not have intrinsic kinase activity, but must use a cytoplasmic kinase. The binding of a ligand to the receptor causes a conformational change in the receptor that leads to activation of the JAK kinase. Janus kinases (JAK) are a commonly used cytoplasmic kinase. The Janus kinases are a family of 4 kinases that are all tyrosine kinases. They are differentially expressed among the tissues in the body.

20. 20 JAK Kinases Different receptors associate with different members of the JAK kinase family, and the different JAK kinases phosphorylate different target proteins, many of which act as transcription factors. JAK’s traditional targets are the Signal Transducers of Activated transcription (STATs). However, they have also been shown to interact with other proteins. The result of these pathways is the synthesis of new proteins, which mediate the cell’s response to the first messenger. Signaling by cytokines—proteins secreted by cells of the immune system that play a critical role in immune defenses—occurs primarily via receptors linked to JAK kinases.

21. 21 G Protein-Coupled Receptors Bound to the inactive receptor is a protein complex located on the cytosolic surface of the plasma membrane and belonging to the family of heterotrimeric (containing three different subunits) proteins known as G proteins. All G proteins contain three subunits, called the alpha, beta and gamma subunits. The alpha subunit can bind GDP and GTP. The beta and gamma subunits help anchor the alpha subunit in the membrane. The binding of a ligand to the receptor changes the conformation of the receptor.

22. 22 G Protein-Coupled Receptors This activated receptor increases the affinity of the alpha subunit of the G protein for GTP. When bound to GTP, the alpha subunit dissociates from the beta and gamma subunits. This dissociation allows the activated alpha subunit to link up with still another plasma membrane protein, either an ion channel or an enzyme. These ion channels and enzymes are termed plasma membrane effector proteins because they mediate the next steps in the sequence of events leading to the cell’s response.

23. 23 G Protein-Coupled Receptors In essence, then, a G protein serves as a switch to couple a receptor to an ion channel or to an enzyme in the plasma membrane. G proteins can either be stimulatory or inhibitory. Once the alpha subunit of the G protein activates its effector protein, a GTP-ase activity inherent in the alpha subunit cleaves the GTP into GDP plus P i. This cleavage renders the alpha subunit inactive, allowing it to recombine with its beta and gamma subunits. There are several subfamilies of plasma membrane G proteins, each with multiple distinct members, and a single receptor may be associated with more than one type of G protein. Moreover, some G proteins may couple to more than one type of plasma membrane effector protein. In this way, a first-messenger-activated receptor, via its G- protein couplings, can call into action a variety of plasma membrane effector proteins such as ion channels and enzymes. These molecules can, in turn, induce a variety of cellular events. To illustrate some of the major points concerning G proteins, plasma membrane effector proteins, second messengers, and protein kinases, the next two sections describe the two most important effector protein enzymes regulated by G proteins—adenylyl cyclase and phospholipase C. In addition, the subsequent portions of the signal transduction pathways in which they participate are described.

24. 24 G Protein-Coupled Receptors There are several subfamilies of plasma membrane G proteins, each with multiple distinct members, and a single receptor may be associated with more than one type of G protein. Examples: Gs, Gi, Gq, Gα Moreover, some G proteins may couple to more than one type of plasma membrane effector protein. G-protein coupled receptors are the most numerous type of receptor family and have a large variety of signaling pathways associated with them.

25. 25 Adenylyl Cyclase and Cyclic AMP Activation of the receptor by the binding of ligand (for example, the hormone epinephrine) allows the receptor to activate its associated G protein (G s ; “stimulatory”). This causes G s to activate its effector protein, the membrane enzyme called adenylyl cyclase (also known as adenylate cyclase). The activated adenylyl cyclase catalyzes the conversion of cytosolic ATP molecules to cyclic 3´,5´-adenosine monophosphate, or cyclic AMP (cAMP). Cyclic AMP then acts as a second messenger.

26. 26 Adenylyl Cyclase and Cyclic AMP The action of cAMP eventually terminates when it is broken down to noncyclic AMP, a reaction catalyzed by the enzyme cAMP phosphodiesterase. Thus, the cellular concentration of cAMP can be changed either by altering the rate of its messenger-mediated generation or the rate of its phosphodiesterase-mediated breakdown. Caffeine and theophylline, the active ingredients of coffee and tea, are widely consumed stimulants that work partly by inhibiting phosphodiesterase activity, which results in prolonging the actions of cAMP within a cell.

27. 27 Adenylyl Cyclase and Cyclic AMP Inside the cell, cAMP binds to and activates an enzyme known as cAMP-dependent protein kinase (PKA). PKA then phosphorylates downstream targets. Examples: epinephrine acts via the cAMP pathway on fat cells to stimulate the breakdown of triglyceride, a process that is mediated by one particular phosphorylated enzyme. In the liver, epinephrine acts via cAMP to stimulate both glycogenolysis and gluconeogenesis, processes that are mediated by phosphorylated enzymes that differ from those in fat cells.

28. 28 G i Proteins Not all G proteins stimulate cAMP; some inhibit adenylyl cyclase. This inhibition results in less, rather than more, generation of cAMP. This occurs because these receptors are associated with a different G protein known as G i (“inhibitory’’). Activation of G i causes the inhibition of adenylyl cyclase. The result is to decrease the concentration of cAMP in the cell and thereby the phosphorylation of key proteins inside the cell.

29. 29 G-protein Coupled Receptors: cAMP Fig. 5-6

30. 30 Signal Amplification Fig. 5-8

31. 31 Fig. 5-9 Actions of cAMP-dependent Kinases

32. 32 Phospholipase C, Diacylglycerol, and Inositol Trisphosphate This system uses the G protein called G q. Activated G q then activates a plasma membrane effector enzyme called phospholipase C (PLC). This enzyme catalyzes the breakdown of a plasma membrane phospholipid known as phosphatidylinositol bisphosphate, abbreviated PIP 2, to diacylglycerol (DAG) and inositol tris-phosphate (IP 3 ).

33. 33 PLC, DAG, IP 3 Both DAG and IP 3 then function as second messengers. DAG activates a class of protein kinases known collectively as protein kinase C (PKC), which then phosphorylate a large number of other proteins, leading to the cell’s response. There are currently 13 known isoforms of PKC which contribute to the large variety of cellular responses observed.

34. 34 IP 3 IP 3 binds to receptors located on the endoplasmic reticulum. These receptors are ligand-gated Ca 2+ channels which when bound to IP 3 open and result in increased cytosolic Ca 2+ concentration. This increased Ca 2+ concentration then continues the sequence of events leading to the cell’s response. One of the actions of Ca 2+ is to help activate some forms of protein kinase C.

35. 35 G-protein Coupled Receptors: DAG & IP 3 Fig. 5-10

36. 36 Control of Ion Channels by G Proteins An ion channel can be the effector protein for a G protein and it can be directly or indirectly regulated. In direct regulation, the G protein interacts with the channel without any second messengers being involved. In indirect regulation, you have involvement of second messengers. Example: PKA phosphorylates a plasma membrane ion channel, thereby causing it to open.

37. 37 Ca 2+ as a Second Messenger Ca 2+ functions as a second messenger in many pathways. Ca 2+ can be either increased or decreased cytosolically to elicit a cellular response (change membrane potential). Ca 2+ also has direct actions on other signaling proteins. By means of active-transport systems in the plasma membrane and cell organelles, Ca 2+ is maintained at an extremely low concentration in the cytosol. Consequently, there is always a large electrochemical gradient favoring diffusion of Ca 2+ into the cytosol via Ca 2+ channels found in both the plasma membrane and the endoplasmic reticulum.

38. 38 Ca 2+ as a Second Messenger A stimulus to the cell can alter this steady state by influencing the active-transport systems and/or the ion channels, resulting in a change in cytosolic Ca 2+ concentration. There are Ca 2+ channels in the plasma membrane that are opened directly by an electrical stimulus to the membrane (voltage gated channels). Extracellular Ca 2+ entering the cell via these channels can, in certain cells, bind to Ca 2+ sensitive channels in the endoplasmic reticulum and open them (Ca 2+ induced Ca 2+ release).

39. 39 Ca 2+ as a Second Messenger Besides the changes to the membrane potential, Ca 2+ also acts by its ability to bind to various cytosolic proteins, altering their conformation and thereby activating their function. One of the most important of these is a protein, found in virtually all cells, known as calmodulin. On binding with Ca 2+ calmodulin changes shape, and this allows calcium-calmodulin to activate or inhibit a large variety of enzymes and other proteins, many of them protein kinases. Other proteins Ca 2+ binds include: troponin, nitric oxide synthase, PYK2.

40. 40 Calmodulin Fig. 5-11

41. 41 Arachidonic Acid and Eicosanoids The eicosanoids are a family of molecules produced from the polyunsaturated fatty acid arachidonic acid (AA) which is present in plasma membrane phospholipids. The eicosanoids include the cyclic endoperoxides, the prostaglandins, the thromboxanes, and the leukotrienes. They are generated in many kinds of cells in response to an extracellular signal. The synthesis of eicosanoids begins when an appropriate stimulus binds to its receptor and activates phospholipase A 2 (PLA 2 ).

42. 42 Arachidonic Acid and Eicosanoids PLA 2 splits off AA from the membrane phospholipids, and the AA can then be metabolized by two pathways. One pathway is initiated by an enzyme called cyclooxygenase (COX) and leads ultimately to formation of the cyclic endoperoxides, prostaglandins, and thromboxanes. The other pathway is initiated by the enzyme lipoxygenase and leads to formation of the leukotrienes. Within both of these pathways, synthesis of the various specific eicosanoids is enzyme-mediated.

43. 43 Arachidonic Acid and Eicosanoids Each of the major eicosanoid subdivisions contains more than one member, as indicated by the use of the plural in referring to them (prostaglandins, for example). On the basis of structural differences, the different molecules within each subdivision are designated by a letter—for example, PGA and PGE for prostaglandins of the A and E types—which then may be further subdivided—for example, PGE 2.

44. 44 Arachidonic Acid and Eicosanoids Eicosanoids may in some cases act as intracellular messengers, but more often they are released immediately and act locally (paracrine and autocrine agents). After they act, they are quickly metabolized by local enzymes to inactive forms. The eicosanoids exert a wide array of effects, particularly on blood vessels and in inflammation. Because AA transduces a signal from a messenger and its receptor into a cellular response (production and secretion of eicosanoids), it is sometimes considered a second messenger as well as a substrate to be converted into other products.

45. 45 Arachidonic Acid and Eicosanoids Some of the most commonly used drugs influence the eicosanoid pathway. Aspirin inhibits cyclooxygenase blocking the synthesis of the endoperoxides, prostaglandins, and thromboxanes. It and other drugs that also block cyclooxygenase are collectively termed nonsteroidal anti-inflammatory drugs (NSAIDs). Their major uses are to reduce pain, fever, and inflammation. The term nonsteroidal distinguishes them from synthetic corticosteroids (hormones made by the adrenal glands) that are used in large doses as anti-inflammatory drugs; these steroids inhibit phospholipase A 2 and block the production of all eicosanoids.

46. 46 AA Pathways Fig. 5-12

47. 47

48. 48 Cessation of Activity in Signal Transduction Pathways Once initiated, signal transduction pathways are eventually shut off because chronic overstimulation of a cell can in some cases be detrimental. The key event is usually the cessation of receptor activation. Responses to messengers are transient events that persist only briefly, and subside when the receptor is no longer bound to the ligand. A major way that receptor activation ceases is by a decrease in the concentration of first messenger molecules in the region of the receptor. This occurs as enzymes in the vicinity metabolize the first messenger, as the first messenger is taken up by adjacent cells, or as it simply diffuses away.

49. 49 Cessation of Activity in Signal Transduction Pathways Receptors can be inactivated in at least three other ways: 1.The receptor becomes chemically altered (usually by phosphorylation), which may lower its affinity for a first messenger, and so the messenger is released. 2.Phosphorylation of the receptor may prevent further G protein binding to the receptor. 3.Plasma membrane receptors may be removed when the combination of first messenger and receptor is taken into the cell by endocytosis.

50. 50 Interactions of Signal Transduction Pathways It is essential to recognize that the pathways do not exist in isolation but may be active simultaneously in a single cell, undergoing complex interactions. This is possible because a single first messenger may trigger changes in the activity of more than one pathway and, much more importantly, because many different first messengers—often dozens—may simultaneously influence a cell. Moreover, a great deal of “cross-talk” can occur at one or more levels among the various signal transduction pathways. For example, active molecules generated in the cAMP pathway can alter the of receptors and signaling molecules generated by other pathways.