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

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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)

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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)

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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