Cell signalling and pharmacology Flashcards

Question

What are protein kinases?

Answer 1

Protein kinases are also switch proteins themselves ie activated/ deactivated by phosphorylation Often organised in sequence in a signal transduction pathway Once activated, can turn phosphorylate and activate the next protein kinase in the sequence

Answer 2

Adenylyl cyclase – cAMP – PKA – multiple specific molecules required for specific response

Answer 3

CAMP is produced from ATP by the enzyme adenylyl cyclase Adenylyl cyclase: consists of 2 transmembrane domains: joined by a catalytic intracellular domain CAMP is degraded from a cyclin nucleotide to a 5’ monophosphate (AMP) by a cAMP phosphodiesterase (very important – controls signalling)

Answer 4

Most responses to cAMP are mediated via cAMP-dependent protein kinase A (ie protein kinase A PKA) Inactive PKA consists of two regulatory (R) subunits and 2 catalytic (c) kinase subunits CAMP binds to the regulatory subunits causing the molecules to dissociate 2 resulting monomeric kinase units are active and can bind phosphorylate target proteins Note : cAMP can also directly bind and activate cAMP-mediated ion channels

Answer 5

Common signalling pathway - PI3-kinase – PIP2 to PIP3 – PDK1 – PKB (Atk) - multiple molecules required for specific response

Answer 6

PIP2 (phosphatidylinositol 4,5 – bisphosphate) Cell membrane phospholipid Found in inner leaflet of lipid bilayer Phosphoinositide comprised of: Hydrophobic diaglycerol (DAG) lipid tail Hydrophilic inositol sugar as head group (inositol triphosphate IP3)

Answer 7

Phospholipase C – PIP2 to DAG and IP3 – Ca2+ - calmodulin – multiple specific molecules required for specific response

Answer 8

Activation of a receptor causes: Activation of phospholipase C (PLC) Cleaves PIP2 into DAG and IP3 DAG activates Protein Kinase C (PKC) (important in growth) IP3 triggers release of Ca2+ (also required for PKC activation)

Answer 9

Variation of Ca2+ concentration in cytosol ie (Ca2+i) constitutes the signal

Answer 10

Influx of Ca2+ from outside cell via Ca2+ channel proteins in the plasma membrane Release of Ca2+ from intracellular stores ie endoplasmic recticulum (ER), sarcoplasmic reticulum (SR) and mitochondria (caused mainly via IP3)

Answer 11

The plasma membrane (pumps out Ca2+) ER, SR and mitochondrial membrane (sequester Ca2+ back into organelle)

Answer 12

Has 4 ca2+ binding sites Activated when (ca2+i) increases above 500nm Ca2_ bound calmodulin binds and activity of its target proteins

Answer 13

Eliminate extracellular signalling molecule Enzymatic degradation Deactivate signal transduction molecules Dephosphorylation by phosphatases Degradation by enzymes

Answer 14

Receptor and signal molecule (Ligand) are internalised: Either the receptor and signalling molecules are separated and the receptor is recycled to surface and ligand destroyed Or The the receptor and ligand are both destroyed

Answer 15

Ensures specificity of response ie lock and key The cell can also influence repsonse by: Regulating the number of receptors Synthesising different isoforms of the receptor Example – growth hormone binds to its receptor through complementary molecular structure

Answer 16

Agonist: A molecule that binds and activates a receptor, inducing signalling and a biological response eg native ligands and drugs Full agonist – full activation Partial agonist – partial activation

Answer 17

Antagonist: A molecule that binds to a receptor, but does not induce signalling and a biological response eg native ligands and drugs

Answer 18

Ion channel linked receptor (ionotropic receptor) G-protein coupled receptor (metabotropic receptors) Enzyme linked receptor Intrinsic enzyme activity Recruit enzyme from cytoplasm

Answer 19

Act as gates Ligand binding causes receptor to change shape and open gate Allows ion flow passively through channel Can be pharmacologically modified by: Channel blockers (physically block channel) Channel modulators (bind to channel and enhance or inhibit opening)

Answer 20

Synaptic vesicles containing neurotransmitter Presynaptic membrane – voltage gated Ca2+ channels Postsynaptic membrane – ligand gated ion channels Note: synaptic transmission end when the neurotransmitter is either taken up by the synaptic terminal or degraded

Answer 21

Largest family of cell surface receptors in biology Bind an enormous range of extracellular signalling molecules 800 GPCRs in human, at least twice as many in other mammals Mediate a wide array of physiological processes (including odorant detection)

Answer 22

All GPCRs have a similar structure: Extracellular ligand binding region Seven alpha helices that span the membrane Intracellular portion interacts with a trimeric G protein

Answer 23

- inactive when bound to GDP - active when bound to GTP - intrinsic GTPase activity * hydrolysis of GTP to GDP switches off protein - exists in 2 forms * within trimeric complex (used by gpcks) * as a single monomeric protein

Answer 24

Trimeric G protein: 3 subunits ie alpha, beta, gamma Galpha unit binds GDP and GTP and has the GTPase activity

Answer 25

Binding of ligand alters the conformation of the receptor Galpha unit binds to the receptor Binding of galpha protein allows release of GDP and its exchange for GTP Alpha subunit is active and dissocitates from the beta and gamma units Both active galpha subunit and betagamma complex can now interact with the effector molecules to relay the signal – focus on galpha subunit Relaying the signal: GTP bound activated Galpha unit binds to an effector molecule altering its activity

Answer 26

Galpha subunit hydrolyses the GTP to GDP – oocurs in seconds – can use RGS protein to aid hydrolysis Galpha dissociates from effector molecule Alpha subunit having returned to its orginal GDP inactive conformation can reassemble with the beta-gamma complex to form iactive trimeric G protein *RGS = regulator of G-protein signalling

Answer 27

Determined by which effector molecule with galpha subunit couples with and the resulting effect There are 23 known classes of Galpha units coupling to various effect molecules, but three most common classes are shown below (which you need to know)

Answer 28

CLASS OF PROTEIN – EFFECTOR MOLECULE – EFFECT ON EFFECTOR MOLECULES GalphaS – adenylyl cyclase – stimulation increase in cAMP GalphaI - adenylyl cyclase – inhibition decrease in cAMP GalphaQ – phospholipuase C – stimulation increase in DAG and IP3

Answer 29

Not shown but REMEMBER: Signalling pathway amplifies original signal PKA can also enter the nucleus and effect transcription factors involved in mediating the longer term coordinated events in this biological response

Answer 30

Cholera toxin binds to GalphaS and fixes it in GTP bound conformation Over stimulation of adenylyl cyclase and cAMP production Downstream signalling effects transporters involved in ion transport leading to water loss Figure shows neutralisation of cholera toxin with nanoparticle decoys for treatment of cholera

Answer 31

LTP – long term potentiation

Answer 32

The most well studied receptors with intrinsic enzyme activity are the receptor tyrosine kinases (RTKs) RTKs consist of: Extracellular domain which binds the ligand (mainly growth factors) Transmembrane domain Intracellular or cytoplasmic domain which contains the tyrosine kinsase site A tyrosine kinase adds phosphate groups from ATP to only tyrosine residues on target proteins Reminder: In animal cells, two classes of protein kinase exists, i.e tyrosine kinases and serine/threonine kinases

Answer 33

Requires dimerization of two receptor monomers Activates the tyrosine kinase in each receptor Kinase phosphorylates tyrosine's on opposite receptor tail i.e transphosphorylation Recruitment/binding of adaptor and/or effector signalling molecules directly to the phosphorylated tyrosine's to initiate signalling

Answer 34

RTKs commonly utilise the monomeric g-protein Ras to relay or transduce the signal: Activated receptor either directly or indirectly (via an adaptor protein) binds and activates the GEF for Ras, thereby activiting this key signalling molecule.

Answer 35

Glucose transporters ie GLUT-4 are stored in walls of cytoplasmic vesicles Insulin induced IRS-1/PI-3 kinase/PKB signalling triggers vesicle translocation to the plasma membrane Vesicles fuse with membrane where they take up glucose and pass it into the cell Note: insulin induced signalling has multiple cellular effects which also include decreasing glycogen metabolism and promoting glycolysis

Answer 36

Cytokine receptors recruit Janus kinase (JAK) to help initiate signal transduction Mechanism of action: Cytokine receptors lack intrinsic kinase activity Recruit soluable tyrosine kinase ie JAK Ligand binding eg prolactin causes: Receptor dimerisation and JAK recruitment and activation JAKs phosphorylate each other and the receptor Recruitment of STAT transcription factor to phosphorylated tyrosine residues on the receptor

Answer 37

Intracellular receptors – also known as nuclear receptors In the absence of ligand, they can be found in the nucleus or the cytoplasm After binding ligand, cytoplasmic receptors will translocate to the nucleus Binds lipid soluble molecules such as steroid hormones or small molecules that can pass through the lipid bilayer Exert effects by affecting gene transcription Ie activated receptor is a ligand activated transcription factor

Answer 38

Nuclear receptors are ligand-activated transcription factors Receptors contain: A ligand binding domain A DNA binding region (binds to ‘reponse’ elements in the promoter region of target genes) N-terminal variable region which can be modified by other molecules to enhance transcriptional abilities

Answer 39

Cortisol is produced in the adrenal glands in response to stress Passes through lipid bilayer and binds to its cytoplasmic nuclear receptor Ligand bound receptor translocates to nucleus Binds to regulatory response elements in target gene to drive gene transcription NOTE: nuclear receptors for some other steroid hormones, thyroid hormones, fatty acids and retinoids are found in the nucleus bound to the response element in gene even in the absence of ligand – bound to transcriptional repressor which is released when ligand binds

Answer 40

Oestradiol (an oestrogen) is synthesised from testosterone by an enzyme called aromatase Oestradiol is hydrophobic and therefore crosses the nuclear membrane Within the nucleus, oestradiol binds to oestrogen receptors (ER) which dimerise, associate with co-activators and form a complex which acts as a transcription factor The transcription factor activates Estrogen Repsonse Elements, altering the transcription of target genes

Answer 41

Plant signalling mechanisms can be relatively rapid through to extremely slow

Answer 42

Similar to animal cells, extracellular communication in plants is designed by the distance travelled by the signalling molecule to reach its target cell Long distance (endocrine) Slow via vascular system ie xylem and phloem Short distance (paracrine) Most common No distance – same cell (autocrine)

Answer 43

Via active transport via transport proteins Passive – freely diffusible Via plasmodesmata

Answer 44

Comprised of cytoplasmic channels linking adjacent cells 30-60nm in diameter (cf gap junctions with 1.5nm diameter) Allows passage of both small molecules and macromolecules Aids electrical signalling between plant cells

Answer 45

Allows relatively rapid long-distance communication eg in response to stress damage Food capture in carnviorous plants

Answer 46

Stimulation of sensory trigger hairs activates mechano-sensitive ion channels Leads to depolarisation of membrane and generation of an action potential Changes turgor pressure in hinge cells causing closure of leaf lobes

Answer 47

Respond to environmental or physiological signals Possess similar transduction components to animals Membrane enzyme linked receptors and intracellular receptors, with and without kinase activity (but negligible existence of GPCRs and G-proteins_ Uses mainly serine/threonine kinases Intracellular signalling molecules eg lipid signalling molecules Ca2+ Signal transduction can illicit rapid responses but mainly relies on altering gene expression (slow response) By positive gene activation Mainly involves inactivation of transcriptional repressor proteins

Answer 48

Natural auxin in plants is indoleacetic acid (IAA) Produced in the seed of embryo, meristems of apical buds and young leaves Functions include stem elongation, root growth, branching, fruit development and apical dominance Actively transported from one cell to the next – long distance and local acting *removal of the dominant apical bud allows lateral buds to grow due to the redistribution of auxin

Answer 49

Mediated by the blue-light photoreceptor, phototropin (see later) Signalling induced by the receptor causes redistribution of auxin to shaded side Induces elongation on the shaded side Shoot bends towards the light

Answer 50

Auxin utilises the mechanism of inactivating repressor proteins to affect gene transcription Auxin binds to its nuclear receptors which are ubiquitin ligases Binding promotes ubiquitinoylation and degradation of repressor protein Suppression of gene transcription in relieved

Answer 51

Functions include fruit ripening and leaf abscission Can pass through cells walls or diffuse through air

Answer 52

Ethylene receptors are found in the membrane of the endoplasmic reticulum and golgi In the absence of ethylene, the ethylene receptor is activating a kinase – promoting the destruction of the transcription regulator Deactivation of the ethylene receptor allows the transcription of ehthylene sensitive genes

Answer 53

Plant photoreceptors and phototropism Plants can detect the direction, intensity and wavelength (colour) of light Light regulates key biological events in plant growth and development Plants contain 2 major classes of photoreceptors

Answer 54

Contains either cryptochromes, phototropin or zeaxanthin as photopigments Cell surface receptor

Answer 55

Phytochromes (red light and far red light) Intracellular receptor

Answer 56

Cryptochromes influence de-etiolation ie development and expansion of leave ie greening after seedling growing in dark/underground Phototropins mediate phototropism by inducing signalling which influences auxin redistribution Phytochromes regulate photoperiodism ie response of plants to the period of day and night Red light (day) Far red light found at extreme end of red spectrum (night)

Answer 57

Exist as 2 subunits and each has - A light detecting pigment or chromophore A region that has kinase activity Exist between 2 states Pr and Pfr depending on proportion of red light and far red light

Answer 58

Translocating to the nucleus Either directly binding to and activating a transcription factor Or indirectly by phosphorylating transcription factors

Answer 59

Apotosis - (Gr. Falling as in leaves) a process seen in multicellular organisms, by which specific cells are killed and removed for the benefit of the organism 60 billion adult human cells per day die via apotosis Apoptosis is essential for animal development Examples include: Removal of redundant structures Embryogenesis (eg sculpting of limbs)

Answer 60

Regulation of cell numbers Too much – degenerative diseases eg Neurodegenerative disorders, ischaemic heart disease or autoimmune diseases Too little – diseases of over-proliferation eg solid tumours, leukaemia

Answer 61

DNA damage When repair mechanisms cannot cope with damage Accumulation of misfolded proteins Causes endoplasmic reticulum stress and cell death Linked with neurodegenerative disorders Cells infected by certain viral agents Limits spread of infection

Answer 62

Ultrastructural changes (note: these changes are irreversible once apoptosis is triggered) Cell shrinkage Chromatin condensation Fragmentation of intracellular contents and membrane blebbing Formation of apoptotic bodies (ABs) Membrane bound portions of cytoplasm and organelles Phagocytic ingestion of Abs and degradation

Answer 63

Apoptotic cells have large membrane bleb and intact membrane and a cell dying of necrosis will have a ruptured membrane

Answer 64

In animal cells, apoptosis is mediated by a family of suicide proteases called caspases (Cysteinyl-aspartate-specific proteases)

Answer 65

Cysteine at active site Cleaves target proteins at specific aspartic acids Synthesised as inactive procaspase Activated by proteolytic cleavage at own aspartic residues

Answer 66

Initiator caspases: Undergo autocleavage Activates other caspases Effector caspases: Activate other effector caspases after cleavage by initiator caspase Cleave cellular proteins

Answer 67

Hallmark cleavage of chromosomal DNA Caspase cleaves a protein that normally blocks endonuclease action: DNA cleavage by endonucleases cuts DNA into internucleosomal units of 180-200 base pairs Apoptotic cells show DNA ‘laddering’ on electrophoresis

Answer 68

Key ‘engulf me’ signal is phosphotidylserine (PS) Phagocytes ie macrophages and neutrophils recognise ‘engulf me’ or ‘cell corpse’ signals on cell surface of apoptotic bodies PS usually found in inner leaflet of plasma membrane In apoptosis, some PS molecules move to outer leaflet Action of caspases activate scramblase (Xrkr8) which mediates PS flipping

Answer 69

Intrinsic pathway (mitochondrial) Lack of trophic factor induced signalling ie growth factor/ survival factor withdrawal NB cells are prevented from dying by ‘trophic’ factors DNA damage (by radiation or toxins) Protein misfolding (ER stress) Whether the intrinsic pathway is activated depends upon the release of cytochrome c from the mitochondria Regulated by a balance between molecules that promote apoptosis and those which inhibit apoptosis

Answer 70

Pro-apoptotic molecules – BAX, BAK, BAD Anti-apoptotic molecules – BCL-2, BCL-XL

Answer 71

BLC-2 inhibits apoptosis by preventing release of cytochrome c from the mitochondria by blocking action of BAX and BAK BAX/BAK promote apoptosis by forming channels in the outer mitochondrial membrane to allow cytochrome c release BCL-2 > BAX: apoptosis prevented BAX > BCL-2 : apoptosis occurs

Answer 72

Increasing the transcription and translation of anti-apoptotic molecules Signal transduction kinases (eg protein kinase B) which are activated by stimulation of trophic receptors, phosphorylate and inactivate pro-apoptotic molecules

Answer 73

Used by cells of the immune system to kill their targets (example cancer cells, pathogen infected cells) Initiated by death ligands on/or secreted by the immune cells, binding to their receptors on the target cell Activated death receptors result in caspase cascade Different initiator caspases used compared with intrinsic route ie procaspase 8 in extrinsic route

Cell signalling and pharmacology Flashcards

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