Cell Signaling Mechanisms PDF

Cell signalling – how cells ‘talk’ to each other ! Intercellular communication Intracellular signalling (signal transduction) Division of labour – cellular specialization human body is a community of some 1014 individual cells many different kinds of cells – arran...

Cell signalling – how cells ‘talk’ to each other ! Intercellular communication Intracellular signalling (signal transduction) Division of labour – cellular specialization human body is a community of some 1014 individual cells many different kinds of cells – arranged in organ systems- with different specialist functions need for coordination so that activities of cells correspond to those needed to maintain overall function of organism Evolution of Cell Signaling Communication among bacteria Pathway similarities suggest that ancestral signaling molecules 1 Individual rod- shaped cells evolved in prokaryotes and were 0.5 mm modified later in eukaryotes 2 Aggregation in The concentration of signaling process molecules allows bacteria to detect population density 3 Spore-forming structure (fruiting body) Fruiting bodies Intercellular communication Chemical communication between cells hormones: endocrine glands, released directly into circulation to reach distal targets growth factors, cytokines: released by various kinds of cells, wound healing, immune system, protective action against virus-infection, cultured cells need growth factors – examples interleukin 2, erythropoietin, platelet–derived growth factor (PDGF), epidermal growth factor (EGF), nerve growth factor (NGF) Neurotransmitters: released from nerve endings to signal to next neuron or muscle contraction – examples acetylcholine, adrenaline/noradrenaline, serotonin, glutamate (others in the CNS)  Modes of intercellular communication (A) (B) (C) Importance of receptors All eukaryotic signalling molecules combine with protein receptors Receptors can be membrane-bound molecules on the surface of cells or, intracellular receptors where they bind lipid soluble signals that are able to enter cells A cell without a receptor for a given signal cannot respond to the signal ! The chemistry of the signalling molecule is of no significance – they do not take part in reactions – they just bind to specific receptors Importance of conformational changes in receptors Receptors exhibit allostery – change shape when bound to signalling molecules (allos = other + stereos = shape) For transmembrane receptors, ligand-conformational change in the receptor causes a conformational change in the cytoplasmic portion – the signal is conveyed (transduced) across the membrane A reminder: proteins are flexible molecules Forces maintaining secondary and tertiary structure are ‘weak’ forces and these are constantly broken and reformed (at body temp) Think about the induced-fit model – protein and ligand adjust its structure to the presence of the other (protein ‘snuggles’ around the ligand !) Intracellular signalling is about getting information across the cell membrane Lipophilic (lipid soluble) signals can traverse the membrane and bind to intracellular receptors steroid hormones, prostaglandins, nitric oxide Hydrophilic (water soluble) signals cannot cross the membrane and will bind to transmembrane receptors proteins (insulin), peptide hormones (glucagon, ADH) So, signalling molecules do not enter into chemical reactions but are molecules of the right shape and properties for binding with their receptor with great specificity by noncovalent bonds Outline of receptor-mediated signalling Water-soluble signalling molecules cannot pass through the lipid bilayer. Bind to external domain of transmembrane receptor Lipid-soluble signalling molecules enter the cell directly and bind to intracellular receptors Stages of cell signalling Reception = binding of signal to receptor Transduction = the system of molecular interactions that transmit signal from receptors to target molecules in the cell (intracellular signal transduction) Response = regulation of transcription or cytoplasmic activities as a result of reception and transduction  The Three Stages of Cell Signalling Earl W. Sutherland (1915-1975) discovered how the hormone adrenaline acts on cells Sutherland suggested that cells receiving signals went through three processes: – Reception EXTRACELLULAR CYTOPLASM FLUID Plasma membrane 1 Reception 1 Receptor Signaling molecule  The Three Stages of Cell Signalling Earl W. Sutherland (1915-1975) discovered how the hormone adrenaline acts on cells Sutherland suggested that cells receiving signals went through three processes: – Reception – Transduction EXTRACELLULAR CYTOPLASM FLUID Plasma membrane Reception Transduction 1 2 Receptor Relay molecules in a signal transduction pathway Signaling molecule  The Three Stages of Cell Signalling Earl W. Sutherland (1915-1975) discovered how the hormone adrenaline acts on cells Sutherland suggested that cells receiving signals went through three processes: – Reception – Transduction – Response EXTRACELLULAR CYTOPLASM FLUID Plasma membrane 1 Reception 2 Transduction 3 Response Receptor Activation of cellular response Relay molecules in a signal transduction pathway Signaling molecule Responses mediated by intracellular receptors Lipid soluble, able to cross the cell membrane Egs steroid hormones, thyroxine hormone, vitamin D Lipid soluble hormones bind to cytosolic receptors/nuclear receptors Lipid soluble hormones regulate expression of specific genes in target cells Alter gene transcription Likely to have long term effects Glucocorticoid hormone signalling eg hormones from the adrenal glands – cortisol receptor exists in the cytoplasm in association with heat shock proteins (Hsps) this masks a peptide sequence on receptor protein (nuclear localisation signal , NLS) binding with hormone causes a conformational change in receptor and Hsps dissociate, revealing NLS receptor is able to enter the nucleus activation of appropriate genes Responses mediated by receptors in the cell membrane For water soluble hormones signalling occurs via membrane-bound receptors Many different kinds, but all have an external binding site, a transmembrane domain, and a cytosolic domain Hormone binding to its specific receptor causes the cytosolic domain to undergo a conformational change This conformational change activates an intracellular signalling pathway This will activate specific genes and/or modulate metabolic systems Three main types of membrane-bound receptors Ionotropic Metabotropic (G-protein coupled receptors: GPCRs) Tyrosine kinase-linked receptors Ionotropic receptors Ligand gated ion channels eg nicotinic acetylcholine receptor Mainly involved in ion transport processes and neurotransmission Usually found in large numbers and have varying specificities Different channels respond to different signals (ligand-gated or voltage-gated) Direction of movement of ions is determined by concentration gradients – act fast so good for bringing about rapid changes Ligand-binding site and ion channel is all part of the same protein structure  Signaling Gate molecule Ions Ligand-gated ion channels closed (ligand) Plasma Ligand-gated membrane ion channel receptor Gate open Cellular response Gate closed G-protein-coupled receptors (GPCRs) Constitute a very large superfamily of receptors – in many different kinds of organisms – most numerous receptors in all eukaryotic genomes (1-5% of total number of genes) Transduce a large variety of stimuli, in humans involved in gustation, olfaction, vision, behaviour and mood regulation, immune system, BP and HR regulation, water balance… Approx. 100 ‘orphan’ GPCRs in the human genome for which no ligand yet identified ! Mutations responsible for a wide range of genetic diseases (hereditary forms of blindness) Targets of the majority of therapeutic drugs (morphine, anti-histamines, Ventolin, ‘β-blockers’....) and drugs of abuse Structure of GPCRs GPCRs consist of a single polypeptide that is folded into a globular shape and embedded in the plasma membrane Seven segments of this molecule span the entire width of the membrane — explaining why GPCRs are sometimes called seven- transmembrane receptors (serpentine receptors) The extracellular loops form part of the pockets at which signaling molecules bind to the GPCR Structure of GPCRs On the cytoplasmic side each receptor is associated with a heterotrimeric G-protein Made up of three subunits a, b and g The alpha subunit of the trimeric inactive g-protein is bound to GDP Receptor activation by ligand binding causes GDP to be exchanged by GTP Alpha subunit dissociates from the bg subunits and activates a signal transduction pathway leading to formation of second messengers Cyclic AMP as second messenger Produced from ATP by enzyme adenylate cyclase Adenylate cyclase is an integral cell membrane protein Activated by alpha-GTP complex that detaches from the heterotrimeric G protein and binds to adenylate cyclase to cause its activation The alpha subunit has GTPase activity (hydrolyses GTP to GDP) In the GDP bound form it detaches from adenylate cyclase and rejoins the bg subunits – reforming the inactive GDP-bound G protein cAMP is hydrolysed to AMP by phosphodiesterase to terminate signal Formation of cAMP PKA- a downstream target of cAMP Many signal molecules trigger formation of cAMP cAMP usually activates another enzyme, protein kinase A (PKA), which phosphorylates various other proteins PKA is a serine/threonine kinase Different types of G-protein receptors GTP-a subunit stimulates adenylate cyclase – the subunit is called Gs (s for stimulatory) Another type of GTP-a subunit can inhibit adenylate cyclase (Gi) Therefore hormones can exert different effect on different cells according to their type of receptor So different G proteins associated with receptors can control different signal transduction pathways Diversity of control options ! Phosphatidylinositol cascade Here a different enzyme is activated by GTP bound alpha subunit (eg acetylcholine and ADH) Activation of phospholipase C causes hydrolysis of PI-(4,5)- bisphosphate into inositol trisphosphate (IP3) and diacylglycerol (DAG) IP3 causes mobilisation of intracellular calcium from ER DAG is the physiological activator of PKC (multiple targets, multiple forms) PI cascade- interactions Signal transduction pathways from tyrosine kinase receptors We will consider the receptor for epidermal growth factor (EGF) But the similar principles apply for other growth factors EGF receptor is a monomer with an external binding site for EGF, a transmembrane domain and a cytosolic domain The cytosolic domains have tyrosine kinase activity (ie able to catalyse the transfer of a phosphate group to tyrosine) Events following EGF receptor binding When EGF binds the receptors undergo dimerization in the membrane Kinase domains come close together and they phosphorylate each other (autophosphorylation) The sequence-specific phosphorylation of the cytosolic domains creates binding sites for other intracellular proteins and causes their activation The Ras pathway of signal transduction Summary: EFG signalling EGF binds to receptor → receptor dimerization and becomes autophosphorylated on tyrosine group GRB-SOS binds to phosphorylated receptor (binding is sequence specific) Receptor-bound GRB-SOS activates Ras, and active Ras activates Raf Raf is a protein kinase that sits at the top of a kinase cascade  The control of Ras protein Ras is a monomeric GTP binding protein, uses the GTP/GDP switch mechanism, but does not have a trimeric structure like the G-proteins Ras is inactivated by its intrinsic slow GTPase activity GTPase activating proteins (GAPs) enhance rate of GTP hydrolysis and thus terminate activation of receptor Fine tuning of responses – Amplification and Specificity Enzyme cascades amplify the cell’s response At each step, the number of activated products is much greater than in the preceding step Different kinds of cells have different collections of proteins These different proteins allow cells to detect and respond to different signals Even the same signal can have different effects in cells with different proteins and pathways Pathway branching and “cross-talk” further help the cell coordinate incoming signals  Signaling molecule Receptor Relay molecules Response 1 Response 2 Response 3 Cell A. Pathway leads Cell B. Pathway branches, to a single response. leading to two responses. Activation or inhibition Response 4 Response 5 Cell C. Cross-talk occurs Cell D. Different receptor between two pathways. leads to a different response. Signaling Efficiency: Scaffolding Proteins and Signaling Complexes Scaffolding proteins are large relay proteins to which other relay proteins are attached Scaffolding proteins can increase the signal transduction efficiency by grouping together different proteins involved in the same pathway Signaling Plasma molecule membrane Receptor Three different Scaffolding protein protein kinases