Transmembrane signalling Flashcards
(35 cards)
Types of signalling and general model
Signalling is to: interact w/ env; convey info in multicellular org; facilitate complex coordination
Autocrine (to oneself), via gap junctions (to direct neighbours), (nearby cells), endocrine (long-distance via bloodstream)
General model includes: specific receptor; transduction mech often w/ amp step; signalling pathway (enzyme/ 2nd messenger); effector protein; termination mechs.
Response to a hormone depends on receptor types exp by cell+ prevalence of downstream signalling pathways. Pathways often converge and interact.
If cells touch, signalling through gap junctions (animals)/ plasmodesmata (plants). If signal lipid-soluble, e.g., steroid hormones+ NO: diffuse into cell, bind specific response elements (usually receptors cytoplasmic) + translocate to nucleus as complex binding target DNA directly.
Specificity achieved by localisation (docking, scaffolds, subcellular compartmentation, local release of volatile signalling molecules), cell-cell variation (receptor/effector exp), combinatorial signalling, crosstalk (-> signal integration)
NO structure and pathway
Systems: nervous, immune, circulatory. Unstable-> local (intracellular/ local by diffusion), short-term effects. ½ life several s, convert->nitrates/nitrites (react O2, H2O). Produced locally (e.g., by vascular endothelium to target underlyings vascular smooth muscle+ maintain vasculature relaxed+ inhibit circulating platelets’ propensity to aggregate) or same cell may synth+ respond to own NO (autocrine signalling)
NOS: Made by NO synthase from L-Arg metabolism using O2 to intro OH onto sidechain-> N-omega-hydroxy arginine-> NO+citrulline. NOS structure+ reaction mech to cyt P450 (use O2, primary seq homology in cofactor binding site regions). Ca2+-CaM-dependent enzyme, so NO synth can amp another signalling event (primary signal-> Ca signal in cell-> NO synth +other things). Ctrls local blood vessel dilation.
Pathway: Guanylyl cyclase (GC) that reacts w/ NO is soluble, heterodimeric+ combines w/ haem group. NO/haem binding restores plana structure pulling coordinating His105 toward haem ring, activating the enzyme to produce cGMP, which activates protein kinase G. rapid cGMP-> GMP degradation by phosphodiesterases balance cGMP production.
Drugs targetting the NO pathway
Drugs targeting NO pathway: anaphylactic shock-> blood pressure drop reversible by NO synth inhibition, blocking guanylyl cyclase activation/cGMP synth/ blood vessel dilation. Arg structural analogues like L-NAME block NOS, but NOS inhibitors an also cause fall in cardiac output+ increased mortality. Vasodilators (nitroglycerine, nitroprusside) release NO free radicals activating GC -> vascular smooth muscle relaxation, vasodilation. Riociguat stimulates sGCs w/out NO, treats pulmonary hypertension. Viagra/Sildenafil maintains cGMP, blocking cGMP PDE- initially to assist vasodilation+ lower blood pressure. 1 end of Viagra molecule- homology w/ guanine. Viagra binds substrate-cleft/PDE 5 directly+ blocks cyclic AMP PDEs (much lower affinity)- enhances existing stimuli from nervous system (prolongs lifetime), no effect in absence of stimuli, so little benefit dilating coronary arteries/ tonically lowering blood pressure. This shows NO is not an important factor in systematic maintenance of vascular tone.
Small hydrophobic molecules
Diffuse directly, don’t need receptor, but insoluble in H2O, bind specific carrier proteins for bloodstream transport. Persist in blood for hrs-days, mediate long-term responses. Include:
* Steroid hormones: cortisol (adrenal glands, stress response), oestradiol+ testosterone (sexual dev+ f(x)), thyroid hormone (from Tyr in thyroid gland-> dev+ metabolism)
* Thyroid hormones: thyroxine-> dev+ metabolism
* Retinoids: retinoic acid from vitamin A, local mediator/ vertebrate development
Receptors= members of nuclear receptor fam- TFs w/ ligand binding, DNA binding+ transcription activation domains. Some steroid receptors inactive in ligand absence due to other binding partners- glucocorticoid receptor binds Hsp90, glucocorticoid binding displaces it-> bind regulatory DNA. Nuclear receptors can also bind coactivatos-> help upreg exp, e.g., coactivators w/ histone acetyltransferase.
Water soluble hormones general points
Water-soluble extracellular signals- require transmembrane receptors
Water soluble hormones include adrenaline+ noradrenaline from Tyr in adrenal gland, act via alpha+ beta-adrenergic receptors (respond differently, often oppositely); peptide hormones (glucagon, insulin, pituitary gland hormones(growth, follicle-stimulating, prolactin)). e.g. neurotransmitters
Eicosanoids
Eicosanoids- lipid-derived (made by ox. Of arachidonic/other polyunsaturated fatty acids), highly active, rapidly metabolised-> mainly local action. Mem lipid hyrdrolysis-> arachidonic acid (phospholipase A2)-> oxidation to PGG2 by cyclo-oxygenase (COX)-> quickly ox to PGH2 (PG+TX precursor) Effects in inflammation, immunity, vascular/renal/GI/reproductive systems. Often assoc. w/ inflam. disease+ cancer. Include:
* Prostaglandins (PG)- opening+ softening cervix/ dilation/blood vessels. Used to induce labour
* Thromboxanes (TX)- platelet aggregation. Prod. By cyclooxygenase pathway (w/ prostaglandins)
* Leukotrienes: contractions/ smooth muscle/ bronchioles. Overprod= major cause/ inflam. in asthma
* Lipoxins- inflammation
Binding Ca2+ to C2 domain promotes phospholipase A translocation cytosol-> membrane, Pi’d only on Ser by MAPKs (ERK1/2+ p38)+ Ca2+ CaMKII/ Mnk1; Ser228= 1st catalytic domain, critical for enz f(x). COX= target/ some anti-inflam. drugs (aspirin- acetylates+ inactivates COX irreversibly; non-steroidal (NSAID ibuprofen, naproxen). Inhibiting synth/PG-> reduce pain, inflam. Paracetamol blocks COX-2, only minor anti-inflam. role.
Growth factors and cytokines
Growth factors:
* Human growth factor (HGH) prod in pituitary, pituitary tumours->xs. Deficiency->growth failure. Artificial HGH in doping agents. HGH anomalies assoc.w/ gigantism+acromegaly
* Nerve growth factor (NGF): neurotrophin fam, reg neuron dev+survival
* Epidermal growth factor (EGF): cell prolif/ epidermis
* Platelet-derived GF/PDGF: proliferation/fibroblasts, contribute to regrowth/damaged tissue
Cytokines: pro-inflam peptides/ proteins/ glycoproteins secreted by many cells of immune sys+ others (glial cell of nervous system)- important in host immune responses. xs-> cytokine storms can-> multisystem organ failure+ death, can be due to infections- H1N1 flu, SARS-COV-2. More common in ppl w/ healthy immune sys
Receptors- general points
Receptor proteins can be enzyme coupled (RTKs), G-protein coupled, ion channels. Often 2+ receptors for 1 ligand+ 2+ intracellular signalling systems per receptor.
Switch proteins- on/off, activate by Pi by protein kinases (protein phosphatases undo). G proteins active bound to GTP, inactive on GTP->GDP hydrolysis. 3 aas w/ pi-able OH groups= ser, thr, tyr. Kinase specificities: ser/thr dual specificity, 99% cellular protein Pi. Tyr kinases. Dual Tyr/Thr kinases unusual, v. specific targets.
Receptor tyrosine kinases: general points
Largest fam/ cell surface receptors, directly linked to intracellular enzymes, Pi substrate on Tyr. N-terminal extracellular ligand-binding domain, single transmem alpha helix, cytosolic C-term domain w/ protein- Tyr kinase activity. Stimulation in 2 steps: receptor dimerization+ (trans)autoPi-> increase protein kinase activity+create binding sites for other proteins downstream in signalling pathway (bind Pi-Tyr). PiTyr act as template to bind other signalling molecules via SH2. Single receptor may dock 2+ SH2-containing proteins-> form signalling complex- molecules co-localise, interact, modulate one another.
Stimulation mech demo: inslulin receptor exists as dimer, subunits linked by disulfide bonds-> use to produce chimeric receptor w/ insulin rec external domains+ EGF rec transmem/ intracel domains- only transmit signal when insulin bound- show dimerization insufficient for signalling (ligand binding-> conf change-> autoPi)
Often involved in growth, repair, survival, dysregulation assoc. w/ cancer. Can become aberrantly active by over exp, activating mutants, oncogenic fusions (permanent fusion/ intra+ extra cellular domains)
RTK examples, modular interaction domains and downstream signalling
E.g.s incl EGF receptor-> cell survival, growth, prolif, differentiation, inductive signal in dev
IGF1- increase growth+ survival. Nerve growth factor action via RTK-> growth of some neurons
Platelet-driven growth factor- survival, growth, prolif, migration
Modular interaction domains: many domains involved in protein: protein interactions found on signalling proteins, e.g., Pleckstrin homology (PH) domain binds certain Pi lipids+ interacts phosphatidylinositol, SH2 binds PiTyr, SH3 bind polyproline. Part of adaptor protein or enzyme. Many signalling pathways require large protein complexes held together by scaffold +/adaptor proteins, containing specialised domains (docking sites)
Downstream signalling v directed+ diverse- branched signal transduction path arises v early in. Downstream signalling via adaptor+ Ras-activating proteins-> activation/ small G protein Ras->activate MAP kinase cascade
Mitogen-driven MAP kinase cascades, scaffold proteins
Mitogen-activated protein kinase/MAPK cascades: MAPKs= fam/ ser/thr kinase proteins w/ 3aa activation motif Thr-x-Tyr, w/ Thr+Tyr Pi by dual-specificity kinase MAPKK, Pi’d by Ser/Thr kinase MAPKKK- Pi cascade amplifies signal. MAPKAPS+ TFs downstream activated by Pi. Overall, RTK->nucleus-> TF Pi/activation.
Scaffold proteins bring together 2+ proteins, contribute to MAPK signalling specificity maintenance
Insulin signalling overview
Insulin signalling: Ligand binds between 2 subunits/ insulin rec (dimer of alphabeta monomers) activating Tyr kinase activity, each beta subunit trans-auto-Pi 3 CTD Tyrs on other beta subunit, opening enzyme active site; Insulin receptor Pi’s IRS-1 on Tyr, IRS acts as scaffold for downstream protein cluster; SH2 of Grb2 binds P-tyr of IRS-1, SOS GAP binds Grb2. SOS activates Ras, activatesRaf-1, activates MEK by Pi Ser, activates ERK (pi Thr+Tyr), erk enters nucleus, activates TFs by Pi- TFs for insulin response can lower gluconeogenesis, glucogenolysis, lipolysis, ketogenesis+ proteolysis; increase glucose uptake, glycolysis, glycogen synth, protein synth, ion uptake (esp K+, PO4,3-)
Indirect protein-Tyr kinases, other enzyme-linked receptors and the JAK-STAT pathway
Indirect protein-Tyr kinases (e.g., cytokine kinases)- similar to RTKs but cytosolic domains have no catalytic activity. Ligand binding-> dimerization+ cross-Pi of associated non-receptor protein Tyr kinases.
Other enzyme-linked receptors incl Ser/Thr kinase receptors (like TGF beta)- binding-> dimerization. Single transmem alpha-helic+ cytosolic domain w/ catalytic activity. In plants, ethylene receptors regulate growth+ dev, enzyme-coupled; empty receptor activates protein kinase that shuts off ethylene-responsive genes; ethylene binds-> receptor kinase inactive, ethylene-responsive genes exp (signalling to remove transcriptional inhibition common in plants)
JAK- STAT pathway (RTK-like): alpha-interferon binds its receptor-> dimerization, trans-auto-Pi bound JAK (Janus kinases after two-faced Greek god) proteins, which Pi STAT proteins bound to receptor via Pi Tyr on receptor via SH2 when JAK activated, then when active translocates to nucleus, acts as TF. JAKs+ STATs specific to one another.
Protein phosphatases, PTPases and PPases
Protein phosphatases reverse protein Pi. ~500 in mammals. 3 groups w/ unrelated catalytic sites: non-specific (acid+ alkaline phosphatases, hydrolyse phosphomonoester bonds in both protein+ non-proteinaceous substrates), PiSer/Thr specific (PPases), PiTyr specific (PTPases)
PTPases (less specific than kinases, same PTPase can de-Pi several related kinases) can be transmembrane receptors (CD45) or intracellular PTPases, activated by Tyr Pi. 4 families, each w/ 1+ phosphatase domain w/ 11-residue signature seq called CX5R motif containing catalytically essential Cys+Arg. During hydrolysis, phosphoryl group transferred Tyr on substrate-> Cys on enzyme-> covalent Cys-Pi intermediate (then hydrolysed). 107 PTPases in humans, similar to #/Tyr Kinases)
PPases: 2 families: PPP+PPM. E.g., PP2A- reg metabolism, DNA rep, transcription+ dev; heterotrimer of scaffold (A- has 11 HEAT repeats in horseshoe shaped arr), reg (B has 8 HEAT like reps), catalytic (C) subunits
Pathogenic bacteria and receptor interactions. Toll-like receptors, Y pestis, Shigella and ELISA tests
Pathogenic bacteria target signalling pathways to block immune response: many gram -ves use type 3 secretion system (T3SS_ to deliver virulence proteins (effectors) across euk mem-> cytoplasm (needle punches pore in, injects bacterial proteins). Effectors switch off immune signalling.
Toll-like receptors (pattern recognition receptors (PRRs) for pathogen-assoc molecular patterns (PAMP) e.g., LPS+ flagellin) activate MAPK pathways-> Pi TFs-> exp pro-inflam cytpkines.
Y pestis (bubonic plague)- effector YopH= phosphatase homologous to euk PTPases, v active, dampens immune response. Overall effect: fever, vomit, acral necrosis, buboes, etc.
Shigella (dysentery)- effector OspF inhibits MAPK (shown by reduces gene exp, block in Erk Pi). Mass spec shows inhibition by OspF cleaving C-OP bond of a Thr (PiThr lyase activity, not found in euk)-> inhibition irreversible as kinase can’t Pi its substrate. Overall effect-> epithelium of colon inflam+ death. OspF removes Pi, along w/ OH (permanent) from Erk+JNK, can follow this mass reduction w/ mass spec.
ELISA can be used to measure amount of cytokine (alt test= fluor/luciferase assay). Reporter assays show that OspF inhibits MAPK (Erk not Pi’d)
G-protein coupled receptors overview (hint- talk about PAR group, heterodimeric G proteins)
GCPRs/serpentine/7TM~ 3.5% human genome. Over ½ therapeutic drugs target these (adrenergic beta blockers, e.g.) Medium affinity, respond @ [signal]=~ 10-7-10-4M. Resting+ active discrete conformations. Binding pocket often quite deep in cluster/ transmem helices forming receptor but can be formed by external loops. Ligand bind-> conf change to active-> allow corresponding heterotrimeric G protein to bind in cell. E.g., PAR group (protease activated recs)- PAR1= thrombin receptor+ Ser prtease-> blood clotting+ platelet activation. Cloning receptor shows that PAR-1 has thrombin-cleavage site close to N-term, functional studies show that thrombin clips off short peptide-> reveal new N-term w/ seq Ser-Phe-Leu-Leu-Arg- this binds receptor (tethered ligand) to activate. Synthetic peptides w/ this seq (thrombin receptor activator peptide/TRAP) stimulate receptor w/out cleavage.
Ligand binds-> transmission/ conf change to intracellular domains. Mutational studies-> ID clusters/ aas close to inner surface of mem crucial to signal transduction. Produce chimeric receptors where 5th+6th helices+ connecting 3rd loop substituted-> redirect receptor to different G protein.
Heterotrimeric G proteins assoc plasma mem, alpha (up to 45kDa- Ras-like G+ helical domains), beta (36- helical domain+ beta propeller) + gamma (7- 2 helical segments) subunits. Alpha+ gamma lapidated, hence attached to mem. Beta gamma difficult to separate, operate as 1 f(x) unit. Binding to active receptor-> GDP to GTP on alpha-> alpha dissociates. Beta/gamma and GTD/alpha can both bind downstream effector proteins.
G-protein families and adrenergic receptors
G protein families: individual mammal G proteins v conserved (often 98%+), highest homology levels in GTP-binding site. ~20alpha, 7beta, 12 gamma subunits known. 3 fams of alpha: Gs-, Gi-, Gq- like- distinct gene products, though alt splice variants/Gs in many cells. Alpha typically 39-41 kDa, Gs= largest (44-45 kDa)+ insert near N-term of either 13/14aa-> larger form
Adrenergic receptors/ diff categories (alpha1/2, beta) couple diff G proteins- alpha1 (phospholipase C)-Gq, alpha2 (adenylate cyclase)-Gi, beta-Gs (adenylate cyclase). Signalling-> increase heart rate+ blood pressure+ glycolysis+ gluconeogenesis, decrease blood flow to peripheral organs+ insulin release. Gs (stimulates adenylate cyclase system) discovered 1st, found necessary for hormonal stimulation/ adenylate cyclase (ATP-> cAMP). The S49 lymphoma cell line cyc- lacks Gs, doesn’t make cAMP- addition of Gs rescued mutation
Adenylate cyclases and cAMP
Adenylate cyclases: fam of 9 proteins (mammals) that have M1+M2 domains, each w/ 6 alpha-helical mem spans, separated by cytoplasmic loop C1 of ~400aa. C1+ cytoplasmic C-term C2= catalytic+ reg sites. Diff ACs in diff tissues, e.g AC8 in brain, adrenal, lung; AC9 in brain, skeletal muscle; AC1 in brain, adrenal.
cAMP: most effects in mammals due to increased cAMP-dependent protein kinase (PKA) activity. PKA= tetramer, 2 reg+ 2 catalytic subunits, dissociates on binding 2 cAMP to each reg site. A-kinase anchor proteins (AKAPs) can bind both reg subunits+ also cytoskeleton components/ organelle mem, tethering enzyme complex to a cellular compartment
Second messengers
Second messengers: Sutherland- Nobel 1971 for cAMP ID as 2nd messenger. Later, cGMP, Ca2+ etc. ID’d-> heteromeric G proteins seen as key signal transducers+ role of phosphoinositides (minor mem lipid fam) in reg intracellular [Ca2+] ID’d. Changes in 2nd messenger levels-> physiological effects of 1st messenger.
Generally 2nd messenger constrained in target cell by being polar, H2O-soluble w/ no means of crossing mem or being v lipophilic so can’t diffuse away from mem where they’re made. This is to prevent nearby cells from unregulated activation by 2nd messengers in neighbouring cells
Phosphodiesterases, type II cAMP PDE
Phosphodiesterases: cAMP breakdown. Several forms specific for cA/GMP/both. Reg by mech incl Pi and Ca2+/calmodulin. Best known-
type II cAMP PDE, activated by cGMP (binds+ lowers Km for cAMP, enhancing catalytic activity)- eg of crosstalk, allows cAMP-dependent processes to be modulated by ligands which activate cGMP synth. E.g., adrenocorticotrophic hormone (pituitary) stimulates aldosterone production by adrenocortical cells via cAMP-dependent pathway in adrenal gland-> increase blood volume. To limit increase in blood pressure, atrial natriuretic factor synth in atria-> activate catalytic receptor form of guanylyl cyclase-> cGMP synth->cGMP binds, activates PDE II-> stop PKA activation+ aldosterone synth
Phospholipase C, PI3K, signalling proteins with PH domains
Phospholipase C- activated by G-proteins-> prod inositol triphosphate (IP3)+ diacylglycerol (DAG) 2nd messengers. IP3 releases Ca2+ from ER, DAG activates PKC (recruits it to plasma mem)
Phosphatidylinositol 3-Kinase/ PI3K- Pi’s 3 position OH of inositol ring/ phosphatidylinositol (exists as dimer- p85 reg+ p110 catalytic subunits- both modular w/ specific sites to interact other signalling molecules). Modular structure allows PI3K to serve as adaptor+ have catalytic role. Activated by GCPRs (e.g., thrombin rec in platelets) or intracellular Tyr kinases (interact SH2/3 domains of p85) or Ras (interacts p110 directly)
Signalling proteins w/ PH domains/other phosphoinositide-binding domains recruited to various mems by interaction w/phosphoinositides produced by PI3Ks (PI3P, PI(3,4)P2, PtdIns(3,5)P2, PtdIns(3,4,5)P3). Relatively low abundance/ phosphoinositides compared to PI+ other me lipids makes them useful signalling molecules.
Effectors downstream of PI3K (with a table), subunits and signalling
Effectors downstream of PI3K incl small GTP-binding proteon Rac, some PKC isoforms, P70 ribosomal S6 kinase. Effects related to cytoskeletal f(x), incl cell motility, vesicle traffic, chemotaxis; also DNA synth+ cell survival.
Subunits+ signalling: 1st thought alpha exclusively bound to effector enzyme+ reg its activity. Now clear betagamma has own signalling roles. Purify/exp single subunits-> diff permutations tested+ signal pathways reconstituted in membrane vesicles so diversity of reg clarified.
Yeast mating signalling
Yeast mating- a and alpha types secrete pheromone binding GPCR of other type. alpha-factor binds Ste2-> release of betagamma/activates G protein; betagamma subunit recruitsMAPK adaptor Ste5+ small G protein Cdc42 (activates MAPKKK). MAPK cascadw-> activate pheromone response elements-> change gene exp-> morphological changes for mating.
Desensitisation of GPCRs
Desensitisation of 7TM receptors by 2 mech:
* Heterologous desensitisation: Pi Ser in CTD (due to cAMP-dependent PKA activity)-> block binding Gs, allows Gi interaction-> signal termination. Can be due to any stimulus activating adenylate cyclase. Pi/ beta-AR turns off activatory pathway+ turns on inhibitory.
* Homologous desensitisation: beta-adrenergic receptor kinase (betaARK) Pi’s group/ Ser/Thr in CTD- cryptic in restic receptor, accessible when activated. Pi allows receptor to recruit inhibitory molecule beta-arrestin- block signal transmission through beta-AR only. betaARK also assists internalisation+ down-reg of receptor
