Binding and activation of receptors (1).pptx

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1. SANA SAIFI M.PHARM TOPIC: BINDING AND ACTIVATION OF RECEPTOR ASSIGNMENT OF MEDICINAL CHEMISTRY

2. INTRODUCTION: Cellular receptors are proteins either inside a cell or on its surface which receive a signal. In normal physiology, this is a chemical signal where a protein-ligand binds a protein receptor. The ligand is a chemical messenger released by one cell to signal either itself or a different cell. The binding results in a cellular effect, which manifests as any number of changes in that cell, including altering gene transcription or translation or changing cell morphology. BINDING OF RECEPTORS

3. Typically, a single ligand will have a single receptor to which it can bind and cause a cellular response . There are several different types of cellular signaling, all of which depend on different ligands and cellular receptors . The major categories of cellular signaling include autocrine , signal across a gap junction, paracrine, and endocrine. Autocrine signaling is when a cell releases a signal that then binds one of its receptors to change its functioning. Signaling across gap junctions is when small signaling molecules move directly across neighboring cells that are attached . Paracrine signaling is communication between cells that are nearby . Endocrine signaling is when cell signals travel to target cell receptors in a different part of the body through the bloodstream

4. . Each type of signaling requires a ligand and a receptor. Cellular receptors can broadly categorize into internal receptors, cell-surface receptors, ion channel receptors, G-protein-coupled receptors (GPCRs), and enzyme-linked receptors . Internal Receptors : These receptors are also known as either intracellular or cytoplasmic . They are found in the cytoplasm of a cell and are often targeted by hydrophobic ligands that can cross the lipid bilayer of the animal plasma cell membrane . They accomplish this by the ligand-receptor complex being able to travel to the nucleus and bind DNA at a gene regulatory site. Testosterone , estrogen, cortisol, and aldosterone are examples of steroid hormones that are hydrophobic and pass through the plasma membrane to target internal receptors ..

5. Ion Channel Receptors When a ligand binds an ion channel receptor, a channel through the plasma membrane opens that allows specific ions to pass through. Ligand binding creates a change in the shape of the receptor that allows specific ions to pass, usually sodium, magnesium, calcium, or hydrogen. Chemically gated ion channels are on dendrites and the cell bodies of neurons . GPCRs GPCRs are a subtype of cell surface receptors that act through a G-protein to start a second messenger cascade, modulating cellular function . The receptor has the ligand-binding site on the outside of the plasma membrane and has a transmembrane portion that can bind to a G-protein in the intracellular space . A G-protein is a heterotrimeric protein with three subunits, alpha, beta, and gamma.

6. The beta and gamma subunits are attached to the membrane by a lipid anchor.  When no ligand is bound to the receptor, the alpha subunit and a GDP are bound to the transmembrane receptor and the beta and gamma subunits . When the ligand binds to the receptor, a conformational change activates the G protein, and a GTP molecule replaces the GDP molecule on the alpha subunit . The G-protein dissociates with the beta and gamma subunits remaining attached by their anchor, and the activated alpha subunit, now bound to a GTP molecule, is freed from the intracellular wall of the plasma membrane . Both the beta-gamma dimer and the alpha-GTP can act to propagate the signal cascade. Some common enzymes and second messengers activated by this cascade include adenylate cyclase , cyclic AMP, diacylglycerol , inositol 1, 4, 5-triphosphate, and phospholipase C. GPCRs can be both activating and inhibiting. GPCRs are involved in many functions of the multicellular organism, including but not limited to growth, endocrine signaling, sensation, and clotting.

7. Cell-Surface Receptors These receptors are also known as transmembrane receptors. These are proteins that are found on the surface of cells and span the plasma membrane. They bind to ligands that cannot themselves pass through the plasma membrane. These are often hydrophilic ligands. These receptors don’t bind DNA to modify gene transcription and translation themselves but rather perform signal transduction; an extracellular signal triggers an intracellular signal, which will usually go to the nucleus to affect cell functioning. Often a cell surface receptor will be specific for that cell type so that the ligand can only affect the functioning of its target cells. Cell-surface receptors come in three main types: ion channel receptors, GPCRs, and enzyme-linked receptors .

8. Enzyme-Linked Receptors This subtype of transmembrane receptors has a catalytic site on the cytoplasmic domain. Often, when the ligand binds these receptors, they dimerize , which activates the receptor’s catalytic site and results in enzymatic activity. There are several types of enzyme-linked receptors; the most common type is the receptor tyrosine kinase . Other examples include receptor serine/threonine kinase, receptor guanylyl cyclase , and receptor tyrosine phosphatases . Receptor tyrosine and receptor serine and threonine kinases dimerize , which causes autophosphorylation to happen at the tyrosine, serine, or threonine sites, respectively. This phosphorylation is what activates the enzymatic activity of the receptor.

9. Activation of receptor The interaction of a receptor with its chemical messenger is only the first step in a complex chain of events involving several secondary messengers, proteins, and enzymes that ultimately leads to a change in cell chemistry. These events are referred to as signal transduction . Unfortunately, a full and detailed account of these processes would fi ll a textbook in itself so the following account is focused mainly on the signal transduction processes that result from activation of G-protein-coupled receptors and kinase receptors. The signal transduction pathways following activation of G-protein-coupled receptors are of particular interest as 30% of all drugs on the market interact with these kinds of receptors. The transduction pathways for kinase receptors are also of great interest as they offer exciting new targets for novel drugs, particularly in the area of anticancer therapy . An understanding of the pathways and the various components involved helps to identify suitable drug targets.

10. Signal transduction mechanism for G-Protein coupled receptor G-protein-coupled receptors activate a signalling protein called a G-protein, which then initiates a signalling cascade involving a variety of enzymes . The sequence of events leading from the combination of receptor and ligand (the chemical messenger) to the final activation of a target enzyme is quite lengthy, so we shall look at each stage of the process in turn.

11. Interaction of the receptor–ligand complex with G-proteins The first stage in the process is the binding of the chemical messenger or ligand to the receptor, followed by the binding of a G-protein to the receptor–ligand complex. G-proteins are membrane-bound proteins situated at the inner surface of the cell membrane and are made up of three protein subunits (α, β, and γ). The α-subunit has a binding pocket which can bind guanyl nucleotides (hence the name G-protein) and which binds guanosine diphosphate (GDP) when the G-protein is in the resting state. There are several types of G-protein (e.g. Gs , Gi /Go, Gq /G 11 ) and several subtypes of these. Specific G-proteins are recognized by specific receptors. For example, G s is recognized by the β- adrenoceptor , but not the α- adrenoceptor .

12. ) Firstly, the receptor binds its neurotransmitter or hormone. As a result, the receptor changes shape and exposes a new binding site on its inner surface. The newly exposed binding site now recognizes and binds a specific G-protein. Note that the cell membrane structure is a fluid structure and so it is possible for different proteins to ‘ float ’ through it . The binding process between the receptor and the G-protein causes the latter to change shape, which, in turn, changes the shape of the guanyl nucleotide binding site. This weakens the intermolecular bonding forces holding GDP and so GDP is released . Therefore, GTP replaces GDP .

13. Binding of GTP results in another conformational change in the G-protein which weakens the links between the protein subunits such that the α-subunit (with its GTP attached) splits off from the β and γ-subunits . Both the α-subunit and the βγ-dimer then depart the receptor. The receptor–ligand complex is able to activate several G-proteins. This leads to an amplification of the signal. Both the α-subunit and the βγ-dimer are now ready to enter the second stage of the signalling mechanism .. We shall first consider what happens to the α-subunit.

14. Signal transduction pathways involving the α-subunit The first stage of signal transduction (i.e. the splitting of a G-protein) is common to all of the 7-TM receptors. However , subsequent stages depend on what type of G-protein is involved and which specific α-subunit is formed . Different α-subunits—there are at least 20 of them—have different targets and different effects : • αs stimulates adenylate cyclase ;

15. • αi inhibits adenylate cyclase and may also activate potassium ion channels ; • αo activates receptors that inhibit neuronal calcium ion channels ; • αq activates phospholipase C We do not have the space to study all these pathways in detail. Instead , we shall concentrate on two—the activation of adenylate cyclase and the activation of phospholipase C .

16. Signal transduction involving G-Proteins and adenylate cyclase Activation of adenylate cyclase by the αs-subunit The αs -subunit binds to a membrane-bound enzyme called adenylate cyclase (or adenylyl cyclase ) and ‘switches’ it on . Th is enzyme now catalyses the synthesis of a molecule called cyclic AMP ( cAMP ) . cAMP is an example of a secondary messenger which moves into the cell’s cytoplasm and carries the signal from the cell membrane into the cell itself .

17. The enzyme will continue to be active as long as the αs - subunit is bound, resulting in the synthesis of several hundred cyclic AMP molecules—representing another substantial amplification of the signal. However , the αs - subunit has intrinsic GTP- ase activity (i.e. it can catalyse the hydrolysis of its bound GTP to GDP) and so it deactivates itself after a certain time period and returns to the resting state . The αs -subunit then departs the enzyme and recombines with the βγ-dimer to reform the Gs -protein while the enzyme returns to its inactive conformation .

18. Activation of protein kinase A cAMP now proceeds to activate an enzyme called protein kinase A (PKA ). PKA belongs to a group of enzymes called the serine–threonine kinases which catalyse the phosphorylation of serine and threonine residues in protein substrates. Protein kinase A catalyses the phosphorylation and activation of further enzymes with functions specific to the particular cell or organ in question, for example lipase enzymes in fat cells are activated to catalyse the breakdown of fat. The active site of a protein kinase has to be capable of binding the region of the protein substrate which is to be phosphorylated, as well as the cofactor ATP which provides the necessary phosphate group.

19. There may be several more enzymes involved in the signalling pathway between the activation of PKA and the activation (or deactivation) of the target enzyme. For example, the enzymes involved in the breakdown and synthesis of glycogen in a liver cell are regulated.

20. Signal transduction involving G-proteins and Phospholipase C G-protein effect on phospholipase C Certain receptors bind G s - or G i -proteins and initiate a signalling pathway involving adenylate cyclase . Other 7-TM receptors bind a different G-protein called a G q -protein , which initiates a different signalling pathway . This pathway involves the activation or deactivation of a membrane-bound enzyme called phospholipase C.

21. . The first part of the signalling mechanism is the interaction of the G-protein with a receptor–ligand complex. This time, however, the G-protein is a G q- protein rather than a G s or G i -protein, and so an αq -subunit is released. Depending on the nature of the released αq -subunit, phospholipase C is activated or deactivated . If activated, phospholipase C catalyses the hydrolysis of phosphatidylinositol diphosphate (PIP 2 ) (an integral part of the cell membrane structure) to generate the two secondary messengers diacylglycerol (DG) and inositol triphosphate (IP 3)

22. Action of the secondary messenger: diacylglycerol Diacylglycerol is a hydrophobic molecule and remains in the cell membrane once it is formed. There , it activates an enzyme called protein kinase C (PKC) which moves from the cytoplasm to the cell membrane and then catalyses the phosphorylation of serine and threonine residues of enzymes within the cell. Once phosphorylated, these enzymes are activated and catalyse specific reactions within the cell . These induce effects such as tumour propagation, inflammatory responses, contraction or relaxation of smooth muscle, the increase or decrease of neurotransmitter release, the increase or decrease of neuronal excitability, and receptor desensitizations .

23. Action of the secondary messenger: inositol triphosphate Inositol triphosphate is a hydrophilic molecule and moves into the cytoplasm. This messenger works by mobilizing calcium ions from calcium stores in the endoplasmic reticulum . It does so by binding to a receptor and opening up a calcium ion channel . Once the ion channel is open, calcium ions flood the cell and activate calcium-dependent protein kinases which, in turn, phosphorylate and activate cell-specific enzymes .

24. The released calcium ions also bind to a calcium binding protein called calmodulin , which then activates calmodulin -dependent protein kinases that phosphorylate and activate other cellular enzymes . Calcium has effects on contractile proteins and ion channels. the release of calcium is crucial to a large variety of cellular functions including smooth muscle and cardiac muscle contraction, secretion from exocrine glands, transmitter release from nerves, and hormone release.

25. Signal transduction involving Kinase –Linked receptors Activation of signalling proteins and enzymes E nzyme-Linked Receptors  • have intrinsic enzymatic activity or are associated with an enzyme (usually a kinase) • play a role in apoptosis, cell differentiation, cell division, cell growth, immune response, inflammation, and tissue repair . Kinases   (Protein Kinases [PKs]) • enzymes that catalyze the phosphorylation of target molecules to cause their activation.  In other words, they add a phosphate group to a molecule/protein/another kinase . Phosphatases  • enzymes that catalyze the dephosphorylation of target molecules. This dephosphorylation usually inactivates the target molecule (effector). Phosphatases act in opposition to kinases.

26. It is the balanced activity of kinases and phosphatases that results in effective, and fast, signaling events to allow for the desired biological effect and enable our bodies to maintain homeostasis.

27. Enzyme Linked Receptor Structure  • composed of three key domains Ligand-Binding Domain  • often has a large  EXTRACELLULAR  ligand-binding domain allowing for easy access and activation to the receptor . Transmembrane Domain  • composed of a series of hydrophobic amino acids (reminder: inner membrane is lipophilic/hydrophobic) that tethers the receptor to the cell membrane . Intracellular “Active Enzyme” Domain  • either intrinsic to the receptor or tightly bound to the transmembrane domain. The majority of the “active enzyme” domains are kinases that phosphorylate the amino acids serine, threonine and tyrosine of proteins.

28. Example  •  Insulin Receptor  • ligand is insulin, which stimulates carbohydrate (glucose) utilization and protein synthesis . Ligand-binding domain  = alpha domain Transmembrane domain  = runs through the membrane anchoring it to the membrane “Active enzyme” domain  = tyrosine kinase domain (intracellular component) activated by phosphorylation

29. Once activated, the insulin receptor leads to a cascade of events eventually resulting in expression of glucose transporters (GLUTs) on the surface of a cell to allow it to bring in glucose for energy utilization.

30. Signal Transduction by Enzyme-Linked Receptors Ligand binding leads to dimerization of two neighboring receptors . Neighboring dimerized receptors auto phosphorylate one another SH2-domain proteins bind to the phosphorylated receptors and are then phosphorylated enabling the continuation of the signal eventually leading to gene transcription. SH2-domain   • protein domain composed of about 100 amino acid residues. SH2-domains most commonly play a role in the signal transduction by receptor tyrosine kinase pathways as you will see later on in this section. They interact with specific target molecules (peptides) with a phosphorylated tyrosine residue.

31. Main Types of Enzyme-Linked Receptors  Receptor Tyrosine Kinases  [ RTK ] • make up the majority of enzyme-linked receptors. Signal transduction through RTK results in specific phosphorylation of  tyrosine  residues on target proteins and subsequent increase in gene transcription and regulation of cell growth, differentiation and survival . Serine/Threonine Kinases  • specifically phosphorylate the hydroxyl side chains of  serine or threonine  amino acid residues. These kinases may have a role in cell proliferation, differentiation, apoptosis and even embryonic development . Tyrosine Kinase-Associated Receptors  [“mixed”] • do not have a tyrosine kinase domain, rather act through cytoplasmic tyrosine kinases.

32. Receptor Tyrosine Kinases  [RTK] • This class of receptors are also considered enzymes that have intrinsic enzymatic activity. When RTK agonists bind to these receptors, their intrinsic enzymatic activity is stimulated. RTKs bind growth factors to signal processes that result in the regulation of cell growth, differentiation and survival through gene transcription. When RTKs are activated by an agonist, they form cross-linked dimers resulting in the activation of the tyrosine kinase by phosphorylation. Remember, kinases specifically act to catalyze the phosphorylation of a target molecule, which in this case is a neighboring RTK ..

33. The dimerized RTKs phosphorylate each other multiple times to result in signal amplification. This process is known as cross-phosphorylation. RTK cross-phosphorylation then leads to the phosphorylation of other proteins that will eventually result in modulation of gene transcription

34. Ras  = G-protein specific to this pathway                     RAF  = proto-oncogene serine/threonine kinase MEK  = mitogen-activated protein kinase                    MAPK  = mitogen-activated protein kinase Ras   • a small G protein ( GTPase ) involved in signal transduction leading to cell division and proliferation. If not regulated properly, Ras proteins can lead to uncontrolled cell division that eventually results in tumor formation . RAF [Rapidly Accelerated Fibrosarcoma ]  • family of protein kinases that are involved with retroviral oncogenes (genes that can potentially cause cancer).

35. Tyrosine-Kinase Associated Receptors  • associate with intracellular proteins that have tyrosine kinase activity. These receptors lack the tyrosine kinase domain that was discussed earlier and, therefore, accomplish tyrosine phosphorylation by cytoplasmic tyrosine kinases instead . Cytokine receptors make up the largest family of receptors that relay signals into the cell by cytoplasmic tyrosine kinases . These particular receptors are associated with the cytoplasmic kinase, Jak (Janus kinase). Jak will go on to activate a gene regulatory protein called STAT (signal transducers and activators of transcription). The pathway is described and depicted below:

36. Key Pathway  •  Jak /Stat pathway  (Janus kinase/signal transducers and activators of transcription ) • The Jak /Stat pathway is the principal pathway for cytokines and growth factors in humans. This pathway is activated by a number of cytokines (most commonly interferons ) and growth factors . Activation stimulates cell proliferation, differentiation, migration and apoptosis. Furthermore, cytokines control the synthesis and release of a number of inflammatory mediators . When a cytokine binds to its enzyme-linked receptor it results in a conformational change leading to phosphorylation of the intracellular active-enzyme domain, eventually leading to the transcription of inflammatory mediators. As with all signal transduction mechanisms, homeostasis is reliant on proper regulation of all these different pathways. Lack of proper regulation of the JAK pathway can cause inflammatory disease, erythrocytosis , gigantism and leukemias .