![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.](https://image.slidesharecdn.com/cellsignalling-2-120126010652-phpapp01/85/Cell-signalling-2-14-320.jpg)
![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.](https://image.slidesharecdn.com/cellsignalling-2-120126010652-phpapp01/85/Cell-signalling-2-16-320.jpg)
![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 ).](https://image.slidesharecdn.com/cellsignalling-2-120126010652-phpapp01/85/Cell-signalling-2-40-320.jpg)
Cell signalling 2
- 1. Cell signalling By: Khuram Aziz M.phill biochemiatry
- 2. Previous discussion What is cell signaling? Signal transduction Receptors Types Functions Steps for signaling
- 3. Today What is g protein coupled receptor Regulation What is g protein Regulatioon Mode of action
- 4. Signal Reception: G Protein-Coupled Receptors
- 5. 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
- 6. 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..)
- 7. 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:
- 8. 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.
- 9. 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
- 10. 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!
- 11. 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
- 12. 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).
- 14. 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.
- 15. 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).
- 16. 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.
- 17. 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
- 18. Another Receptor – G Protein Cycle
- 19. Structure, function and mechanisms of G-Proteins
- 20. 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)
- 21. 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.“
- 22. 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
- 23. 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)
- 24. 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.
- 25. 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).
- 26. 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
- 27. 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.
- 28. 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.
- 29. 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 .
- 30. 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.
- 31. 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.
- 32. 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 .
- 33. 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.
- 34. 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.
- 35. 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 .
- 36. 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.
- 37. & 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 .
- 39. 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 .
- 40. 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 ).
- 41. 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.
- 42. Fig 15.3 The G Protein Cycle
- 46. G Protein-Linked Receptors note how activation is reversible
- 47. G Protein-Linked Receptors the more ligand binding, the more K + in cytoplasm
- 48. 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.
- 49. 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.
- 50. 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)
- 51. 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
- 52. 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
- 53. How G-protein-coupled receptors work (1) extracellular space cytosol heterotrimeric G-protein ‘ 7TM’ - receptor GDP GDP GTP Ligand N
- 54. How G-protein-coupled receptors work (2) inactive GDP P active N GTP N
- 55. 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
- 56. Some G-proteins are inhibitory -Adrenoceptor 2 -Adrenoceptor AC active AC inactive s GTP i GTP
- 57. -Subunits of G proteins may have regulatory activity, too Muscarinic (M 2 ) acetylcholine receptor K ir AC inactive K + i GTP
- 58. 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
- 59. 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
- 60. Thanks
Editor's Notes
- #60: D3 is somewhat mysterious – some references say it drops cAMP. Need to find out more about that.