G protein-Coupled Receptors Flashcards
1
Q
What is the most plentiful membrane receptor?
A
G protein-coupled receptors (GPCRs).
2
Q
How do GPCRs exert their affects?
A
By acting on G-protein, GTPase.
3
Q
What are GPCRs also known as?
A
7TM receptors.
4
Q
What is an orphan receptor?
A
A receptor found that does not have a known matching ligand found.
5
Q
Where are GPCRs expressed?
A
In only specific tissues and cell types.
6
Q
What is the effect of a GPCR?
A
To induce FAST BIOCHEMICAL CHANGE within target cells.
Ex: Noradrenalin inducing muscle cells to break down glycogen to glucose units.
7
Q
What is unique about GPCRs?
A
Very few GPCR mediated signals induces changes in gene expression.
8
Q
Which receptor would I use if I did not want to alter gene expression?
A
GPCR.
9
Q
What are the therapeutic drugs acting through GPCRs?
A
- Antidepressants
- Antihypertensives
- Asthma drugs
- Anti-inflammatories
10
Q
What are the NON-therapeutic drugs acting through GPCRs?
A
- Amphetamines (serotonin)
- Cannabinoids
- Cocaine (dopamine D3)
- Ketamine (dopamine D2 receptor)
- LSD (serotonin receptor)
- Opium alkaloids (natural ligands are peptides)
11
Q
What is the best known GPCR?
A
Rhodopsin, a receptor of light and energy in the retina.
12
Q
What is a second best known GPCR?
A
B-adrenergic receptor: receptor for noradrenalin the fight or flight hormone.
13
Q
What happens when these two well known receptors are activated?
A
Activation causes specific biochemical changes in cytoplasm.
14
Q
What is the composition of a GPCR?
A
Single polypeptides.
15
Q
What are the three classes of GPCR?
A
- Class A (short extracellular domain), rhodopsin like
- Class B (long extracellular domain), secretin like
- Class C (extra long extracellular domain), metabotropic glutamate/pheromone
16
Q
What are the three components or domains of the GPCR?
A
- Extracellular domains (N-terminal portion), loops between 2&3, 4&5, 6&7. Stabilized by S-S bonds that are often glycosylated (carb attached to OH group).
- Transmembrane domain, consisting of 7TM segments.
- Intracellular or cytoplasmic domain (C-terminal domain), loops between 1&2, 3&4, and 5&6. There are cys residues near C-terminus that may be modified with palmitoyl group acting as the anchor due to increasing affinity for membrane.
17
Q
What happens when a ligand binds to an extracellular site?
A
Causes a change in the cytoplasmic domain.
18
Q
What happens when the cytoplasmic domain changes from ligand binding on extracellular site?
A
Allows for interaction with signalling transducing G protein.
19
Q
What must be known in order to understand signalling of GPCRs?
A
The structure of GPCR.
20
Q
What is used to determine the structure of GPCR?
A
X-ray crystallography.
21
Q
What was the GPCR in question when developing crystallography methods?
A
B-adregenic receptor.
22
Q
What are key characteristics of the B-adregenic receptor?
A
FLEXIBLE. Some molecules even become activated when there is no agonist (noradrenalin).
23
Q
What is the problem with this flexibility?
A
Create a mixed population of molecules therefore, difficult to crystallize.
24
Q
What was the most flexible region of the B-adregenic receptor?
A
The intracellular domain including the C-terminus.
To lock all receptors in one conformation would be to create a single population of receptors and the ability to crystallize.
25
What was another issue of trying to crystallize the B-adregenic receptor?
It has hydrophobic surfaces that interact with membrane lipids skewing or giving inaccurate structure.
26
How do we compensate for this?
Attach a Fab fragment from a monoclonal antibody to the flexible loop (5/6) showing not to interfere with receptor response. (Loop is part of receptor).
27
What were the components or modifications of the GPCR?
Complex contained;
1. Inverse agonist (carazolol ring).
2. Membrane lipid and detergent.
3. Extracellular oligosaccaride groups removed.
4. 48 residues of polypeptide cut out, removing most flexible region.
28
What parts were still undefined despite this modification?
1. Extracellular loop (although not many modifications were done to it).
2. Even parts of the intracellular 5/6 C-terminus loop.
TM and 1/2, 3/4 loops were clear.
29
Was the ligand recognizable?
The flat carazolol ring (at the binding site) was recognizable.
30
Where is the binding site?
Near the extracellular surface, where helices 3,6,7 come together to form binding pocket.
31
What were these residues (3,6,7) involved in before?
1. Noradrenalin binding.
| 2. Where the 11-cis-retinal, prosthetic group is located on rhodopsin structure.
32
What was the second X-ray crystallography approach for determining GPCR structure?
1. Also used carazolol ligand to stabilize the inactive conformation.
2. Replaced third intracellular loop with lysozyme enzyme.
33
Why was the lysozyme enzyme used to replace the third intracellular loop?
Will crystallize itself, and would possible direct crystallization of the fusion protein.
34
What else did the second method of XRD also implement?
Lipid mixture containing cholesterol.
35
What were the results of this method?
Structure demonstrated all residues except for N-terminus and C-terminus sections.
36
What was seen?
1. Two cholesterol molecules.
2. Carazolol.
3. Palmitic membrane. anchor of Cys341.
37
What was the receptor that had most residues and loops defined?
B1-adrenergic receptor from turkey.
38
What ligand did they use for B1-adrenergic receptor?
Inverse agonist, cyanopindolol.
39
Which residues from knowing this B1 receptor were found to contribute to the ligand binding site of B2?
Helices 3, 5, 6, and 7 PLUS second extracellular loop.
40
What is the conclusion of the ligand binding pocket?
The ligand pocket must close down or tighten when agonist is present.
41
What happens when ligand pocket closes?
Movement in helix 5, shifting loops of cytoplasmic side.
42
What is the GPCR created from the gene that controls asthma susceptibility?
GPRA.
43
What is unique about GPRA?
Has two alternatively spliced forms (isoforms) when transcribed.
44
What occurs in the mutant GPRA?
B isoform strongly expressed in airway epithelia and bronchial smooth muscle and hardly expressed in normal tissue.
45
What gene, that is associated with a defect in cerebral cortex development, specifies an orphan GPCR?
GPR56.
46
Where are the mutations of this gene?
N-terminal region, which probably prevents the GPR56 proteins from inserting correctly on the membrane.
47
What is the condition called when puberty remains incomplete?
IHH. Inherited hypogonadotropic hypogonadism.
48
Where do individuals of IHH show mutation?
Orphan GPCR, GPR54.
49
What is the ligand for GPR54?
Kisspeptin or metastin.
50
What does kisspeptin and its receptor release?
GnRH by the hypothalamus.
51
How do GPCRs trigger a biochemical response inside the target cell?
Through regulatory GTPases.
52
What did the first GTPase discovered do?
The first GTPases coupled GTP hydrolysis to energy requiring steps of protein synthesis, used GTP.
53
What are regulatory GTPases called?
G proteins.
54
What are the two conformational states of GTPases?
1) Active state, binding GTP.
| 2) Inactive state, binding GDP.
55
What is the E.S complex?
The active state, wanting to be active in order to convert the substrate into product.
56
What is the E.P complex?
The inactive state as the product has already been made. We don't need to make any more product.
57
What remains bound to the active site of regulatory GTPases?
The GDP product (product inhibition-we have product and therefore do not need more), unless something is done to remove it.
58
What factors may stimulate the release of GDP from the active site of GTPase?
GEFs, guanine nucleotide exchange factors.
59
What factors may stimulate the catalytic rate?
GTPase activating proteins, GAPs.
60
Who can bind once GDP is gone?
Other guanine nucleotide exchange factors.
61
What is more common in the cell GTP or GDP?
GTP is 10x more abundant.
62
What is bound to happen when something such as a GEF removes the GDP from the GTPase active site?
The G protein will become activated as the active site will be replaced with abundant GTP.
63
Draw cycle of GTPase GTP and GDP.
See lecture.
64
What is common to the GTPase superfamily?
All proteins of this family contain the GTP-hydrolyzing domain.
65
What are the three classed of G protein?
1. Heterotrimeric G proteins (Signal transducing partners of GPCRs).
2. Small (Ras-type) G proteins (Key component of GF signalling pathway).
3. GTPase of protein initiation and elongation (trafficking in cytoplasm).
66
What are the three subunits of the heterotrimeric G proteins?
1. a subunit: catalytic, containing GTP hydrolyzing G domain, and has membrane anchor.
2. B subunit: regulatory.
3. Gamma subunit: regulatory and membrane anchor.
67
Going even deeper, what are the subfamilies of the Galpha subunit?
These, once again, are the catalytic G proteins, and therefore will include the stimulatory, the inhibitory, and Gq and G12.
68
What does Gs do?
Stimulatory, will be activated by hormones and odour receptors.
Activate adenylyl cyclase to make intracellular cAMP.
Inhibited by cholera toxin.
69
What does Gi do?
Inhibitory, will be activated by somatostatin receptor. Inhibits adenylyl cyclase.
Includes transducers.
Activate K channels and inhibit Ca channels.
Inhibited by pertussis toxic,
70
What does Gq do?
Activated by vasopressin, angiotensin, TSH receptors.
Activates phospholipase C b-isoforms.
Insensitive to pertussis or cholera toxin.
71
G12?
Regulates H/Na exchange and Ca currents.
| Insensitive to pertussis and cholera toxin.
72
What are toxins used for?
Used as a tool for characterizing heterotrimeric G proteins based on catalytic results.
73
What do the enzymes of cholera and pertussis toxins do?
Transfer an ADPribose unit from NAD+ to particular side chains of Ga polypeptides.
74
What are the negative effects of cholera toxin?
Induces the loss of water and salt leading to an electrolyte imbalance due to open membrane channels from permanent active state.
75
Where is the ADPribose attached in cholera toxin?
To an active site in arginine in Ga proteins of subunit Gs.
76
What happens when the ADPribose binds to this arginine active site?
GTP usually binds here, will become permanently bound, therefore, GTPase activity is lost and the G protein stays in the active state.
77
What is pertussis toxin?
Whooping cough.
78
Where is the ADPribose attached?
At a cysteine in Ga proteins of class Gi, which keeps Gi in an inactive state-in turn, adenylyl cyclase remains active.
79
What is the second component of pertussis toxin?
It includes a bacterial adenylyl cyclase in which the two components of the cyclase and ADPribose come together to produce high levels of cytoplasmic cAMP in airway cells, leading to excessive mucous and airway congestion.
80
Draw G-protein mechanism.
Lecture.
81
What is the E1 component affected by the active form of the Ga protein?
Three possibilities of the effector.
1. Adenylyl cyclase which catalyzes ATP+H2O to cAMP and PPi. Activated by Gas, inhibited by Gai.
2. Phospholipase C which catalyzes phosphatidyl-inossitol-P2 to diabcylglycerol and inositol-P3. Activated by Gaq.
3. Cyclic GMP phosphodiesterase which catalyzes cGMP+H2O to 5-GMP. Activated by Gai like transducin.
82
What is adenylyl cyclase?
Membrane embedded protein with:
1. 9 isoforms.
2. 12 transmembrane domains.
3. two catalytic regions that come together to form the active site.
83
How may the different isoforms of adenylyl cyclase be activated?
1. Gas
2. Gbgamma complex
3. Ca-calmodulin complex
4. protein kinase C
84
What does forskolin do?
Activator of all adenylyl cyclase forms.
85
How may the different isoforms of adenylyl cyclase be inhibited?
1. Gai, Ca
| 2. Gbgamma complex
86
What is phospholipase C (PLC) and what are its isoforms?
C: designates the bond hydrolyzed between phosphate and glycerol.
B isoform: Activate by Gai proteins.
Gamma isoform: Activated by receptor protein kinases.
Delta isoform: activated by calcium.
87
What is another target of Ga protein?
Guanylyl cyclase. GTP tp cGMP and PPi.
- membrane bound forms activated by Ga GPCRs
- soluble forms are activated by other cytoplasmic mechanisms.
88
How is the cGMP phosphodiesterase activated?
Activated by active from of Ga protein transducin (which itself is activated by light acting on rhodopsin).
Will drop cytoplasmic cGMP, allowing CNG (cyclic nucleotide gated) cation channels to close and thus hyperpolarizing the cells.
This will in turn affect neurotransmitter release at the synapse leading to transmission of an impulse to optical centre of brain.
89
Which enzymes stimulate GTPase activity of G proteins and return them to their inactive state?
GAPs.
90
How are GPCRs switched off?
Phosphorylation (catalyzed by protein kinases) on Ser or The of intracellular domain receptor.
But will only occur if the ligand is bound.
91
What are the enzymes that phosphorylate and in tun inactivate the GPCR?
1. Protein kinase A (cAMP dependent). GPCR activate camp release, thus high levels of camp will allow a protein kinase A to turn off GPCR.
2. GRK (not cAMP dependent), must be at the membrane to do the job.
92
What happens when receptor is phosphorylated?
1. Moved from membrane to cytoplasm (translocation), dephosphorylated and reinstalled in the membrane in active state (RECYCLED).
2. Arrestin proteins may bind to phosphorylated cytoplasmic domain and block further interaction with G-protein. (This is useful for rhodopsin).
93
What happens when GRK or arrestins are knocked out in mice?
1. Essential developmental process is affected and mouse dies as an embryo.
2. Just b-2-aresstin, supersensitive opioid receptor and unusually sensitive to morphine as a painkiller.
