Post on 18-Jan-2016
1
Receptors
Seminar No. 9
2
Feature Lipophilic hormones Hydrophilic hormones
Chemical type
Water solubility
Transport protein
Plasma half-life
Membrane penetration
Receptor location
2nd messenger example
Q. 1
3
Feature Lipophilic hormones Hydrophilic hormones
Chemical typesteroids, iodothyronines,
calcitriol, retinoids
amino acid derivatives,
polypeptides
Water solubility no yes
Transport protein yes no
Plasma half-life long (hours, days) short (minutes)
Membrane penetration yes no
Receptor location intracellular cell membrane
2nd messenger examplehormone-receptor
complex cAMP, Ca2+ ....
4
Q. 2
5
A. 2
• Allosteric protein (in membrane or cytosol)
• It has two domains:
• ligand-binding domain (with binding site for signal
molecule) – changes conformations of receptor
• effector domain – starts biological response to ligand
(production of second messenger etc.)
6
Q. 3
7
A. 3
Receptor Transporter
Common features:
Protein, Binding selectivity to ligand, Changes in conformation
Different features:
Transfers signal Transfers substance
8
Q. 4
9
A. 4
• signal molecule (ligand) - carries specific information into cell
• has extremely low concentration in blood (10-9 – 10-15 mol/l)
• binds to corresponding receptor
• signal molecule is usually quickly inactivated
• agonist – ligand which after binding to receptor transduces signal
• antagonist – ligand which after binding to receptor blocks signal
transduction no biological response
10
Q. 5
11
Amplification of signal:
1 signal molecule
10 000-100 000 molecules of second messenger
A. 5
The second messenger transfers information to other intracellular systems and then is quickly inactivated
12
feed back
metabolisminactivation
Hormone synthesis
Hormone storage
Hormone secretion
Transport in ECF
Receptor in target cell
Biological response
regulation effects
secretion impuls
excretioncell condition
Factors involved in biological action of hormones
13
Q. 6
14
A. 6 Examples of second messengers
• Hydrophilic – cAMP, IP3
• Lipophilic – diacylglycerol (DAG)
• Inorganic – Ca2+, NO
15
Q. 9
2nd messenger Inactivation
cAMP
IP3
Ca2+
NO
16
A. 9
2nd messenger Inactivation
cAMP
IP3
Ca2+
NO
cAMP + H2O AMP
IP3 + H2O IP2 + Pi
↓↓ [Ca2+ ] in cytosol
oxidation to nitrate ion (NO3-)
17
Three types of membrane receptors
Ion channelsin synapses, activated by neurotransmitters, very quick response
Receptors activating G-proteinsstimulate or inhibit adenylate cyclase /phospholipase C
Receptors with enzyme activity guanylate cyclase - atrial natriuretic factorstyrosine kinase - insulin
Two types of receptors:
membrane and intracellular
18
Q. 11
Acetylcholine formation / inactivation
19
Acetylcholine formation
N
CH3
CH3
CH2CH2OHCH3 N
CH3
CH3
CH2CH2CH3 O C CH3
O
cholinacetylcholincholine acetylcholine
food
or
synthesis
from serine
acetyl-CoA
Inactivation ?
20
Acetylcholine inactivation
Acetylcholine + H2O Choline + acetic acid
acetylcholinesterase
21
GABA formation / inactivation
22
GABA formation
COOHCHCH2
NH2
CH2HOOC
glutamát
CH2CH2
NH2
CH2HOOC
GABA gama-aminobutyric acid
CO2-
glutamate
Inactivation ?
23
GABA inactivation
CH2C CH2 COOHO
HGABA
succinate semialdehyde
succinate
oxid. deamination
- NH3
CAC
oxidation
CH2HOOC CH2 COOH
24
Q. 12 + 13
25
A. 12 + 13
• Excitatory neurotransmiters open cationic channels
depolarization (more positive membrane potential)
• Inhibitory neurotransmiters open anionic channels
hyperpolarization (more negative potential)
26
Nicotinic acetylcholine receptor
• transmembrane protein = channel for Na+ and K+
• heteropentamer (α2βγδ)
• α-subunits have two binding sites for acetylcholine (ACH)
• nicotine (= xenobiotic) is agonist of this receptor
27
Q. 15 – Four events on postsynaptic membrane and corresponding changes of membrane potential
11
44
23
3
2
28
A. 15 Four events on postsynaptic membrane
1. ACH binds to receptor channel opens influx of Na+ and efflux
of K+ membr. potential changes (-60 -40 mV)
2. partial depolarization of membrane opens voltage-dependent Na+-
channel further influx of Na+ depolarization of postsyn.
membrane ( +20 mV)
3. this depolarization opens K+-channel (volt. dep.) efflux of K+
membrane potential returns to normal value (-60 mV) =
repolarization
4. Na+,K+-ATPase gets ion distribution to normal state
(Na+ OUT, K+ IN)
29
GABA receptor
• channel for chloride ion (Cl-)
• has the binding site for GABA channel opens Cl- ions
get into cell hyperpolarization ( -80 mV) decrease
of excitability
• benzodiazepines and barbiturates (synthetic substances)
have similar effects like GABA, they are used as anxiolytics
and/or sedatives
• endozepines – endogenous peptides have opposite effects,
close the channel (are responsible for anxiety feelings)
30
Diazepine Benzo[f]diazepine
12a
bf
N
N
N
N4
diazepine is a seven-membered unsaturated heterocycle
with two nitrogen heteroatoms in the positions 1,4
31
Benzodiazepines
N
NCl
OCH3
N
NO2N
OCH3
F
N
NCl
OCH3
diazepam
anxiolytic / sedative
flunitrazepam
hypnotic
tetrazepam
myorelaxant
structural modifications lead to different pharmacological effects
32
Barbiturates
N
N
H
H
O
O
O
R2
R1
derivates of 2,4,6-trioxoperhydropyrimidine
allobarbital:
R1 = R2 = -CH2-CH=CH2
33
Receptors with adenylate cyclase system
(Scheme on p. 4)
Describe the pathway of
signal
34
Signal molecule binds to receptor
Receptor activates G-protein
Activated G-protein (α-unit with GTP) activates effector = adenylate cyclase
Adenylate cyclase produces the second messenger = cAMP
Four molecules of cAMP bind to two R(regulatory) units of protein kinase A
Two C(catalytic) units of protein kinase A catalyze protein phosphorylation
Phosphorylated protein = biological response to signal molecule
cAMP is inactivated by phosphodiesterase: cAMP + H2O AMP
35
G-Protein linked receptors
• extracellular part of receptor has a binding site for hormone
• intracellular part has a binding site for G-protein
• G-proteins are heterotrimers (αβγ)
• in resting state, α-unit has GDP attached
• after binding hormone (α-GDP)βγ makes complex with
receptor GDP is phosphorylated to GTP
• activated G-trimer dissociates: (α-GTP)βγ α-GTP + βγ
• α-GTP interacts with effector (enzyme) activated/inhibited
enzyme second messenger (↑ or ↓)
36
O
OH
O
OH
AdeninP
O
O
OP
O
O
OP
O
O
OO
OH
Adenin
O
P OO
O
cAMP
ATP
What reaction is catalyzed
by adenylate (adenylyl) cyclase?
What is adenylyl ?
37
Adenylyl cyclase reaction
O
OH
O
OH
AdeninP
O
O
OP
O
O
OP
O
O
OO
OH
Adenin
O
P OO
OcAMP ATP
- diphosphate
cAMP = cyclic 3’,5’-adenosine monophosphate
38
AMP is called also adenylic acid
N
N
N
N
NH2
O
OH
O
OH
P
O
O
O
N
N
N
N
NH2
O
OH
O
OH
P
O
O
adenylate is anion adenylyl is acyl
39
Adenylate (adenylyl) cyclase (AC)
• membrane bound receptor
• catalyzes reaction: ATP cAMP + PPi
• Gs protein stimulates AC conc. of cAMP ↑
• Gi protein inhibits AC conc. of cAMP ↓
40
Q. 19
41
A. 19
• Protein kinase – phosphorylation by ATP
Protein-OH + ATP Protein-O-P + ADP
• Protein phosphatase – hydrolysis of phosphate ester
Protein-O-P + H2O Protein-O-H + Pi
42
General scheme of phosphorylation
OH P
O
O
O O
O
O
P RibO
O
O
P O
Ade
+
ATP (4-)substrát
kinasa
++
fosforylovaný substrát (2-) ADP (3-)
P
O
O
O RibO
O
O
P O
Ade
HO P
O
O
O
protein kinase
substrate (protein)
phosphorylated protein (2-)
Which AA are phosphorylated?
43
Three amino acids have a hydroxyl group in the side chain
• Serine (3 C, primary alcohol hydroxyl)
• Threonine (4 C, secondary alcohol hydroxyl)
• Tyrosine (3 + 6 C, phenolic hydroxyl)
write structural formulas
44
Phosphatidyl inositol system
(Scheme on p. 4)
Describe the pathway of
signal
45
Signal molecule binds to receptor
Receptor activates G-protein
Activated G-protein (α-unit with GTP) activates effector = phospholipase C
Phospholipase C catalyzes the hydrolysis of PIP2 and produces two second messengers: DG + IP3
DG activates protein kinase C
IP3 opens calcium channel in ER Ca2+ concentration in cytoplasm increases and Ca2+ ions are associated with calmodulin (CM)
Ca2+-CM complex activates caldomulin-dependent kinases
Phosphorylated intracellular proteins = biological response to signal molecule
46
The structure of PIP2
OH
HOO
OOH
P
P
C
O
CH
CH2O
O
C
O
CH2 O P
O
O
O
describe the structure
47
Q.
What is the source of inositol in human body?
48
The origin of inositol
HO OH
OH
OHHO
HO
Exogenous source: any plant food (inositol hexaphosphate = phytic acid)
Endogenous source: glucose-6-P (side path of metabolism)
49
Main types of G-proteins
G-protein Effector Intracell. signal Signal change
Gs adenylate cyclase cAMP
Gi adenylate cyclase cAMP
Gq phospholipase C DG + IP3 + Ca2+
50
Q. 20
51
A. 20
Enzyme Activator
Protein kinase A
Protein kinase C
Protein kinase G
cAMP
DAG and Ca2+
cGMP
!
52
Receptors with guanylate cyclase
• second messenger = cGMP
• activates protein kinase G (PKG)
Q. 23
53
A. 23
• By the action of NO• In smooth muscle cells (chapter 14)
54
Insulin receptor
• has four subunits (α2β2)
• extracellular α-units bind insulin
• intracellular β-units have tyrosine kinase activity
phosphorylation of tyrosine phenolic hydroxyl of
intracellular proteins including insulin receptor itself
(autophosphorylation) cascade of further events
biological response
55
Intracellular receptors:
- cytoplasmatic- nuclear
for non-polar signal molecules
steroids, iodothyronines, calcitriol, retinoids
56
Intracellular receptors• make complex with hormone
• activate or repress the transcription of genes
hydrophobic hormone penetrates cell membrane
inactive receptor in cytoplasm in complex withhsp dimer and other proteins
active complex receptor-hormone
active complexes dimerize are translocated into nucleus
57
Q. 30
58
A. 30
• HRE = hormone response elements
• regulatory DNA sequences, bind complexes of hydrophobic
hormones with their intracellular receptors
• They act as enhancers or silencers
• Located at the beginning of regulatory DNA region
• 5’-----HRE ---Promoter-------3’
59
Steroid and thyroid hormones
• insoluble in water in ECF are transported in complex with
transport proteins
• hormone themselves diffuse easily across cell membrane
• they are bound to cytoplasmatic or nuclear receptors
• in nucleus, the hormone-receptor complex binds to HRE
(hormone response element) in regulation sequence of DNA
• this leads to induction of mRNA synthesis = transcription of
gene
60
Cholinergic synapses
• neurotransmitter: acetylcholine
• two types of receptors
• nicotinic rec. (ion channel) – e.g. neuromuscular junction
• muscarinic rec. (G-prot.) – e.g. smooth muscles
61
Cholinergic receptors
Feature Nicotinic receptorMuscarinic receptors
M1, M3 M2
Receptor type
2nd messenger
Antagonist
Locations
Ion channel
∆ψ*
tubocurarine
neuromuscular juct.
Gq
DAG, IP3
atropine
brain
Gi
cAMP
atropine
myocard
* the change of membrane potential
62
Q. 31
63
A. 31
• Presynaptic membrane contains voltage-gated calcium channels
• influx of Ca2+ activates protein kinase which phosphorylates synapsin
and other proteins
• this triggers the fusion of presynaptic vesicles (contaning
acetylcholine) with cell membrane and exocytosis of acetylcholine
• acetylcholine is liberated into synapse
64
Q. 32
65
A. 32
acetylcholine + H2O choline + acetic acid
hydrolysis of ester
66
Q. 33
What is nicotine?
67
Nicotine is the main alkaloid of tobacco (Nicotiana tabacum)
N
N
CH3
3-(1-methylpyrrolidine-2-yl)pyridine
more basic
pKB = 6.2
less basic
pKB = 11
1
2
3
1
2
68
Q. 34
What is muscarine?
69
A. 34 Muscarine is quaternary ammonium alkaloid in some mushrooms
OH3C
HO CH2 N
CH3
CH3
CH3 CH2 N
CH3
CH3
CH3CH2HO
muskarin kandicin
Amanita muscaria (fly agaric)
muscarine
tetrahydro-4-hydroxy-N,N,N,5-
tetramethyl-2-furanmethanammonium
70
Q. 35
71
A. 35
• nicotine binds to acetylcholine nicotinic receptors in the brain and other
tissues including cells of adrenal medulla
• activation of nicotinic receptor change of membrane potential
exocytosis of vesicles with adrenaline secretion of adrenaline
metabolic processes typical for acute stress (see Seminar No. 6)
Other effects of nicotine:
• increases the secretion of saliva and gastric juice
• increase intestinal peristalsis
• vasoconstriction
72
Inhibitors of acetylcholinesterase
• Reversible – carbamates (N-substituted esters of carbamic acid), e.g. fysostigmine, neostigmine
• they are used to improve muscle tone in people with myasthenia gravis and routinely in anesthesia at the end of an operation to reverse the effects of non-depolarising muscle relaxants. It can also be used for urinary retention resulting from general anaesthetia
• Irreversible – organophosphates, very toxic compounds (chemical warfare agents)
73
Carbamates – General formulas
OHCH2N
O
OCH2N
O
R OCN
O
R
R
R
Complete the names of the structures
74
Carbamates – General formulas
OHCH2N
O
OCH2N
O
R OCN
O
R
R
R
carbamic acid
(hypothetic compound)
alkyl carbamate
(ester)
N-disubstituted
alkyl carbamate
75
Organophosphates
P
S
OH
OHHO P
O
OH
FHO P
O
OH
CNHO
P
O
OH
CH3HO P
O
F
CH3HO P
O
F
CH3O
H3C
H3C
thiophosphoric acid fluorophosphoric acid cyanophosphoric acid
kys. methylfosfonová kys. methylfluorofosfonová sarin methylphosphonic acid methylfluorophosphonic acid sarin
76
Q. 36
ENZYME Ser OHP
O
OO
F
CH3
H3C
H3C
CH3
irreversible
phosphorylation
of enzyme
77
P
O
OO
O
CH3
H3C
H3C
CH3Ser ENZYME
- HF
inactivated enzyme
ENZYME Ser OHP
O
OO
F
CH3
H3C
H3C
CH3
78
Adrenergic synapses
• neurotransmitter: noradrenaline
• four types of receptors: α1, α2, β1, β2
• all of them are G-protein linked receptors
• occur in various cells and tissues
79
Adrenergic receptors
Feature α1 α2 β1 β2
G-protein
2nd messenger
Occurence*
Gq
DG, IP3
smooth muscle
Gi
↓ cAMP
brain
Gs
↑ cAMP
myocard
Gs
↑ cAMP
smooth m.
* Example of occurence
80
Q. 37
Describe the synthesis of noradrenaline.
81
The formation of DOPA and dopamine
COOH
CHH2N
CH2
OH
COOH
CHH2N
CH2
OH
OH
hydroxylace
O2, BH4
tyrosin DOPA(3,4-dihydroxyfenylalanin)
dekarboxylace
- CO2
CH2
CH2
OH
OH
NH2
dopamin(katecholamin)
hydroxylation decarboxylation
tyrosine DOPA
3,4-dihydroxyphenylalanine dopamine
82
Noradrenaline and adrenaline
CH2
CH2
OH
OH
NH2
hydroxylace na C-2
O2, askorbát
CH2
CH
OH
OH
NH2
OH
dopamin noradrenalin
N-methylace
SAM
CH2
CH
OH
OH
NH
OH
CH3
adrenalin
Cu2+
prefix nor- means N-demethyl
hydroxylation at C2 N-methylation
dopamine noradrenaline adrenaline
O2, ascorbate