Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Endomembrane system wikipedia , lookup
Organ-on-a-chip wikipedia , lookup
NMDA receptor wikipedia , lookup
Purinergic signalling wikipedia , lookup
List of types of proteins wikipedia , lookup
Paracrine signalling wikipedia , lookup
G protein–coupled receptor wikipedia , lookup
Signaling via G-Protein-Linked Surface Receptors: Cell- This is largest family of cell-surface receptors >100 members have already been defined in mammals Though these receptors bind different hormones and may mediate different cellular responses, they form a class of receptors that functions similarly and exhibits the following properties: They consist of a single polypeptide chain that threads back and forth across the lipid bilayer seven times Fig 15.17 Alberts 3rd Ed These seven α-helices contains ~ 22-24 hydrophobic amino acid residues Four extra cellular loops and four intracellular loops Cytosolic loops and C-terminal segment, which face the cytosol, are important for interaction with a Gprotein Fig 20.10 Lodish 3rd Ed The signal transducing G-Protein associated with the receptor functions as an on-off molecular switch, which is in the off state when it binds GDP. Binding of ligand to the receptor causes the G protein to release its bound GDP and to bind GTP, converting the Gprotein to the on state Fig 15.14 Alberts 3rd Ed Fig 15.15 Alberts 3rd Ed The activated G-protein, with a bound GTP, binds to and activates/inhibits an effector enzyme, which catalyzes formation of 2nd messenger Hydrolysis of GTP, bound to the G-protein, switches the G-protein back to the inactive i.e. in off state To illustrate the operation of this important class of receptors, we will take example of epinephrine/nor-epinephrine receptors and will try to understand structure-function relationship and their associated signal-transducing G-proteins Effector system in this case is adenyl cyclase which synthesizes cAMP as 2nd messenger Binding of Epinephrine (EP) to β-and αAdrenergic Receptors Induces TissueSpecific Responses Mediated by cAMP: Epinephrine and nor-epinephrine were originally recognized as products of the medulla/core of the adrenal gland They are also called adrenaline and nor-adrenaline Embryologically nerve cells drive from the same tissue as adrenal medulla cells They are also synthesized by neurons of central and peripheral nervous system Both hormones are charged compounds that belong to the catecholamines, active amines containing the compound catechol: Location: Fig 25.9 Brum Synthesis: Fig 24.10 Zubay In times of stress, such as fright or heavy exercise, all tissues have an increased need for glucose and fatty acids These principle metabolic fuels can be supplied to the blood in seconds by the rapid breakdown of glycogen in liver i.e. glycogenolysis and of triacylglycerol in adipose storage cells i.e. lipolysis Epinephrine was first hormone to be isolate, characterized and synthesized Tyrosine hydroxylase: The amount and activity are regulated by cAMPdependent mechanism that are responsive to neurotransmitter (acetylcholine) CaM kinase II: -Present in all animal cells but especially enriched in nervous system and highly concentrated in synapses (neurotransmitters) -Ca 2+ influx through voltage-gated Ca 2+ channel in plasma membrane stimulates cell to secrete neurotransmitters - Both secretion and resynthesis of the neurotransmitters are stimulated when cell is activated In mammals, the liberation of glucose and fatty acids can be triggered by: Binding of epinephrine and nor-epinephrine to βadrenergic receptors on the surface of hepatic and adipose cells Epinephrine bound to similar β-adrenergic receptors on heart muscle cells increases the contraction rate, which increases the blood supply to the tissues Epinephrine bound to β-adrenergic receptors on smooth muscle cells of the intestine causes them to relax Another type of adrenergic/epinephrine receptor, the αadrenergic receptor, is found on smooth muscle cells lining the blood vessels in the intestine tract, skin and kidneys Binding of epinephrine to α-adrenergic receptors causes the arteries to constrict, cutting off circulation to the peripheral organs All these diverse effects of one hormone are directed common end: to a Supplying energy for the rapid movement of major locomotor muscles in response to bodily stress All of the very different tissue-specific responses induced by binding of epinephrine to β-adrenergic receptors are mediated by a rise in the intracellular level of cAMP resulting from activation of adenylate cyclase cAMP as a second messenger modifies the rate of different enzyme catalyzed reactions in specific tissues generating various metabolic responses Binding of numerous other hormones to their receptors also leads to a rise in intracellular cAMP and characteristic tissuespecific metabolic responses Table 20.5 Lodish 3rd Ed Two types of experiments have been used to establish the identity of the β-adrenergic receptor Fig 20.11 Lodish 3rd Ed Evidence that β-adrenergic receptor mediates induction of epinephrine-initiated cAMP synthesis was studies with receptors purified by affinity chromatography Fig 20.12 Lodish 3rd Ed Fig 2.17 Lodish 3rd Ed Fig 15.4 Lodish 3rd Ed Similar results were obtained by transfecting cloned cDNA encoding the β-adrenergic receptor into receptor-negative cells Analogs Provide Information about Essential Features of Hormone Structure and are Useful as Drugs: Studies with chemically synthesized analogs of epinephrine and other natural hormones have provided additional evidence that saturable cell-surface receptors are physiologically relevant Analogs are of two kinds: Agonists that mimic the function of a hormone by binding to its receptor and cause the normal response Antagonists that bind to the receptor but do not activate hormone-induced effects Therefore antagonist acts as an inhibitor of the natural hormones/agonists by competing for binding sites on the receptor, thereby blocking the physiological response of the hormones Comparison of the molecular structure and activity of various catecholamine agonists and antagonists have been used to define: the parts of the hormone molecule necessary for binding to β-adrenergic receptors as well as the parts necessary for the subsequent induction of a cellular response Table 20.6 Lodish 3rd Ed/Table 20.2 Lodish 4th Ed Such studies indicate that the side chain containing the NH groups determine the affinity of the ligand for receptor while Catechol ring is required for the ligand - induces increase in cAMP level Two types of β-adrenergic receptors have been identified in humans: Cardiac muscle cells possess β1-receptors which promote increased heart rate and contractility by binding catecholamines with the rank order of affinities isoproterenol > norepinephrine > epinephrine β-blockers like practolol are used to slow heart contractions in the treatment of cardiac arrhythmias and anginas These β1-selective antagonists usually have little effect on β1-adrenergic receptors on other cell types The smooth muscle cells lining the bronchial passages possess β2-receptors which mediate relaxation by binding catecholamines with the rank order of affinities isoproterenol >> epinephrine > norepinephrine Agonist selective for β2-receptors like terbutaline, are used in the treatment of asthma because they specifically mediate opening of bronchioles, the small airways in the lungs Studies with Mutant β-adrenergic Receptors Identify Residues that Interact with Catecholamines: Mutant forms of the β-adrenergic receptor generated by site-specific mutagenesis Expressed in culture cells and Checked their ability to bind the agonist isoproterenol Based on such studies, the model has been proposed Fig 15.11 Lodish 6th Ed Turned on to ill effect Nature (1993) 365:603-604 (LH receptor-7A Talk) LH Receptor 7A from Talk Trimeric Signal -Transducing Gs Protein Links β Adrenergic Receptors and Adenylate Cyclase: As explained, β-adrenergic receptors on different types of mammalian cells mediate distinct tissue specific responses, but the initial response following binding of epinephrine is always the same i.e. An elevation in the intracellular level of cAMP Such a rapid response requires balancing a rapid synthesis of the molecules with its rapid breakdown or removal Fig 15.20 Alberts 3rd Ed Increase in cAMP occurs as a result of activation of adenylate cyclase a membrane bound enzyme Fig 15.21 Alberts 3rd Ed Fig 13.14 Lodish 5th Ed Cycling of Gs between Active and Inactive Forms: A current model of how Gs couples receptor activation to adenyl cyclase activation? Fig 15.13 Lodish 6th Ed Important evidence supporting this model has come from studies with a non-hydrolysable analog of GTP called GMPPNP in which a PNH-P replaces the terminal phosphodiester bond in GTP Although this analog can not hydrolyzed, it binds to Gsα like GTP The addition of GMPPNP and an agonist to an erythrocyte membrane preparation results in a much larger and longer-lived activation of adenylate cyclase than occurs with an agonist and GTP Once GDP bound to Gsα is displaced by GMPPNP, it remains permanently bound to Gsα Because the Gsα.GMPPNP complex is as functional as the normal Gsα.GTP complex in activating adenylate cyclase, as a result the enzyme is in a permanently active state Gsα Belongs to GTPase Super Intracellular Switch Proteins: Family of Understanding that how Gsα subunits cycle between the active and inactive forms has come from studies of a related and extremely important intracellular signal transduction protein called Ras Like Gsα, Ras alternates between an active on state with bound GTP and inactive off state with a bound GDP In the on state Ras binds to and activates specific effector proteins that control the growth and differentiation of cells Both Gsα and Ras are members of a family of intracellular switch proteins collectively called as the GTPase super family Other members of this family include the Rabs which regulate fusion of vesicles within the cells Fig 16.43 Lodish 3rd Ed Fig 14.20 Lodish 3rd Ed Ras (~ 170 amino acids) is smaller than Gsα (~ 300 amino acids) and its three dimensional structure is similar to that of the part of Gsα that binds GTP Fig 5.18 Alberts 3rd Ed Fig 5.20 Alberts 3rd Ed - Abundant molecule in bacterial cell, where it serves an elongation factor in protein synthesis, loading each amino-acyl tRNA molecule to ribosome. - tRNA molecule forms a tight complex with GTP-bound form EF-Tu Cycling of Ras protein between the inactive formith bound GDP and the active form with bound GTP Fig. 20.17 Lodish 3rd Ed Based on the similarity in the three dimensional structure of Ras and the Gsα subunit of trimeric G proteins and discovery of the role of GAP in cycling of the Ras protein, a model of Gsα has been proposed Fig. 20.18 Lodish 3rd Ed Fig. 13.8 Lodish 5th Ed Adenylate Cyclase is Stimulated and Inhibited by Different Receptor-Ligand Complexes: Fig 15.21 Lodish 6th Ed/Fig 20.20 Lodish 3rd Ed Some Bacterial Toxins Irreversibly Modify G Proteins: Confirmation of the GTP cycle came from a study of certain bacterial toxins The function of cholera toxin, a peptide produced by the bacteria Vibrio cholerae was elucidated first The classic symptom of cholera is massive diarrhea, caused by water flow from the blood through the epithelial cells into small intestine; death is often due to dehydration The study showed that cholera toxin irreversibly activates adenylate cyclase in the intestinal epithelial cells, causing a high level of cAMP Later studies showed that the toxin irreversibly activates adenylate cyclase in a large number of cell types Like diphtheria toxin, cholera toxin consists of two types of peptide chains One chain is enzyme that penetrates the cell surface membrane and enters the cytosol, where it catalyzes the covalent addition of an ADP-ribosyl group from intracellular NAD+ to α-subunit of the Gs protein This irreversibly modified Gs subunit can activate adenylate cyclase normally but can not hydrolyze bound GTP to GDP Thus GTP remains bound to Gsα and Gs is always in the activation mode: adenylate cyclase is continuously turned on As a result the level of cAMP in the cytosol rises 100-fold or more Fig 19.17 Lodish 2nd Ed In the intestinal epithelial cells, this rise apparently causes certain membrane proteins to permit a massive flow of H2O from the blood into intestinal lumen Other bacterial toxin link ADP-ribose to other Gproteins and have proved invaluable in unraveling the functions of these transducing molecules For example, the pertussis toxin, secreted by the “whooping cough” bacterium: Bordetella pertussis, adds ADP-ribose to the α subunit of Gi In this case, Giα linked ADP-ribose can not inactivate adenylate cyclase Pertussis toxin also adds ADP-ribose to and inactivates the α subunits of several other G proteins Table 19.6 Lodish 2nd Ed Analogous Regions in All Seven-Spanning Receptors Determine G-Protein and Ligand Specificity: Although G protein linked receptors are thought to span the membrane seven times and hence their three dimensional structures are predicted to be similar, their amino acid sequences generally are quite dissimilar Fig 20.10 Lodish 3rd Fig 15.11 Lodish 6th Ed For example, the sequences of the closely related β1and β2-adrenergic receptors are only 50% identical The sequence of the α- and β- adrenergic receptors exhibit even less homology The specific amino acid sequence of each receptor determines which ligand it binds and which Gproteins interacts with Studies with recombinant chimeric receptor proteins, containing part of an α2 receptor and part of a β2receptor localized certain functional domains with specific regions of receptor sequence Fig 15.12 Lodish 6th Ed Degradation of cAMP is also Regulated: The level of cAMP usually controlled by the hormoneinduced activation of adenylate cyclase Another point of regulation is the hydrolysis of cAMP to 5’AMP by cAMP phosphodiesterase This hydrolysis terminates the effect of hormone stimulation Fig 15.20 Alberts 3rd Ed Many cAMP phosphodiesterases are activated by increase in cytosolic Ca2+ which are often induced by neuron or hormone stimulation Role of cAMP in the Regulation of Cellular Metabolism: In earlier part, we saw that hormone stimulation of Gs protein-linked cell-surface receptors leads to an elevation of the level of cAMP Recall that cAMP is the second messenger for many hormones and that the effects of elevated cAMP differ markedly in various types of cells Table 20.5 Lodish 3rd Ed Now, we will discuss how cAMP affects enzymatic activity, thereby regulating cellular metabolism? cAMP and Other Second Messengers Activate Specific Protein Kinases: The diverse effect of cAMP are thought to be mediated through the action of cAMP-dependent protein kinases (cAPKs: also referred to as kinase A or PKA) These kinases catalyze transfer of the terminal phosphate group from ATP to specific serine or threonine of selected proteins Fig 5.14 Alberts 3rd Ed Fig 15.24 Alberts 3rd Ed Fig 5.12 Alberts 3rd Ed Fig 15.23 Lodish 6th Ed A-kinases are found in all animal cells and thought to account for all the effects of cAMP in most of these cells The substrates for A-kinase differ in different cells types, explaining why the effects of cAMP vary depending on the target cell Epinephrine Stimulates Glycogenolysis in Liver and Muscle Cells: As mentioned previously that cAMP-dependent protein kinases induce many effects depending on the particular substrate proteins that they phosphorylate The first cAMP-mediated cellular response to be discovered i.e. release of glucose from glycogen (glycogenolysis) has been studied the most This reaction occurs in muscle and liver cells stimulated by epinephrine/agonist of β-adrenergic receptors Before describing how cAPKs regulate glycogen metabolism? We first review the pathways of glycogen synthesis and degradation Fig 20.24 Lodish 3rd Ed The fate of glucose 1-phosphate resulting from degradation of glycogen differs in liver and muscle cells In muscle cells, glucose 1-phosphate produced from glycogen is converted by phophogluco-mutase to glucose 6-phosphate This is metabolized via the Embden-Meyerhoff glycolytic pathway to generate ATP which is source of energy for contraction In contrast, glucose from glycogen is not major source of ATP in the liver Rather, the liver stores and releases glucose primarily for use by other tissues Unlike muscle cells, liver cells contain a glucose 6phosphatase, which hydrolyzes glucose 6-phophate to glucose The free glucose is immediately released into the blood and transported to other tissues, particularly the muscles and brain cAMP-Dependent Protein Kinase the Enzymes of Glycogen Metabolism: Regulates In liver and muscle cells, the epinephrine-stimulated elevation in the cAMP level enhances the conversation of glycogen to glucose 1-phophate in two ways: by inhibiting glycogen synthesis by stimulating glycogen degradation Fig 20.25 Lodish 3rd Ed Fig 20.26 Lodish 3rd Ed Fig 15.25 Alberts 3rd Ed Fig 15.26 Alberts 3rd Ed Kinase Cascade Permits Multi - Enzyme Regulation and Amplifies Hormone Signal: The set of protein phosphorylations and dephophorylations just described constitute a cascade, a series of reactions in which the protein catalyzing one step is activated or inhibited by the product of a previous step Although such a cascade may seem over-complicated, it has at least two advantages for the cell: First, a cascade allows an entire group of enzyme catalyzed reaction to be regulated by a single type of molecule As we have seen, the three enzymes in the glycogenolysis cascade: • cAMP-dependent protein kinase • Glycogen phosphorylase kinase and • Glycogen phosphorylase are regulated directly or indirectly by cAMP • • Second, a cascade provides a huge amplification of an initially small signal. For example: Blood levels of epinephrine as low as 10-10 M can stimulate glycogenolysis and release of glucose resulting in an increases of blood glucose levels by as much as 50% An epinephrine stimulus of this magnitude generates an intra-cellular cAMP concentration of 10-6 M, an amplification of 104 M • Because three more catalytic steps precede the release of glucose, another 104 M can occur Fig 20.27 Lodish 3rd Ed Fig 15.42 Alberts 3rd Ed • In striated muscle, the concentrations of the three successive enzymes in the glycolytic cascade – PKA, glycogen phosphorylase kinase and glycogen phosphorylase - are in a 1:10:240 ratio, which dramatically illustrates the amplification of the effects of epinephrine and cAMP Growth Hormone Cascade: The synthesis of many hormone is regulated by a cascade of hormones Fig 24.20 Zubay Some G-Protein-Linked Receptors Activate the Inositol Phospholipid Signaling Pathway by Activating Phospholipase C-β Another crucial enzyme Fig 19.4 Lodish 2nd Ed A role of phospholipids in signal transduction was first suggested in 1953 When it was found that some extracellular signaling molecules stimulate the incorporation of radioactive phosphate into phosphotidylinositol (PI), a minor phospholipid in cell membrane Later it was shown that this incorporation results from the breakdown and subsequent synthesis of inositol phospholipids The inositol phospholipids found to be most important in signal transduction were two phosphorylated derivatives of PI: PI phosphate (PIP) PI biphosphate (PIP2) These are thought to be located mainly in the inner half of the plasma membrane lipid bilayer Fig 15.29 Alberts 3rd Ed Although PIP2 is less plentiful in animal cell membranes than PI, it is the hydrolysis of PIP that matters most Fig 15.30 Alberts 3rd Ed The chain events leading to PIP2 breakdown begins with the binding of a signaling molecule of a Gprotein-linked receptor in the plasma membrane Fig 15.33 Alberts 3rd Ed More than 25 different cell-receptors have been shown to utilize this transduction pathway Table 20.7 Lodish 3rd Ed We will first discuss how a rise in cytosolic Ca2+ ions induces various metabolic responses and Then consider how many hormones acting through IP3 cause this rise in the cytosolic Ca2+ level We also examine the role of DAG in regulating other cellular functions as all these 2nd messengers interact in complex circuits to regulate crucial aspects of the grow growth and metabolism of cells Ionophores - An interesting group of low molecular weight compounds (up to several thousand daltons) compounds synthesized by bacteria facilitates the translocation of inorganic ions across membranes of other cells