Types of Receptors, GABA, GLR,EGF-R, Tyrosine Kinase

1. Receptors:
Structure and Function

2. The role of the receptor
• Globular proteins
• Located mostly in the cell membrane
• Receive messages from chemical messengers coming from
other cells (CNS)
• Transmit a message into the cell leading to a cellular effect
• Different receptors specific for different chemical
messengers
• Each cell has a range of receptors in the cell membrane
making it responsive to different chemical messengers

3. The Role of the receptor
Cell
Nerve
Messenger
Signal
Receptor
Nerve
Nucleus
Cell
Response

4. The Role of the receptor
 Neurotransmitters: Chemicals released from nerve
endings which travel across a nerve synapse to bind
with receptors on target cells, such as muscle cells or
another nerve. Usually short lived and responsible for
messages between individual cells
 Hormones: Chemicals released from cells or glands
and which travel some distance to bind with receptors
on target cells throughout the body
 Note: Chemical messengers ‘switch on’ receptors
without undergoing a reaction

5. The role of the receptor
•Receptors contain a binding site (hollow or cleft on the receptor surface)
that is recognised by the chemical messenger
•Binding of the messenger involves intermolecular bonds
•Binding results in an induced fit of the receptor protein
•Change in receptor shape results in a ‘domino’ effect
•Domino effect is known as signal transduction, leading to a chemical
signal being received inside the cell
•Chemical messenger does not enter the cell. It departs the receptor
unchanged and is not permanently bound

6. The Binding Site
Cell
Membrane
Cell
Receptor
Messenger
message
Induced fit
Cell
Receptor
Messenger
Message
Cell
Messenger
Receptor

7. The Binding Site
• A hydrophobic hollow or cleft on the receptor surface
- equivalent to the active site of an enzyme
• Accepts and binds a chemical messenger
• Contains amino acids which bind the messenger
• No reaction or catalysis takes place
ENZYME
Binding site

8. The Binding Site
• Binding site is nearly the correct
shape for the messenger
• Binding alters the shape of the
receptor (induced fit)
• Altered receptor shape leads to
further effects - signal transduction
Messenge
r
Induced fit
M

9. How does the Binding Site Change Shape?
 Before –
 Intermolecular bonds not optimum length for
maximum binding strength
 After –
 Intermolecular bond lengths optimised
Phe
Ser
O
H
Asp
CO2 Induced
Fit
Phe
Ser
O
H
Asp
CO2

10. Induced Fit
• Binding interactions must be strong enough to hold the
messenger sufficiently long for signal transduction to take
place
• Interactions must be weak enough to allow the
messenger to depart
• Implies a fine balance
• Designing molecules with stronger binding interactions
results in drugs that block the binding site - antagonists
M
M
ER R
M
ER
Signal transduction

11. Main Types of Receptors
• ION CHANNEL RECEPTORS
• G-PROTEIN-COUPLED RECEPTORS
• KINASE-LINKED RECEPTORS
• INTRACELLULAR RECEPTORS

12. Ion Channel Receptors
• Receptor protein is part of an ion channel protein complex
• Receptor binds a messenger leading to an induced fit
• Ion channel is opened or closed
• Ion channels are specific for specific ions (Na+, Ca2+, Cl-, K+)
• Ions flow across cell membrane down concentration gradient
• Polarises or depolarises nerve membranes
• Activates or deactivates enzyme catalysed reactions within cell

13. Ion Channel Receptors
Hydrophilic
tunnel
Cell
membrane
Induced fit
and opening
of ion channel
ION
CHANNEL
(open)
Cell
Cell
membrane
MESSENGER
Ion
channel
Ion
channel
Cell
membrane
RECEPTOR
BINDING
SITE
Cell
Lock
Gate
Ion
channel
Ion
channel
Cell
membrane
Cell
membrane
MESSENGER

14. Ion Channel Receptors
Transmembrane Proteins
TM2 of each protein subunit ‘lines’ the central pore
Protein
subunits
TM4
TM4TM4
TM3
TM3
TM3
TM3
TM3 TM2
TM2
TM2TM2
TM2
TM1
TM1
TM1
TM1
TM1
TM4 TM4

15. Gating
Five glycoprotein subunits
traversing cell membrane
MessengerReceptor
Induced
fit
‘Gating’
(ion channel
opens)
Binding site
Cell
membrane
Cell
membrane

16. gating
• Chemical messenger binds to receptor binding
site
• Induced fit results in further conformational
changes
• TM2 segments rotate to open central pore
Closed
Transverse view
TM2
TM2
TM2
TM2
TM2
Cell
membrane
TM2 TM2
Open
Transverse view
TM2
TM2
TM2
TM2
TM2

17. Gating
• Fast response measured in msec
• Ideal for transmission between nerves
• Binding of messenger leads directly to ion flows
across cell membrane
• Ion flow = secondary effect (signal transduction)
• Ion concentration within cell alters
• Leads to variation in cell chemistry

18. • Receptor binds a messenger leading to an induced fit
• Opens a binding site for a signal protein (G-protein)
• G-protein binds, is destabilised then split
messenger
G-protein
split
G-PROTEIN-COUPLED RECEPTORS
induced
fit
closed open

19. • G-protein subunit activates membrane bound enzyme
• Binds to allosteric binding site
• Induced fit results in opening of active site
• Intracellular reaction catalysed
Intracellular
reaction
active site
(closed)
active site
(open)
Enzyme Enzyme
G-PROTEIN-COUPLED RECEPTORS

20. Transmembrane
helix
C -Terminal chain
G-Protein
binding region
Variable
intracellular loop
Extracellular
loops
Intracellular loops
N -Terminal chain
HO2C
NH2
VII VI V IV III II IMembrane
G-PROTEIN-COUPLED RECEPTORS

21. LIGAND BINDING SITE -
varies depending on receptor type
A) Monoamines: pocket in TM helices
B) Peptide hormones: top of TM helices + extracellular loops
+ N-terminal chain
C) Hormones: extracellular loops + N-terminal chain
D) Glutamate: N-terminal chain
Ligand
B DCA

22. The alpha subunit of the G-protein is activated by...
separating from the gamma and beta subunits.
the G-protein changing conformation.
binding to the calcium ions.
replacing the GDP with GTP.
replacing the GTP with GDP.
The activated alpha subunit then binds to...
the calcium ion channel.
the calcium ions.
the gamma and beta subunits.
GDP
GTP

23. As a result...
the calcium channel opens and calcium ions leave the cell.
the calcium channel opens and calcium ions enter the cell.
the calcium channel closes.
the calcium ions bind to calmodulin.
the calcium ions bind to the ligand receptor site.
The G-protein changes conformation when the GTP replaces the GDP on the alpha subunit.
True
False
The combination of the calcium and the calmodulin produces the response of the cell to the ligand.
True
False

24. Bacteriorhodopsin & Rhodopsin Family
• Rhodopsin = visual receptor
• Many common receptors belong to this same family
• Implications for drug selectivity depending on similarity (evolution)
• Membrane bound receptors difficult to crystallise
• X-Ray structure of bacteriorhodopsin solved - bacterial protein similar
to rhodopsin
• Bacteriorhodopsin structure used as ‘template’ for other receptors
• Construct model receptors based on template and amino acid
sequence
• Leads to model binding sites for drug design
• Crystal structures for rhodopsin and b2-adrenergic receptors now
solved - better templates

25. Bacteriorhodopsin & Rhodopsin Family
Common ancestor
Endothelins
Opsins, Rhodopsins
Tachykinins
Monoamines
alpha beta
H2 1
muscarinic
H12 4 15 3 2A 2B 2C D1A D1B D5D4 D3 D2 3 2 1
Bradykinin,
Angiotensin.
Interleukin-8
Muscarinic Histamine -Adrenergic Dopaminergic b-Adrenergic
Receptor
types
Receptor
sub-types

26. • Receptor types and subtypes not equally distributed amongst tissues.
• Target selectivity leads to tissue selectivity
Heart muscle b1 adrenergic receptors
Fat cells b3 adrenergic receptors
Bronchial muscle 1& b2 adrenergic receptors
GI-tract 1 2 & b2 adrenergic receptors
RECEPTOR TYPES AND SUBTYPES

27. Enzyme linked receptors
Tyrosine kinase - linked receptors
• Bifunctional receptor / enzyme
• Activated by hormones
• Overexpression can result in cancer

28. • Protein serves dual role - receptor plus enzyme
• Receptor binds messenger leading to an induced fit
• Protein changes shape and opens active site
• Reaction catalysed within cell
• Overexpression related to several cancers
closed
messenger
induced
fit
active site
open
intracellular reaction
closed
messenger
Tyrosine kinase-linked receptors

29. N H2
C O2H
Cell membrane
Catalytic binding region
(closed in resting state)
Ligand binding region
Extracellular
N-terminal
chain
Intracellular
C-terminal
chain
Hydrophilic
transmembrane
region (-helix)
Tyrosine kinase-linked receptors

30. Reaction catalysed by tyrosine kinase
N C
O
Protein Protein
OH
Tyrosine
residue
Tyrosine
kinase
Mg++
ATP ADP
N C
O
Protein Protein
O
Phosphorylated
tyrosine
residue
P

31. Epidermal growth factor receptor (EGF- R)
Inactive EGF-R
monomers
Cell
membrane
Binding site for EGF
EGF - protein hormone - bivalent ligand
Active site of tyrosine kinase
Induced fit
opens tyrosine kinase
active sites
Ligand binding
and dimerisation
OH
OHOH
HO
Phosphorylation
ATP ADP
OP
OPOP
PO
EGF

32. • Active site on one half of dimer catalyses phosphorylation of Tyr
residues on other half
• Dimerisation of receptor is crucial
• Phosphorylated regions act as binding sites for further proteins and
enzymes
• Results in activation of signalling proteins and enzymes
• Message carried into cell
Epidermal growth factor receptor (EGF- R)

33. Insulin receptor (tetrameric complex)
Insulin
Cell
membrane
Insulin binding site
Kinase active site
OP
Phosphorylation
ATP ADP
OP
OP
PO
Kinase active site
opened by induced fit
OH
OHOH
HO

34. Growth hormone receptor
Tetrameric complex constructed in presence of growth hormone
Growth hormone binding site
Kinase active site
Kinase active site
opened by induced fit
GH
OH
OH
OH
HO
kinases
GH receptors
(no kinase activity)
GH binding
&
dimerisation
OP
OPOP
PO
ATP ADP
Activation and
phosphorylation
OH
Binding
of kinases
OHOH
HO

35. Intracellular receptors
•Chemical messengers must cross cell
membrane
• Chemical messengers must be hydrophobic
• Example-steroids and
steroid receptors
Zinc
Zinc fingers contain Cys residues (SH)
Allow S-Zn interactions
CO2H
H2N
DNA binding region
(‘zinc fingers’)
Steroid
binding region

36. Cell
membrane
Intracellular receptor Mechanism
4. Binds co-activator protein
1. Messenger crosses membrane
2. Binds to receptor
3. Receptor dimerisation
5. Complex binds to DNA
6. Transcription switched on or off
7. Protein synthesis activated or inhibited
Messenger
Receptor
Receptor-ligand
complex
Dimerisation
Co-activator
protein
DNA

37. Oestrogen receptor
Oestradiol
H12
Oestrogen
receptor
Binding
site AF-2
regions
Dimerisation &
exposure of
AF-2 regions
Coactivator
Nuclear
transcription
factor
Coactivator
DNA
Transcription
Intracellular receptors

38. Cyclic
nucleotide
Known binding proteins
Pathway/Biological
association
cAMP
1.protein kinase A
2.cyclic nucleotide-gated ion
channels
3.Epac
4.Catabolite Activator
Protein (CAP)
1.smooth muscle relaxation
2.photo/olfactory receptors
3.glucagon production in
pancreatic beta cells
4.lacoperon regulation in E.
coli
cGMP
1.cGMP-dependent protein
kinase (PKG)
2.cyclic nucleotide-gated ion
channels
1.smooth muscle relaxation
2.photo/olfactory receptors
cCMP
1.cGMP kinase I
2.protein kinase A
1.smooth muscle relaxation

39. A simple intracellular signaling pathway activated by an extracellular signal molecule

41. Signaling through G-protein-coupled cell-surface receptors (GPCRs)
and small intracellular mediators

42. Trimeric G proteins disassemble to relay signals
from G-protein-linked receptors

43. Activation of a G protein by an activated GPCR

44. Some G-proteins regulate the production of cyclic AMP
A nerve cell in culture responding to the neurotransmitter serotonin, which acts
through a GPCR to cause a rapid rise in the intracellular concentration of cyclic
AMP

45. The synthesis & degradation
of cyclic AMP
Many extracellular signals
work by increasing cAMP
concentration, and they do
so by increasing the activity
of adenyl cyclase rather than
decreasing the activity of
phosphodiesterase.
All receptors that act via
cAMP are coupled to a
stimulatory G protein (Gs),
which activates adenyl
cyclase.

46. Cyclic-AMP-dependent protein kinase (PKA) mediates most of the effects
of cyclic AMP
Mammalian cells have at least two types of PKAs:
• type I is mainly in the cytosol,
• whereas type II is bound via its regulatory subunit and special anchoring
proteins to the plasma membrane, nuclear membrane, mitochondrial outer
membrane, and microtubules.

47. • Cyclic AMP induced responses
could be rapid or slow.
• In skeletal muscle cells, PKA
induces a rapid response by
phosphorylating enzymes
involved in glycogen
metabolism.
• In an example of a slow
response, cAMP activates
transcription of a gene for a
hormone, such as somatostatin
*CREB: cAMP-response element-binding
protein

48. Some G proteins activate the inositol phospholipid signaling pathway by
activating phospholipase C-b
The effects of IP3 can be mimicked by a Ca2+ ionophore (A23187 or ionomycin
and the effects of diacylglycerol can be mimicked by phorbol esters

49. The hydrolysis of PI(4,5)P2 by phospholipase C-b

50. Ca2+ functions as a ubiquitous intracellular messenger
Three main types of Ca2+ channels that mediate Ca2+ signaling:
1. Voltage dependent Ca2+ channels in the plasma membrane
2. IP3-gated Ca2+-release channels
3. Ryanodine receptors

51. The concentration of Ca2+ in the cytosol is kept low in resting cells by
several mechanisms

53. Ca2+/ Calmodulin-dependent protein kinases (CaM-kinases)
mediate many of the actions of Ca2+ in animal cells
The structure of Ca2+/ Calmodulin

54. The stepwise activation of CaM-kinase II

55. BCDD

56. GABA A receptor
 It is an ionotropic receptor and ligand-gated ion channel.
 Its endogenous ligand is γ-aminobutyric acid (GABA), the
major inhibitory neurotransmitter in the central nervous
system.
 Upon activation, the GABA A receptor selectively conducts
Cl− through its pore.
 Cl- will flow out of the cell if the internal voltage is less
than resting potential and Cl- will flow in if it is more than
resting potential (i.e. -75 mV).
 This causes an inhibitory effect on neurotransmission by
diminishing the chance of a successful action potential
occurring.
 The reversal potential of the GABAA-mediated inhibitory
postsynaptic potential (IPSP) in normal solution is −70 mV,
contrasting the GABAB IPSP (-100 mV).

57. GABA A receptor
 The active site of the GABAA receptor is the binding site for
GABA and several drugs such as muscimol, gaboxadol, and
bicuculline.
 The protein also contains a number of different allosteric
binding sites which modulate the activity of the receptor
indirectly.
 These allosteric sites are the targets of various drugs
 benzodiazepines,
 nonbenzodiazepines,
 neuroactive steroids,
 barbiturates,
 alcohol (ethanol),
 inhaled anaesthetics, and
 Picrotoxin

58. GABA A receptor
 GABAA receptors occur in all organisms
that have a nervous system.
 To a limited extent the receptors can be
found in non-neuronal tissues.
 Due to their wide distribution within the
nervous system of mammals they play a
role in virtually all brain functions.

59. GABA A STRUCTURE
 The much sought structure of a
GABAA receptor was finally
resolved, with the disclosure of the
crystal structure of human β3
homopentameric GABAA receptor.
 The majority of GABAA receptors
are heteromeric and the structure
did not provide any details of the
benzodiazepine binding site.
 This was finally elucidated in 2018
by the publication of a high
resolution cryo-EM structure of a
human α1β2γ2 receptor bound
with GABA and the neutral
benzodiazepine flumazenil.

60.  GABAA receptors are pentameric transmembrane receptors which consist
of five subunits arranged around a central pore.
 Each subunit comprises four transmembrane domains with both the N-
and C-terminus located extracellularly.
 Some isoforms may be found extrasynaptically.
 The ligand GABA is the endogenous compound that causes this receptor
to open; once bound to GABA, the protein receptor changes
conformation within the membrane, opening the pore in order to allow
chloride anions (Cl−) to pass down an electrochemical gradient.
GABA A STRUCTURE

61. GABA A STRUCTURE
 the reversal potential for chloride in most neurons is close to or
more negative than the resting membrane potential,
 Hence activation of GABAA receptors tends to stabilize or
hyperpolarise the resting potential, and can make it more difficult
for excitatory neurotransmitters to depolarize the neuron and
generate an action potential.
 The net effect is typically inhibitory, reducing the activity of the
neuron.
 However, depolarizing responses have been found to occur in
response to GABA in immature neurons due to a modified Cl-
gradient.
 These depolarization events have shown to be key in neuronal
development.
 In the mature neuron, the GABAA channel opens quickly and thus
contributes to the early part of the inhibitory post-synaptic potential
(IPSP).
 The endogenous ligand that binds to the benzodiazepine site is
inosine.

62. GABA A Function
 The molecular target of the benzodiazepine class of
tranquilizer drugs.
 Bind to distinct benzodiazepine binding sites situated at the
interface between the α- and γ-subunits of α- and γ-subunit
containing GABAA receptors.
 While the majority of GABAA receptors (those containing
α1-, α2-, α3-, or α5-subunits) are benzodiazepine sensitive,
there exists a minority of GABAA receptors (α4- or α6-
subunit containing) which are insensitive to classical 1,4-
benzodiazepines,[10] but instead are sensitive to other
classes of GABAergic drugs such as neurosteroids and
alcohol.

63. GABA A Function
 In order for GABAA receptors to be sensitive to the action of benzodiazepines
they need to contain an α and a γ subunit, between which the benzodiazepine
binds.
 Once bound, the benzodiazepine locks the GABAA receptor into a conformation
where the neurotransmitter GABA has much higher affinity for the GABAA
receptor, increasing the frequency of opening of the associated chloride ion
channel and hyperpolarising the membrane.
 This potentiates the inhibitory effect of the available GABA leading to sedative
and anxiolytic effects.[citation needed]
 Different benzodiazepines have different affinities for GABAA receptors made
up of different collection of subunits, and this means that their pharmacological
profile varies with subtype selectivity.
 For instance, benzodiazepine receptor ligands with high activity at the α1
and/or α5 tend to be more associated with sedation, ataxia and amnesia,
whereas those with higher activity at GABAA receptors containing α2 and/or α3
subunits generally have greater anxiolytic activity.[12]
 Anticonvulsant effects can be produced by agonists acting at any of the GABAA
subtypes, but current research in this area is focused mainly on producing α2-
selective agonists as anticonvulsants which lack the side effects of older drugs
such as sedation and amnesia.

64. GABA A Function
 In order for GABAA receptors to be sensitive to the action of benzodiazepines they need to contain an α and
a γ subunit, between which the benzodiazepine binds. Once bound, the benzodiazepine locks the GABAA
receptor into a conformation where the neurotransmitter GABA has much higher affinity for the GABAA
receptor, increasing the frequency of opening of the associated chloride ion channel and hyperpolarising the
membrane. This potentiates the inhibitory effect of the available GABA leading to sedative and anxiolytic
effects.[citation needed]
 Different benzodiazepines have different affinities for GABAA receptors made up of different collection of
subunits, and this means that their pharmacological profile varies with subtype selectivity. For instance,
benzodiazepine receptor ligands with high activity at the α1 and/or α5 tend to be more associated with
sedation, ataxia and amnesia, whereas those with higher activity at GABAA receptors containing α2 and/or
α3 subunits generally have greater anxiolytic activity.[12] Anticonvulsant effects can be produced by
agonists acting at any of the GABAA subtypes, but current research in this area is focused mainly on
producing α2-selective agonists as anticonvulsants which lack the side effects of older drugs such as
sedation and amnesia.
 The binding site for benzodiazepines is distinct from the binding site for barbiturates and GABA on the
GABAA receptor, and also produces different effects on binding,[13] with the benzodiazepines increasing
the frequency of the chloride channel opening, while barbiturates increase the duration of chloride channel
opening when GABA is bound.[14] Since these are separate modulatory effects, they can both take place at
the same time, and so the combination of benzodiazepines with barbiturates is strongly synergistic, and can
be dangerous if dosage is not strictly controlled.
 Also note that some GABAA agonists such as muscimol and gaboxadol do bind to the same site on the
GABAA receptor complex as GABA itself, and consequently produce effects which are similar but not
identical to those of positive allosteric modulators like benzodiazepines.[citation needed]

65. Glucagon-like peptide-1 receptor

66. GLP1R
 The GLP1R is a receptor protein.
 Binds glucagon-like peptide-1 (GLP1) and glucagon as its
natural endogenous agonists.
 Found on beta cells of the pancreas and on neurons of the brain.
 It is involved in the control of blood sugar level by enhancing
insulin secretion.
 In humans it is synthesized by the gene GLP1R, which is
present on chromosome 6.
 It is a member of the glucagon receptor family of G protein-
coupled receptors.

67. GLP1R Structure
 The GLP-1 receptor sequence contains a large hydrophilic
extracellular domain and seven hydrophobic
transmembrane domains.
 The GLP-1 receptor protein has three potential N-linked
glycosylation sites, and glycosylation may modulate
receptor function.
 GLP1R is composed of two domains:
 one extracellular (ECD) that binds the C-terminal helix of
GLP-1 and
 One transmembrane (TMD) domain that binds the N-
terminal region of GLP-1.
 In the TMD domain there is a fulcrum of polar residues that
regulates the biased signaling of the receptor while the
transmembrane helical boundaries and extracellular surface
are a trigger for biased agonism.

68. GLP1R Function
 Activated GLP1R stimulates the adenylyl cyclase pathway which
results in increased insulin synthesis and release of insulin.
 Consequently, GLP1R has been a target for developing drugs
usually referred to as GLP1R agonists to treat diabetes mellitus.
 Exendin-4 is one of the peptides used therapeutically to treat
diabetes, and its biological binding mode to the GLP-1R has been
demonstrated using genetically engineered amino acids.
 GLP1R is also expressed in the brain where it is involved in the
control of appetite.
 Furthermore, mice that over express GLP1R display improved
memory and learning.
 Stretch responsive vagal neurons in the stomach and intestines
also express GLP1R.
 GLP1R neurons particularly and densely innervate stomach
muscle and can communicate with additional organ systems
changing breathing and heart rate due to activation.

69. Signal Transduction Pathway
GLP1R
 The GLP-1 receptor is functionally coupled to AC via Gs.
 Through activation of AC, cAMP is formed and activates
PKA.
 Ligand stimulation of the receptor also increases the
cytoplasmic concentration of Ca2+;
 this is thought to be executed both through Na+-
dependent uptake of extracellular Ca2+ and through
release of Ca2+ from intracellular Ca2+ stores.
 The increased cytosolic Ca2+ in conjunction with the
activated PKA stimulates the translocation and exocytosis
of insulin-containing secretory granules

70. Agonists & Antagonists
Agonists
 Exendin-4 or Exenatide, was the first FDA approved
“incretin mimetic”;
 Liraglutide, a long-acting GLP-1 receptor agonist, was 2nd .
 Other agonists are
 CJC-1131, a GLP-1 analog engineered for covalent coupling
to albumin;
 Albiglutide, a recombinant human albumin-GLP-1 protein;
 ZP10, an exendin-4 derivative;
 Taspoglutide
 Dulaglutide
Antagonists
 Exendin (9–39) and T-0632, a small non-peptide ligand,
are antagonists.

71. Serotonin

73. Serotonin deficit

77. Glucocorticoid receptor (GR, or
GCR)
 The GR is also known as NR3C1
(nuclear receptor subfamily 3, group C,
member 1)
 Binds to cortisol and glucocorticoids.
 The GR is expressed in almost every cell
in the body and regulates genes
controlling the development, metabolism,
and immune response.