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1 Cell Communication (GPCRs) (Chapter 16, part 1) General principles of cell signalling GPCRs and second messengers

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PowerPoint PresentationGeneral principles of cell signalling GPCRs and second messengers
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Signal transduction
• Cell signalling requires a signal molecule (from a signalling cell) and a receptor protein (on a target cell).
• The receptor recognizes the signal (= signal reception).
• The cell responds specifically to the signal molecule (= signal transduction).
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Hormones: Steroid hormones
Histamine
Autocrine signalling
• Signal molecule binds back to a receptor on the same cell type. • This receptor is called an autoreceptor. It is sensitive to the signalling molecule
released by the cell in whose membrane the autoreceptor sits. (feedback loop, threshold sensor).
• Autocrine  signalling  can  coordinate  groups  of  identical  cells  (“community effect”),   e.g. cell fate decisions during development.
• Amplified autocrine cell signalling by factors stimulating cell survival or proliferation is one of the hallmarks of cancer.
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”lateral  inhibition”
The same signal molecule (acetylcholine) can induce different responses in different target cells
• In heart muscle and sailvary glands, acetylcholine binds to similar receptors (muscarinic AchR, a GPCR), but these activate different responses (decreased contraction, increased secretion)
• In skeletal muscle, a different acetylcholine receptor (nicotinic AchR, an ion channel) enables these cells to react differently (increased contraction)
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Signal molecules bind either to cell-surface receptors or to intracellular proteins
Some hormones (hydrophobic signal molecules) can cross the plasma membrane and bind to intracellular receptors
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The steroid hormone cortisol acts by activating a gene regulatory protein
(a intracellular/nuclear receptor protein)
Nitric oxide (NO) triggers smooth muscle relaxation
• NO acts as local mediator in many tissues • Endothelial cells release NO (made from arginine by NOS) • NO activates soluble guanylyl cyclase in muscle cells • Increased cGMP induces muscle cell relaxation
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Extracellular signals alter the activity of a variety of cell proteins to change the behavior of the cell
Signalling cascade
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Many intracellular signalling proteins act as molecular switches
Protein phosphorylation can only occur at serine (S), threonine (T) or tyrosine (Y) residues
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trimeric G-protein GPCR
GPCRs are excellent drug targets
• In humans, about 40% of all medical drugs act on GPCRs.
• GPCRs play a central role in human physiology.
• GPCRs are feasible targets for the development of non-peptide agonists and antagonists
• In humans, there are about 800 genes coding for GPCRs (about 400 of them are olfactory receptors).
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2012 nobel prize in Chemistry for GPCR research
• R. Lefkowitz cloned the first GPCR gene in 1986 (the β2-adrenergic receptor), worked on signal transduction and regulation of adrenergic receptors, his lab discovered GPCR kinases (GRKs) and E-arrestins
• B. Kobilka worked on GPCR structure, his lab solved the 3D- structures of the β2-adrenergic receptor in inactive, active and G protein-bound states
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Gether U. (2000) Endocrine Reviews 21(1): 90–113
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Gether U. (2000) Endocrine Reviews 21(1): 90–113
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The D-subunit switches itself off by hydrolyzing its bound GTP
•The activated D subunit can activate a target protein
•The activated EJ complex can activate another target protein
•After a few seconds the intrinsic GTPase activity of the D subunit hydrolyzes GTP to GDP
•This inactivates the D subunit and the trimeric complex is reformed, thereby also inactivating the EJcomplex
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heart contracts less frequently
Signalling of the activated EJ complex: an acetylcholine receptor in heart muscle
cells opens a K+ channel in the PM
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Most G proteins activate the synthesis of intracellular messenger molecules
“second  messengers”
1. cAMP pathway: cAMP 2. Inositol phospholipid pathway: IP3 and DAG, Ca2+
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cAMP activates the A-kinase (cAMP- dependent protein kinase, PKA)
A-kinase phosphorylates serines or threonines on certain intracellular proteins, thus altering their activity.
cAMP is synthesized by adenylyl cyclase and degraded by cyclic AMP phosphodiesterase
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The cAMP concentration rises rapidly in response to an extracellular signal
Blue: low cAMP concentration
Red: high cAMP concentration
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PKA: cAMP- dependent protein kinase
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Second messengers:
• IP3 -> Ca2+
• DAG
The activated PKC (protein kinase C, Ca2+-dependent) can further phosphorylate many different intracellular proteins
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• In the human olfactory epithelium there are about 10 million olfactory sensory neurons
• Humans have about 350 different olfactory receptors
Smell depends on GPCRs that regulate ion channels
Olfactory GPCRs in cell membrane
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Olfactory transduction in the cilia of olfactory sensory neurons
• The odorant binds a specific odorant receptor (OR) • The activated OR activates Golf • Golf activates adenylyl cyclase to increased cAMP • cAMP opens cAMP-gated Na+-channels (olfactory CNG
channels) • influx of Na+ depolarizes the olfactory receptor neuron
and initiates an action potential
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olfactory sensory neurons
• cGMP-gated channels are located in the outer segment of vertebrate photoreceptor cells
• CNG channels are tetramers The different CNG channel subunits adopt similar topologies with 6 transmembranal segments (S1-S6) and a pore forming region between S5 and S6.
• The amino (N) terminus and the carboxy (C) terminus, containing the cNMP- binding site, are located intracellularly.
• When cyclic nucleotides bind, conforma- tional rearrangements lead to the opening of the pore.
• The channels are permeable for Na+ (-> de-polarization), K+ and Ca2+ ions.
cAMP- or cGMP-binding site
1/2 of the prize 1/2 of the prize
USA USA
Columbia University New York, NY, USA; Howard Hughes Medical Institute
Fred Hutchinson Cancer Research Center Seattle, WA, USA; Howard Hughes Medical Institute
b. 1946 b. 1947
The Nobel Prize in Physiology or Medicine 2004 "for their discoveries of odorant receptors and the organization of the olfactory system"
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(yellow) and the protein opsin
Extracellular signal: light
opsin (a GPCR)
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Vision depends on photoreceptors in the eye • light activates the GPCR
rhodopsin
• transducin activates a phosphodiesterase (PDE) to hydrolyze cGMP
• cGMP normally keeps cGMP-gated Na+- channels open.
• the drop in cGMP closes more of the cGMP-gated Na+-channels
• the photoreceptor cell hyperpolarizes
• less glutamate is released to the bipolar cell in light
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CNG channels in phototransduction
• In darkness, open CNG channels in the cone outer segment conduct a depolarizing current carried by Na+ and Ca2+ ions.
• Upon illumination, CNG channels close and the cone hyperpolarizes.
low cGMP level (active PDE)
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Ways how a cell can become desensitized to a signal molecule (adaptation)
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• Arrestin binds to the phosphorylated receptor
• This prevents the receptor from binding to its G protein
• It can also direct its endocytose (arrestin as adaptor protein)
• Arrestin can also redirect the receptor complex to other signalling pathways or act as scaffolding protein
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downregulation
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2. Usually low expression levels
3. Conformational instability
1. A source that contains the GPCR in high amounts.
2. Replacement of the intracellular loop 3 (ICL3) with a well-folded, polar protein (T4 lysozyme)
3. Binding of a monoclonal antibody (Fab5) to stabilize the ICL3
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>50 insect genomes have been sequenced many  more  are  in  the  pipeline  (“i5k”)  
Our aim: - to advance our understanding of insects - to contribute to crop protection - to fight diseases transmitted by insects
Our focus: - insect endocrinomics - GPCRs and neuropeptides - ligand/receptor matching (deorphanization)
reproduction
development
feeding
behaviour
30%
70%
Center for Functional and Comparative Insect Genomics (Biol. Inst., University of Copenhagen)
similar to HTS (high throuput screening) in drug discovery
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Our bioassay using G16 in CHO cells: Ca2+- induced bioluminescence
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0.4
0.6
0.8
1.0
Lu m
in es
ce nc
Control cells without proctolin receptor
Cells expressing the proctolin receptor
proctolin
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Cloning and matching of (new) receptors and (new) ligands in various insects
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Functional in vivo studies by gene knock-down
Identification of new functions by localization of neuropeptides and receptors
Systematic comparison of the hormonal set-up of insects: endocrinomics
Bachelor or master projects availabe:
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