Signaling Across Biomembranes PDF

5 SIGNALING ACROSS BIOMEMBRANES ILOs By the end of this lecture, students will be able to 1. Classify the different types of membranous receptors 2. Describe the mechanism of the signalling transduction pathways 3. Deduce importance of signalling across biomembranes in health a...

5 SIGNALING ACROSS BIOMEMBRANES ILOs By the end of this lecture, students will be able to 1. Classify the different types of membranous receptors 2. Describe the mechanism of the signalling transduction pathways 3. Deduce importance of signalling across biomembranes in health and disease What is signaling? Signals evolving from cells are mostly released as chemicals/molecules in the extracellular fluid around them. They can be transported to distant targets in the body (endocrine signaling by hormones), to adjacent cells (paracrine signaling) or can even act on the same cell (autocrine signaling). The information conveyed by such signals are converted into several cellular responses within the target cells such as metabolism, gene expression, cell division and differentiation..etc. Stages of cell signaling Divided into 3 stages: Reception, Transduction, Response ( Figure 1) 1. Reception: Is the process where a cell recognizes the signaling molecule (a ligand) by a specific protein (a receptor) (on its surface or within its cytosol or nucleus). Binding of the ligand to its specific receptor has to occur in order to start signaling process. Once bound, the receptor initiates a process that amplifies the signal or integrates it with inputs from other receptors. Response ends by loss of binding of the ligand to its receptor. 2. Transduction: Is the cascade of events initiated by receptor-ligand binding which changes the conformation of the receptor causing its activation, hence permitting the flow of information to passes along sequential steps of molecular relays (Signal transduction). In such Signal Transduction Pathway, each relay molecule changes and activates the next group of molecules in the pathway causing amplification of the signal. Also along such pathway there is always upstream molecules and events that come earlier in the relay chain, followed by downstream ones that come later. N.B.Relay Molecules These are mostly proteins/enzymes but are sometimes non-protein or ions. They help transmit the signal from one relay molecule to the other until cellular response is reached. With some receptors, enzymes are the first effector proteins/relay [e.g adenyl cyclase, guanyl cyclase, phospholipase C] enzymes. Such enzymes are activated downstream of ligand binding to such receptors. In turn, they will either switch on/off other sets of proteins directly or achieve that through non-proteins [as cyclic AMP (cAMP), cyclic GMP (cGMP), phospholipid derivatives as inositol phosphate] or ions [as 1  Calcium]. These become considered as secondary messengers transmitting information further downstream. Relay proteins of transduction cascade are switched on/off by a phosphorylation/dephosphorylation process. Phosphorylation is achieved by addition of phosphate to serine or threonine residues by the action of “kinase family” of enzymes. While dephosphorylation is achieved by their removal by the action of “phosphatase family” of enzymes. Although in most cases, phosphorylation acts to switch on (activate) transmission in the face of dephosphorylation which turns it off, yet the reverse is often true where dephosphorylation can switch on transmission. 3. Response: Is the final step in a signal transduction pathway that activates a cellular response; contraction, secretion, motility, metabolic reaction, growth, transcription regulation and differentiation…etc) Figure 1. Steps of cell signaling TYPES OF RECEPTORS These are either present on Cell-Surface or Intracellular. I) Cell-Surface Receptors [Cell Membrane Receptors] Are membrane-anchored proteins that bind to ligands on the outside surface of the cell without need to cross the plasma membrane. Such ligands include large, hydrophilic molecules [as peptides and proteins] including growth factors, cytokines, some hormones or certain neurotransmitters Structurally: they have three different domains: an Extracellular Ligand-binding Domain for reception of ligand, a Hydrophobic Intra-membranous Domain and an Intra-cytosolic Domain for transmission of signals. Many types exist, but the commonest are: Ligand-gated ion channels, G protein-coupled receptors, and enzyme-linked receptors. A) Ligand-Gated Ion Channels ( Figure 2) Are ion channels that can open in response to the binding of a ligand to the receptor. 2  The hydrophilic channel within it, permits crossing of ions [Na+, K+, Ca 2+, Cl-] without having to touch the hydrophobic core of membrane phospholipid bilayer. The reverse is also true, as some channels are initially opened and ligand binding induces their closure. Their cellular response is rapid occurring on milliseconds. They are commonly found in excitable tissues as neurons, muscles and some secretory cells. Figure 2.Ligand gated ion channels Clinical Implication Gene mutation of chloride channels present on surface of epithelial cells in lungs, intestines, skin, and pancreas, results in Cystic Fibrosis. The disease presents as defective ion transport that leads to presence of very high concentrations of Na+ and Cl– in sweat and of highly viscous mucus that obstructs pancreatic and bile ducts and airways of the lungs. b) G Protein-Coupled Receptors (GPCRs) Are a large family of cell surface receptors that share a:  Common structure [seven different protein segments crossing the membrane] present in an inactive state in the cell membrane when ligand is absent.  Method of transmission of signal to inside the cell [by a protein called G protein ] Structurally; a G protein is heterotrimeric, i.e. made up of three subunits (α, β, γ). It is inactive “off” when attached to guanosine diphosphate (GDP) in absence of ligand. When ligand binds it becomes active (on) as guanosine triphosphate (GTP) becomes attached instead. G protein has an intrinsic GTPase activity that can break down (hydrolyze) GTP to GDP rendering it inactive as seen in Figure 3 Figure 3.G protein coupled receptors 3 There are many types of G protein that are categorized depending on the function of its α subunit.The most abundant of these are: Gs: Stimulates adenylate cyclase i.e the cyclic AMP pathway and Ca2+ Channels Gi: Inhibits adenylate cyclase Gq: Stimulates phospholipase C i.e. the phosphoinositide pathway. G0: Inhibits Ca2+ channels Clinical Implication Toxins produced by microorganisms as Bordetella pertussis (pertussis toxin) and Vibrio cholerae (cholera toxin) modify functions of certain G proteins producing diseased states. The Cholera toxin; is an enzyme that covalently modifies the α subunit of Gs to sustain it in a persistently activated state. This increases cAMP, in intestinal epithelium to induce large efflux of Cl and water into the gut producing the severe and potentially fatal diarrhea associated with - cholera. The Pertussis toxin; similarly, activates α subunit of Gi in airway epithelium. This inhibits exchange of GDP for GTP. As a consequence, adenylate cyclase is not inhibited and the increased cyclic AMP will lead to development of fluid imbalance and severe, life-threatening congestion characteristic to whooping cough in airway of patients infected with such organism. c) Enzyme-Linked Receptors Are receptors of many growth factors, cytokines and hormones and have a major role in regulation of cell growth, proliferation and differentiation. Structurally, beyond their extracellular ligand-binding domain, they posses a short hydrophobic transmembrane helix linked to an intracellular effector domain that possess an intrinsic enzyme activity or is associated directly with an intracellular enzyme. Enzyme linked receptors have many types; categorized according to nature of the linked effector enzyme. Examples include: 1-Receptor Tyrosine Kinase (RTK) The receptor is present at the cell surface in the form of a monomer (1 unit).When the ligand [e.g. insulin and most growth factors] binds the receptor, two receptors dimerize, which leads to activation of their intrinsic tyrosine kinase activity. This in turn induces auto-phosphorylation (on tyrosine units on the receptor) to further phosphorylate down-stream adapter proteins and other kinases.(Figure 4) RAS (small G-protein) is a downstream signal of RTK, that regulates normal cell proliferation and growth. Insulin receptor is a special RTK with special structure that regulates many cellular metabolic processes, mainly carbohydrate metabolism. (Refer to endocrine module) 4 2-Receptor Serine/Threonine Kinase When their ligand [few growth factors] binds, the sequence proceeds like that with RTK, but serine or threonine residues are autophosphorylated instead of tyrosine. 3-Tyrosine-Kinase Associated Receptors Their ligand [most cytokines] binds to dimerize two receptors. Thsis receptor doesn’t have an intrinsic kinase activity, but rather activates the associated proteins that have tyrosine kinase activity to phosphorylate downstream adapter proteins and other kinases 4-Receptor Guanylyl Cyclase Their ligand (Natriuretic Peptides] bind to activate guanyl cyclase with production of cGMP as a secondary messenger. Figure 4. Receptor tyrosine kinase Clinical Implication: The small RAS G-protein that is downstream of RTK can switch to become oncogenic under the influence of many cellular injurious insults. Consequently, it becomes constitutively active promoting uncontrolled cellular growth and cancerous transformation. Recent understanding of signaling via enzyme-linked receptors has permitted development of drugs in fields of regenerative medicine & tissue repair and in control of immune disorders and cancer. 5 6 6 Drugs Modulating Cell Signalling ILOs: By the end of this lecture, students will be able to 1. Classify how drugs can generally act while being outside the cell membrane. 2. Appraise how they can exactly modulate cell signalling mainly via targeting membrane receptors. 3. Analyse the plotted relation between drug concentration/dose and its response when constructing a graded dose response curve. 4. Distinguish the two concepts of agonism and antagonism and their subtypes in relation to changes in receptor signalling functions. 5. Correlate the modality of drug receptor interaction to their utility for achieving a therapeutic benefit. From what was so far studied, it was mentioned that drugs (like any other biochemical substance) are variably capable of modulating the: ▪ Actions of enzymes (extracellular or intracellular after diffusion or transport). (Refer to enzymes) ▪ Action on voltage-gated ion channels. (Refer to Transport) However, for most of the drugs in clinical use, the major target of drug action are the receptors whether being Cell-Surface Receptors (Cell-Membrane) (Refer to signalling across biomembranes) or intracellular (Refer to gene expression). Also, another target of drug action is on some membrane transporters (Refer to Victorian Transport) THE ACTION OF DRUGS IN MODULATING THE EFFECT OF RECEPTOR TRANSDUCTION PATHWAYS ACROSS THE CELL MEMBRANE, i.e., this is by modifying transduction of extracellular signals into intracellular responses. Such action falls into three categories: A. Drugs Modulating Ligand-Gated Ion Channels (Ionotropic Receptors) By binding instead of the ligand, drugs will alter flow of ions passing in the channels coupled to such receptors. Doing so, drugs can modulate diverse functions in excitable tissues as neurotransmission, neuronal or cardiac excitability, cardiac or muscle contractility. They can achieve this as in: a. Inhibition of influx of Na and efflux of K, in response to activation of Nicotinic Receptors at the neuromuscular junction to help in muscle relaxation with anaesthetics during operations. b. Stimulation of influx of chloride, in response to Gamma-amino Butyric Acid (GABA) receptor A to suppress neuronal function to help in treatment of convulsions or induction of sleep. A. Drugs Modulating G-Protein Coupled Receptors (Metabotropic) By binding instead of the ligand to these receptors, drugs will ignite or switch off the transduction cascade. Such cascade is initiated by the specific G-protein in question and its coupled 2nd messenger that links to their specific protein kinase, to affect many other Page 1 of 4  down-stream effectors regulating diverse cellular functions. Since these receptors exist in most tissues, most of the available drugs do work through their modulation to yield a therapeutic response triggered mainly by: a. Gs proteins linked to β2 adrenergic receptors, to activate adenylate cyclase (AC)→ increase cAMP → activate protein kinase A (PKA) to induce bronchodilation in bronchial asthma. b. Gi proteins linked to α2 adrenergic receptors, to inhibit AC → decrease cAMP → inhibit PKA, to reduce blood pressure in cases of hypertension c. Gq proteins, linked to α1 adrenergic receptors, to activate PLC → increase IP3 & DAG → activate PKC to increase blood pressure in hypotension induced by anaesthesia during surgery. B. Drugs Modulating Enzyme-Linked Receptors Most advancement in therapies targets these receptors that belong to growth factors, cytokines, some peptides, etc. acting mostly to control autoimmunity, inflammations, and cancer. When drugs act in link to these receptors they can: ▪ Bind to receptor instead of ligand to activate or inhibit down-stream receptor activation as the use of Exogenous Insulin to stimulate intrinsic tyrosine-kinase receptor to control Type I more than Type II Diabetes. ▪ Bind to the ligand itself to prevent its activation to its receptors as during the use of anti-TNFα monoclonal antibodies [mAb] to control Rheumatoid or Inflammatory Bowel disorders ▪ Binding to the down-stream signalling of the receptor directly as when immune modulators is used to treat some cancers. DRUG-RECEPTOR INTERACTIONS Upon interaction of a drug with its receptor the following is considered: Drug AFFINITY to show how well a drug recognises and binds to its receptor. Drug POTENCY to measure the required amount(quantity)(concentration)(dose) of drug necessary to produce an effect of a given intensity when reaching and binding with variable affinity to its receptor. Drug EFFICACY to measure the effectiveness (inherent ability) (intrinsic activity) of a drug to elicit a maximum achieved response, when given at the highest practical concentration. Accordingly, When a drug binds to its specific receptor and the formed bound complex fails to elicit any desired response and meanwhile prevent the endogenous ligand from binding to such receptor, the drug is then called an ANTAGONIST; denoting that it only possesses affinity but no intrinsic activity. Page 2 of 4  When a drug (that possesses an intrinsic inherent ability) binds to its receptor and the formed bound complex elicits a desired response, the drug then is called an AGONIST; denoting that it possesses both affinity and efficacy. If the Relationship between the log Concentration (or the dose) of an Agonist and the evoked-response is plotted, an S-shaped curve develops and is called: The Graded Dose-Response Curve: Figure 1 The curve, clears that: The response increases with increasing the agonist dose, till it plateaus at a maximum concentration (Emax); we are speaking then of drug efficacy. Accordingly, the concentration giving half-maximal response; is then considered Effective Concentration 50 (EC50). The potency of a drug is considered high if an effect of a given intensity is evoked by a small dose of a drug. If higher doses are needed, the potency of drug is low. There are different types of agonistic and antagonistic drugs that act on a receptor. A. Types of AGONISTS (Figure 2) If the response of that agonist ▪ Mimics the response of endogenous ligand, i.e has the same efficacy (inherent activity = 1) as natural ligand and is giving a maximum response, then this denotes that the drug is a FULL AGONIST. ▪ Is incapable of reaching the response of endogenous ligand; i.e has lower efficacy (inherent activity > zero & < 1) and is giving sub-maximal response, then this denotes that the drug is a PARTIAL AGONIST. ▪ Stabilizes a spontaneously activated receptor setting it back to its inactive state, by utilizing its inherent activity (less than zero), to exert a pharmacological effect opposite to that triggered by the agonist; the drug is then called an INVERSE AGONIST. N.B.: On clinical use, no clear distinction can be made between inverse agonists and antagonists. Therefore, many of the inverse agonists are commonly classified as antagonists. B. Types of ANTAGONISTS If the response of that antagonist: Page 3 of 4 ▪ Is blocking the agonist ability to bind to the specific binding site (active site) of its receptor. They have high affinity (competing with agonist for binding) - have zero inherent activity (maintaining receptor in inactive state) According to nature of binding, the drug is either: a. COMPETITIVE REVERSIBLE ANTAGONIST: its binding is non-permanent (reversible) and can be overcome by increasing the concentration of the agonist relative to it. b. COMPETITIVE IRREVERSIBLE ANTAGONIST: its binding is permanent because it forms a covalent bond with the active site. It cannot be overcome by increasing the concentration of the agonist. Its action only disappears after the body synthesis new receptors. ▪ Is blocking the agonist ability to activate its receptor by binding at a different site (allosteric site). They do not interfere with binding of the agonist to its active site (have no affinity to it) and are not overcome by increasing concentration of the agonist. They have zero inherent activity (maintaining receptor in inactive state). They are called; NON-COMPETITIVE ANTAGONIST [ALLOSTERIC ANTAGONIST]. N.B. Other Modalities of Antagonism: 1. Physiological Antagonism: Achieving antagonistic effect by acting on a different receptor. As using adrenaline that induces vasoconstriction by action on α1-adrenergic receptors to antagonize the vasodilator action of histamine produced by its action on H1 receptors. 2. Chemical Antagonism: Achieving antagonistic effect by reacting chemically to form an inactive complex thus abolishing the action of the drug in question. As using Protamine sulphate that forms an inactive complex with heparin when there is need to stop haemorrhage induced by it. Page 4 of 4