DR.CH.PAVANI 1ST YR PG DEPT OF PHARMACOLOGY GUNTUR MEDICAL COLLEGE

1. Dr. Pavani
1st yr post graduate
Department of Pharmacology
G-PROTEIN COUPLED
RECEPTORS

2. CONTENTS
• Introduction
• Structure
• Classification
• Alternative mechanisms for GPCR activation
• G- proteins and their role
• Targets for G- proteins
• Further development in GPCR biology
• GPCR related diseases
• GPCR binding drugs
• Summary
• References

3. NOBEL PRIZE IN MEDICINE /PHYSIOLOGY
Martin rodbell shared the 1994 Nobel Prize in Physiology or
Medicine with Alfred G. Gilman for their discovery of G-proteins and
the role of these proteins in signal transduction in cells.

5. Introduction
Around 800 GPCR are found in humans
olfaction (~400).
 taste (33)
 light perception (10)
 pheromone signalling (5)
 ~350 non-sensory GPCRs mediate signalling
 Around 100-200 orphan GPCRs are known in
human.

6. GPCR
• First GPCR to be fully characterised was the β
adreno receptor(1986).
• X-ray crystallography -to study the molecular
structure of these receptors.
• Flourescence methods -to study the kinetics of
ligand binding and changes associated with
activation.

7. CRYSTAL STRUCTURE
OF RHODOPSIN
• First X-ray crystal structure of
GPCR - 2000
• 72 structures of GPCRs -
Protein Data Bank.
• Rhodopsin, is by far the best
structurally defined GPCR.
• GPCR signaling can be
activated in 200-500 millisec

8. STRUCTURE OF GPCR
• Single polypeptide with
– an extracellular N-terminus
– an intracellular C-terminus
– seven hydrophobic transmembrane α helices (TM1-TM7)
linked by
• three extracellular loops (ECL1-ECL3)
• three intracellular loops (ICL1-ICL3).
• The third intracellular loop interacts with the G-protein

9. STRUCTURE OF GPCR

10. FAMILY RECEPTORS STRUCTURAL
FEATURES
A:Rhodopsin
family
•Largest group
•receptors for most amine
neuro transmitters,
neuropeptides, purines etc
Short extra
cellular tail
B:Secretin/Glucag
on receptor family
•Receptor for peptides
including glucagon, secretin,
calcitonin
Intermediate
extra cellular tail
C:Metabotropic
glutamate
receptor
•Small group.
•Metabotropic glutamate
receptor, GABA b receptors
Long extra
cellular tail

11. Protease activated receptors
• Alternative mechanisms for GPCR activation
Thrombin
by snipping off the N-terminal tail of the receptor
expose 5 or 6 residues
bind intramolecularly
activate the PAR

12. MECHANISM OF PAR-1

13. • PAR molecule can be activated only once
• As cleavage cannot be reversed
• Continuous resynthesis of receptor is
necessary.

14. RESYNTHESIS OF RECEPTOR
Inactivation
further proteolytic cleavage
frees the tethered ligand,
Desensitisation, involving
phosphorylation
receptor is
internalised and degraded
to be replaced by newly
synthesised protein.

15. There are 4 known protease-activated
receptors
• PAR1(thrombin)
• PAR2( trypsin)
• PAR3(thrombin)
• PAR4(thrombin)
PAR are located in myocytes , neurons
and platelets and also in endothelial cells

16. FUNCTION OF PAR
• a) Coagulation cascade( contributes to hemostasis)
• b) Inflammatory cells(play a role in inflammatory
pain)
• c) Digestive tract

17. G- proteins and their role
Actually called G-proteins because of their interaction
with the guanine nucleotides, GTP and GDP.
Recognise activated GPCRs and
pass on the message to effector systems
generate a cellular response

18. α( 21 sub units)
G-proteins β (6 sub units)
γ(12 sub units)
• Subunits are anchored to the membrane through a
fatty acid chain, coupled to the G-protein through a
reaction known as prenylation.

19. Function of GPCR
• Resting state,
– G protein exists as αβγ trimer
– GDP is occupying site on α sub unit
– May or may not be precoupled by receptor

20. • Activated state
Activated by agonist molecule
Agonist –induced interaction of αβγ
GDP replaced with GTP
Dissociation of G protein
α-GTP βγ subunits

21. Active forms + ion channels and enzymes
Activation /Inhibition of target
Single agonist-receptor complex
Many G protein molecules activated (amplification)
Product is often a second messenger

22. Signalling termination
GTP
hydrolysis GTPase
α- GDP
α-GDP dissociates from effector
reunites with βγ
αβγ

23. Function of GPCR

24. G protein sub types and their functions

25. Ligands
• Muscarinic receptors of Ach
• Norepinephrine
• Eicosanoids(TX,LT,PG)
• Peptide hormones
• Opioids
• Amino acids such as GABA
• Dopamine
• Serotonin(5-HT2A receptor)

26. TARGETS FOR G-PROTEINS
• Adenylyl cyclase cAMP formation
• Phospholipase C IP,DAG formation
• Ion channels Calcium and potassium channels
• Rho A/Rho kinase Signaling pathways regulation
• MAP controls cell functions

27. cAMP
• cAMP (cyclic 3′,5′-adenosine monophosphate)is
synthesised from ATP
PDE
• cAMP 5’AMP(inactivated)
• Drugs + GPCR increase /decrease adenylcyclase
Increase /decrease cAMP
Regulate cell functions

28. β-adrenoceptor activation
affects glycogen and fat metabolism
energy is made available as glucose
fuel for muscle contraction
Protein
kinase
cAMP

29. cAMP SYSTEM
Adenylyl cyclase can be activated
directly by
drugs such as Forskolin, which is used
experimentally to
study the role of the cAMP system

30. Phosphodiesterases
– 11 PDE subtypes exist
– PDE3 and PDE4 are cAMP-selective
– while others (e.g. PDE5) are cGMP-selective
– Nonselective PDE inhibitors- methylxanthines(theophylline
and caffeine)

31. PDE inhibitors:
• Vinpocetine-PDE1
• Oxindole-PDE2
• Milrinone - heart failure - PDE3
• Rolipram -asthma -PDE4
• Sildenafil- PAH-PDE5
• Quinazoline-PDE7
• Papaverine-PDE10

32. PHOSPHOLIPASE C/IP3 SYSTEM
• Second messenger system
• First discovered in the 1950s by Hokin and Hokin
• They centred interest on the mechanism of salt secretion by
the nasal glands of seabirds.
• Found that secretion was accompanied by increased turnover
of phosphoinositides(PI)

33. • Michell and Berridge found that many hormones that
produce an increase in free intracellular Ca2+
concentration also increase PI turnover
• PIP2 (phosphatidylinositol (4,5) bisphosphate)is the
substrate for phospholipase Cβ (PLCβ), which splits it
into diacylglycerol (DAG) and inositol (1,4,5)
trisphosphate (IP3)
• both function as second messengers

34. Phospholipase C cycle
hormone + receptor
activate receptors (GPCR)
PLC activation
cleaves (PIP2)
IP3 DAG
calcium release from the ER activate PKC
ca2+ controls the activity of PKC key role in signal
transduction

35. Phospholipase C cycle

36. Ion channels
• Major function of GPCR
– to control ion channel function without involving second
messengers.
• Direct G protein–channel interaction,
βγ subunits of Gi and Go proteins
+
ion channel
controlling K+ and Ca2+ channels

37. Rho/Rho kinase system
• Activated by certain GPCRs and also by non-GPCR
mechanisms
• Couple to G proteins of the G12/13 type
Rho–GDP ( resting form/inactive)
GDP–GTP exchange occurs,
Rho is activated,
activates Rho kinase

38. Rho kinase phosphorylates substrate proteins
Control cellular functions
Smooth muscle contraction,proliferation,synaptic
remodeling,angiogenesis
Rho kinase inhibitors (e.g. Fasudil) are in development

39. MAP KINASE SYSTEM
• Involves several signal transduction
• pathways activated by
 cytokines
 growth factors acting on kinase-linked receptors
 ligands activating GPCRs

40. • coupling of GPCRs to MAP kinases involve
• G protein α and βγ subunits
• Src and arrestins
• MAP kinase system controls
• Gene expression
• cell division
• apoptosis
• tissue regeneration

41. GPCR DESENSITISATION
• Decrease in drug response due to frequent
administration
• Continuous stimulation of cells by agonist leads to a
state of desensitization.
• Also called as Refractoriness
Downregulation
Adaptation

42. TYPES OF DESENSITISATION:
HOMOLOGOUS DESENSITISATION:
– Desensitize stimulated receptor as well as other
inactive receptors on the same cell.
– Receptor decreases its response to an agonist at
high concentration
– Helps organisms to maintain homeostasis

43. HETEROLOGOUS DESENSITISATION:
– also known as cross-desensitization.
– unresponsiveness of cells to one or
more agonists to which they are normally
responsive.
– heterologous desensitization occurs rapidly at low
agonist concentrations.

45. GPCR OLIGOMERISATION
• GPCRs exist and function as monomeric
protein
• First overturned by work on the GABAB
receptor.
• Two subtypes of GPCR exist, encoded by
different genes, and the functional receptor
consists of a heterodimer of the two.

46. • Such dimers have two agonist binding sites,
one on each subunit
• Only one is functional
• Signalling is transmitted through the dimer to
the other receptor in the dimer which couples
to the G protein

47. Pregnant women with hypertension
increased expression of B2(BRADYKININ) receptors
number of these dimers increase
increased sensitivity to the vasoconstrictor action of
angiotensin.
First instance of the role of dimerisation in human
disease

48. GPCR related diseases
Abnormal G protein signalling can result by
• Bacterial toxins(cholera & pertussis)
• Gene mutations
– Loss of function
– Gain of function
• Altered GPCR folding

49. • Retinitis pigmentosa (Rhodopsin mutation),
• Hypothyroidism & Hyperthyroidism (mutation in
TSHR gene
• Nephrogenic diabetes insipidus(Vasopressin
receptor mutation)
• Several fertility disorders(mutation in FSH/LH
receptor)
• Carcinomas(abberant GPCR expression)

50. • To date, over 600 inactivating and almost 100
activating mutations in GPCR have been
identified which are responsible for more than 30
different human disease.
• The number of human disorders is expected to
increase given the fact that over 160 GPCR have
been targeted in mice

51. GPCR BINDING DRUGS
• α1 (Oxymetazoline)and α2 agonist(Clonidine)
• β1 (Dobutamine) and β2 agonist(Terbutaline)
• Anti histaminics (Chlorpheniramine)
• Cholinergics (Pilocarpine) and Anti-cholinergics(Atropine)
• Anti emetics (Promethazine)
• H2 antagonists(Ranitidine)

52. SUMMARY

53. REFERENCES
• H.P.Rang; How drugs act: Molecular aspects; Rang and Dale’s
Pharmacology; 8th edition
• Goodman&Gilmans The pharmacological basis of
therapeutics; 13th edition
• BertramG.Katzung; Pharmacokinetics & Pharmacodynamics;
BASIC AND CLINICAL PHARMACOLOGY; 13th edition
• R.S.Satoskar; General considerations and pharmacokinetics;
PHARMACOLOGY AND PHARMACO THERAPEUTICS;25th
edition.