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Cell signalling   2

Cell signalling 2



communication and signalling and transduction by Khuram aziz

communication and signalling and transduction by Khuram aziz



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  • D3 is somewhat mysterious – some references say it drops cAMP. Need to find out more about that.

Cell signalling   2 Cell signalling 2 Presentation Transcript

  • Cell signalling
    • By:
    • Khuram Aziz
    • M.phill biochemiatry
  • Previous discussion
    • What is cell signaling?
    • Signal transduction
    • Receptors
    • Types
    • Functions
    • Steps for signaling
  • Today
    • What is g protein coupled receptor
    • Regulation
    • What is g protein
    • Regulatioon
    • Mode of action
  • Signal Reception: G Protein-Coupled Receptors
    • G-protein-Coupled Receptors may dimerize or form oligomeric complexes within the membrane.
    • Ligand binding may promote oligomerization, which may in turn affect activity of the receptor.
    • Various GPCR-interacting proteins ( GIPs ) modulate receptor function. Effects of GIPs may include:
      • altered ligand affinity
      • receptor dimerization or oligomerization
      • control of receptor localization , including transfer to or removal from the plasma membrane
      • promoting close association with other signal proteins
  • Neurotransmitter receptors
    • Ligand – gated channels:
      • Nicotinic acetylcholine receptor
      • NMDA-type glutamate receptor
      • Glycine receptor
      • GABA A receptor
      • Serotonin receptor (5-HT 3 )
    • G protein-coupled receptors:
      • Muscarinic acetylcholine receptor (several types)
      • Catecholamine receptors
      • Histamine receptors (H 1 , H 2 )
      • 5-HT receptors other than 5-HT 3
      • GABA B receptors
      • ‘ Metabotropic’ glutamate receptors
      • Peptide receptors (Endorphin, cholecystokinin..)
  • The G Protein-Coupled Receptor (GPCR) Superfamily
    • Largest known receptor family –
    • Constitutes > 1% of the human genome.
    • Comprises receptors for a diverse array of molecules: neurotransmitters, odorants, lipids, neuropeptides, large glycoprotein hormones.
    • Odorant receptor family alone contains hundreds of genes.
    • Mammalian GPCRs: nearly 300 different kinds – grouped into 3 main subfamilies:
    • Each GPCR family contains some orphan receptors, which have been identified as members of the GPCR superfamily by homology cloning but whose activating ligand is unknown.
    • But high throughput screening has recently added to the advances in being able to identify the ligand.
    • GPCRs Interact guanine nucleotide-binding proteins (aka G-proteins)
    • Largest family of membrane proteins in the human genome
    • Eukaryotic trans membrane receptors
    • Seven helices spanning the membrane 
    • Roles:
    •                 - Light and smell processing                 - Behavior and mood                 - Immune response                 - Autonomic nervous system transmission                 - Blood pressure                 - Heart rate                  - Digestive processes                 - CRITICAL FACTOR IN MANY DISEASES!
    • Five different classes (based on sequence and function):
    •                 - Class A: Rhodopsin-like receptors                 - Class B: Secretin receptor family                 - Class C: Metabotropic glutamate/pheromone                 - Class D: Fungal pheromone receptors                 - Class E: Cyclyic AMP receptors
  • Almost all Receptors Comprise a Number of Subtypes
    • Dopamine receptors - 5 subtypes
    • 5-HT receptors – 13 subtypes
    • mGlu receptors - 8 subtypes
    • Acetylcholine receptors – 5 subtypes
    • Identified by their pharmacological and functional characteristics, rather than by strict sequence homology:
    • - Some receptors for the same ligand show remarkably little homology ( e.g ., histamine H3 and H4 have the lowest recorded homology (~ 20 %) to other histamine receptors H1 and H2).
  • Regulation of G protein-coupled receptor function
    • Desensitization/resensitization – a decrease in responsiveness during continuous drug application or a right-shift in a drug dose-response curve.
    • After removal of the drug, receptor activity recovers, although the speed and extent of this resensitization can depend on the duration of agonist activation.
    • Rapid desensitization (sec-min) results from receptor phos, arrestin binding, and receptor internalization.
    • Long-term desensitization (down-regulation) involve changes in receptor and/or G protein levels, and their mRNA stability and expression.
    • Long-term changes in [GPCR]s and [accessory proteins]s known to be induced by chronic drug treatment and involved in several pathologies.
    • Phosphorylation
    • 2 nd messenger kinase
    • G protein receptor kinase (GRK)
    • Arrestin
    • β -arrestin binding to phosphorylated GPCR is required to decrease GTPase activity prior to desensitization.
    • Receptor trafficking, internalization, and recycling (covered earlier; see Protein trafficking and LGIC slides).
    • Mechanisms of long-term down regulation
    • Long-term (> 1 hr) treatment with agonist induces the loss of total cellular receptor number in addition to the decr in surface receptor number.
    • e.g ., antidepressants ( e.g ., fluoxetine) incr [5HT] synapse  decr 5HT receptor density.
    • Receptor endocytosis: C-terminal domain determines whether they enter the recycle pathway or the lysosomal pathway:
    • - 2 distinct motifs:
    • 1. PDZ-domain interats with NHERF in a phos-dependent manner.
    • 2. A short sequence that interacts with NSF ( N -ethylmaleimide sensitive factor).
    • Arrestin has also been shown to be important for recycling:
    • e.g., V2 vasopressin receptor, which continues to bind arrestin while in endosomes, does not recycle back to plasma membrane.
  • D D D D α α β α γ
    • Agonist binding
    • and G protein
    • activation
    (2) Phosphorylation P P (3) Arrestin binding Arrestin P P Arrestin P P Clathrin
    • Clustering in
    • clathrin-coated
    • pits
    (5) Endocytosis Endosomes Arrestin P P D (7) Recycling
    • Dissociation of agonist:
    • Dephosphorylation
    • Sorting between cycling
    • and lysosomal pathways
    (8 ) Traffic to lysosomes Lysosomes Mechanisms of Receptor Regulation
  • Another Receptor – G Protein Cycle
  • Structure, function and mechanisms of G-Proteins
  • What are G-proteins?
    • G proteins bind GTP: guanosine triphosphate. Control and amplify intracellular signaling pathways
    • Exist in two states 1) bound GTP: active
    • 2) bound GDP: inactive
    Fig. 15.1 Examples of GTPase proteins Ras, Cdc-42 (hormone, GF, drug)
  • 1994 Nobel Prize in Medicine, Alfred Gilman and Martin Rodbell, for their „discovery of G-Proteins and the role of these proteins in signal transduction in cells.“
  • G-Protein = Guanine-nucleotide binding protein (GNBD) Guanine Ribose Phosphates 1 2 5 4 3 α   1 3 4 2 6 5 7 8 9 Guanosine Ester Anhydride Guanosine-triphosphate - GTP
  • G-Protein families
    • Heterotrimeric G-Proteins (Transducin, G  i , G  q …), in 7-TM receptor signalling
    • Initiation, elongation, termination factors in protein synthesis (IF1, EF-Tu, EF-TS)
    • Signal recognition particle (SRP) and its receptor , translocation of nascent polypeptide chains in the ER
    • Ras-like GTPases (Ras, Rap, Rho, Ran, Rab, Arf, Arl, Sar), molecular switches in signal transduction
    • Dynamin superfamily of GTPases , remodelling of membranes
    • + 60 further distinct families
    • Leipe et al., JMB (2002)
  • GTPases and disease.
    • Damage to these small GTPase switches can have catastrophic consequences for the cell and the organism.
    • Several small GTPases of the Rac/Rho subfamily are direct targets for clostridial cytotoxins.
    • Further, Ras proteins are mutated to a constitutively-active (GTP-bound) form in approximately 20% of human cancers.
  • G-proteins are tightly regulated
    • 3 types of accessory proteins that modulate cycling of G-proteins between GTP/GDP
    • 1. GAPs : GTPase-activating proteins. Stimulate GTP hydrolysis. Inactivate G-protein. Example of a GAP: PLC 
    • 2. GEFs : Guanine nucleotide-exchange factors: G-protein-coupled receptors (GPCR). Stimulate dissociation of GDP (inactive) from G-protein so GTP can bind (active).
    • 3. GDIs : Guanine nucleotide-dissociation inhibitors. Inhibit release of bound GDP (maintain G-protein in inactive state).
  • The heterotrimeric G proteins transmit signals from a variety of cell surface receptors to enzymes and channels
    • Stimulated by receptors
    • Act on effectors
    • Regulated by nucleotide
    • exchange and hydrolysis
    • The signal is usually passed from a 7-helix receptor to an intracellular G-protein .
      • Seven-helix receptors are thus called GPCR , or G - P rotein- C oupled R eceptors.
      • Approx. 800 different GPCRs are encoded in the human genome.
    • G-proteins are heterotrimeric , with 3 subunits  ,  ,  .
    • A G-protein that activates cyclic-AMP formation within a cell is called a stimulatory G-protein , designated G s with alpha subunit G s  .
    • G s is activated, e.g., by receptors for the hormones epinephrine and glucagon .
    • The  -adrenergic receptor is the GPCR for epinephrine.
    • These domains include residues adjacent to the terminal phosphate of GTP and/or the Mg ++ associated with the two terminal phosphates.
    Structure of G proteins: The nucleotide binding site in G  consists of loops that extend out from the edge of a 6-stranded  -sheet .   Three switch domains have been identified, that change position when GTP substitutes for GDP on G  .
    • GTP hydrolysis occurs by nucleophilic attack of a water molecule on the terminal phosphate of GTP.
    • Switch domain II of G  includes a conserved glutamine residue that helps to position the attacking water molecule adjacent to GTP at the active site.
    • The  subunit of the heterotrimeric G Protein has a  -propeller structure, formed from multiple repeats of a sequence called the WD-repeat .
    • The  -propeller provides a stable structural support for residues that bind G  .
    • It is a common structural motif for protein domains involved in protein-protein interaction.
    • The family of heterotrimeric G proteins includes also:
      • transducin , involved in sensing of light in the retina.
      • G-proteins involved in odorant sensing in olfactory neurons.
    • There is a larger family of small GTP-binding switch proteins , related to G  .
    • Small GTP-binding proteins include (roles indicated):
      • initiation & elongation factors (protein synthesis).
      • Ras (growth factor signal cascades).
      • Rab (vesicle targeting and fusion).
      • ARF (forming vesicle coatomer coats).
      • Ran (transport of proteins into & out of the nucleus).
      • Rho (regulation of actin cytoskeleton )
    • A ll GTP-binding proteins differ in conformation depending on whether GDP or GTP is present at their nucleotide binding site.
    • Generally, GTP binding induces the active state.
    • A GAP may provide an essential active site residue, while promoting the correct positioning of the glutamine residue of the switch II domain.
    • Frequently a (+) charged arginine residue of a GAP inserts into the active site and helps to stabilize the transition state by interacting with (  ) charged O atoms of the terminal phosphate of GTP during hydrolysis.
    Most GTP-binding proteins depend on helper proteins : GAPs , G TPase A ctivating P roteins, promote GTP hydrolysis.
    • G  of a heterotrimeric G protein has innate capability for GTP hydrolysis.
    • It has the essential arginine residue normally provided by a GAP for small GTP-binding proteins.
    • However, RGS proteins , which are negative regulators of G protein signaling, stimulate GTP hydrolysis by G  .
      • An activated receptor (GPCR) normally serves as GEF for a heterotrimeric G-protein.
      • Alternatively, AGS (Activator of G-protein Signaling) proteins may activate some heterotrimeric G-proteins, independent of a receptor.
      • Some AGS proteins have GEF activity.
    GEFs , G uanine N ucleotide E xchange Factors, promote GDP/GTP exchange.
  •  &  subunits have covalently attached lipid anchors that bind a G-protein to the plasma membrane cytosolic surface. Adenylate Cyclase (AC) is a transmembrane protein, with cytosolic domains forming the catalytic site. The   subunit of a G-protein ( G  ) binds GTP , & can hydrolyze it to GDP + P i .
    • The sequence of events by which a hormone activates cAMP signaling:
    • 1. Initially G  has bound GDP , and     &   subunits are complexed together.
    • G  ,  , the complex of  &  subunits, inhibits G  .
    • 2. Hormone binding , usually to an extracellular domain of a 7-helix receptor (GPCR), causes a conformational change in the receptor that is transmitted to a G-protein on the cytosolic side of the membrane.
    • The nucleotide-binding site on G  becomes more accessible to the cytosol, where [GTP] > [GDP].
    • G  releases GDP & binds GTP ( GDP-GTP exchange ).
    • 3. Substitution of GTP for GDP causes another conformational change in G  .
    • G  -GTP dissociates from the inhibitory  complex & can now bind to and activate Adenylate Cyclase.
  • Fig 15.3 The G Protein Cycle
  • G Protein-Linked Receptors
  • G Protein-Linked Receptors
  • G Protein-Linked Receptors
  • G Protein-Linked Receptors note how activation is reversible
  • G Protein-Linked Receptors the more ligand binding, the more K + in cytoplasm
    • Regulation at the G protein level
    • Regulator of G protein signaling (RGS = GAPs = GTPase activating proteins) family of proteins (> 20 members) regulate the rate of GTP hydrolysis in the G α subunit.
    • Can also attenuate G protein actions that are mediated by βγ subunits, because they can alter the number of βγ available by enhancing the affinity of G α subunits for the βγ after GTP hydrolysis  incr rate of reformation of the heterotimer.
    • Regulation at the G protein level (cont’d)
    • RGS proteins also important in regulating the temporal characteristics of G protein actions.
    • E.g ., RGS proteins accelerate the decay of agonist-induced activation of GIRK (G protein regulated inward rectifying K channels).
    • E.g ., RGS proteins accelerate desensitization of adrenergic receptor-induced N-type Ca 2+ channel currents.
    • ADH - Promotes water retention by the  kidneys  (V2 Cells of Posterior Pituitary)
    • GHRH - Stimulates the synthesis and release of GH (Somatotroph Cells of Anterior Pituitary)
    • GHIH - Inhibits the synthesis and release of GH (Somatotroph Cells of Anterior Pituitary)
    • CRH - Stimulates the synthesis and release of ACTH (Anterior Pituitary)
    • ACTH - Stimulates the synthesis and release of Cortisol (zona fasiculata of adrenal cortex in kidneys)
    • TSH - Stimulates the synthesis and release of a majority of T4 (Thyroid Gland)
    • LH - Stimulates follicular maturation and ovulation in women; Stimulates testosterone production and spermatogenesis in men
    • FSH - Stimulates follicular development in women; Stimulates spermatogenesis in men
    • PTH - Increases blood calcium levels (PTH1 Receptor: Kidneys and Bone; PTH2 Receptor: Central Nervous system, Bones, Kidneys, Brain)
    • Calcitonin - Decreases blood calcium levels (Calcitonin Receptor: Intestines, Bones, Kidneys, Brain)
    • Glucagon - Stimulates glycogen breakdown (liver)
    • hCG - Promotes cellular differentiation; Potentially involved in  apoptosis
  • How G-protein-coupled receptors work (1) extracellular space cytosol    heterotrimeric G-protein ‘ 7TM’ - receptor GDP GDP GTP Ligand    N
  • How G-protein-coupled receptors work (2) inactive    GDP P active N  GTP   N
  • How G-protein-coupled receptors work (3) ATP inactive inactive active cAMP cAMP Protein kinase A Phosphorylation of multiple target proteins active Adenylate cyclase    GTP
  • Some G-proteins are inhibitory  -Adrenoceptor  2 -Adrenoceptor AC active AC inactive  s GTP  i GTP
  •  -Subunits of G proteins may have regulatory activity, too Muscarinic (M 2 ) acetylcholine receptor K ir AC inactive K +    i GTP
  • G  -proteins regulate diverse effector systems  q phospholipase C  PIP 2 IP 3 + DAG protein kinase C  phosphorylation of multiple proteins Ca ++ ER  t cGMP phosphodiesterase  cGMP   s adenylate cyclase  protein kinase A  cAMP   i1 adenylate cyclase  protein kinase A  cAMP 
  • Many transmitters have multiple GPCR with different downstream signaling mechanisms Norepinephrine,  1 IP 3 + DAG  epinephrine  2 cAMP   1 ,  2 cAMP  Dopamine D 2 - D 4 cAMP  D 1 , D 5 cAMP  Acetylcholine       IP 3 + DAG   2 , M 3 cAMP 
    • Thanks