You are on page 1of 43


Dr Mohan Amberkar

Activation and inactivation
Structure and role of G-proteins
Classification of G-proteins
Targets of G-proteins

-Adenylyl cyclase
-Phospholipase C
-Ion channels
-Rho A/Rho kinase
-MAP kinase
Recent developments in GPCR biology


Brian Kobilka and Robert Lefkowitz

“crucial for understanding how GPCR
Cell surface proteins
Major class of drug targets
7-TM receptors,Heptahelical
receptors,Serpentine receptors,GPLR’s..
GPCR family
Senses the outside molecules and activates
inside the signal trasduction pathway
cellular responses.


Beta adrenoreceptor in 1986
All the receptors identified have now been cloned
Aspiring receptor has to be cloned
Charecteristic structure:

- 7 transmembrane alpha helices
- extracellular N-terminal
- intracellular C-terminal

5 .

6 .

glucagon. .CCK. -Ligand binds to transmembrane helices (amines) or to extracellular loops (peptides) B: Secretin/Glucagon receptor family Receptors for peptide hormones. Ca2+-sensing receptors Others : D: Fungal mating pheromone receptors E: Camp receptors F: Frizzled/smoothened receptors 7 .Family Receptors Structural Features A: Rhodopsin family -The largest group. etc.leptin etc -Intermediate extracellular tail incorporating ligand-binding domain C: Metabotropic glutamate receptor/calcium sensor family -Smallest group -Long extracellular tail -Metabotropic glutamate receptors. many neuropeptides. calcitonin. -Short extracellular (N terminal) tail. incorporating ligand-binding domain GABAB receptors.Receptors for most amine neurotransmitters. cannabinoids.gastrin. prostanoids. purines. including secretin.

GRAFS Classification Glutamate receptor family Rhodopsin receptor family Adhesion receptor family Frizzled receptor family Secretin receptor family 8 .

9 .

Single site mutagenesis ligand mapping design synthetic ligands X ray crystallography-molecular structure of GPCR’s Fluorescence methods-kinetics of ligand binding 10 .

Methods of Activation Agonist binding Protease activated receptors(PAR’s) .thrombin snips of the N-terminal tail TETHERED AGONIST Activation of PAR’s PAR molecule can be activated only once 11 .

INACTIVATION Further Proteolytic cleavage Densensitization involving phosphorylation Internalization and degradation 12 .

13 .

G Protein Family of membrane resident protein  Recognizes the activated GPCR’s Passes the message to the effector system Generates cellular response Interacts with guanine nucleotides(GTP & GDP) 14 .

Structure Consists of 3 subunits-alpha.beta and gamma Alpha subunit: -Guanine nucleotide binds to the alpha subunit -Has enzymatic activity Beta and gamma subunit: -Remain together as a complex Anchored through an alpha fatty acid chain ‘PRENYLATION’ Freely diffusable 15 .

16 .

g. causing increased cAMP formation Activates phospholipaseC. increasing production of second messengers inositol trisphosphate and diacylglycerol •Activate potassium channels •inhibit voltage-gated calcium channels •activate GPCR kinases •activate mitogen-activated protein kinase cascade 17 . decreasing cAMP formation receptors Gαo As for Gαs. catecholamines. peptide and prostanoid receptors Gβγ subunits All GPCRs Stimulates adenylyl cyclase. histamine. serotonin) Gαi As for Gαs. also opioid. cannabinoid Inhibits adenylyl cyclase. also opioid. cannabinoid ?Limited effects of α subunit (effects mainly due to βγ subunits) receptors Gαq Amine.SUBTYPE ASSOSIATED RECEPTORS MAIN EFFECTORS Gαs Many amine and other receptors (e.

activation of 2 bacterial toxins(enzymes) Cholera toxin Pertussis toxin 18 .SPECIFICITY??? Receptor and effector selectivity Molecular variations within the alpha subunits Specific recognition domains Functional variations: .

Cholera toxin Acts only on Gs Persistent activation Symptoms of cholera 19 .

Pertussis toxin Block Gi and Go Prevents Gi to bind to adenylyl cyclase Increases cAMP 20 .

Activation of GPCR Conformational change in the receptor Assosiaton of G-protein with the receptor GDP-GTP exchange Dissosiation of the trimer Active forms of G-protein Diffuse and assosiate with various enzymes and ions channels Activation of TARGET/EFFECTOR systems 21 .

Targets for G-proteins 1) Adenylyl cyclase 2) Phospholipase C 3) Ion channels 4) Rho A/Rho kinase 5) Mitogen-activated protein kinase(MAP kinase) 22 .

23 .Aenylyl cyclase/cAMP system Sutherland and his colleagues “second messengers in signal transduction” ATP AC PDE’s PDE’s cAMP 5AMP There are 9 different molecular isoforms of adenylyl cyclase.

cAMP Protein kinases Various effects Energy metabolis m Contractile proteins Cell Cell division division Cell differentiatio n Ion transpo rt Ion channels 24 .

25 .

cAMP Protein kinase Increase activity of voltage gated calcium channels in heart muscle cells Phosphorylation of these channels Increase calcium entry Increasing the force of contraction of the heart 26 .

cAMP Protein kinases Inactivation of myosin light chain kinase Smooth muscle relaxation 27 .

Sildenafil(PDE5) 28 . Forskolin and fluoride ions 2. Methylxanthines 3. Rolipram(PDE4) 4.cAMP Decrease  M2 receptor of cardiac muscle  Alpha 2 receptors in smooth muscles  Opioid receptors Increase 1. Milrinone(PDE3) 5.

plays a key role 29 .Phospholipase C/Inositol phosphate system In 1950’s by Hokin and Hokin Michell and Berridge .alpha agonists acting on smooth muscles and salivary glands .member of PI family.Increase in free Ca increase PI turnover Eg: .muscarinic agonists .vasopressin acting on liver cells PIP2.

30 .

opioid analgesics open K+ channels channels inhibit Ca 31 .ION CHANNELS GPCR can act on ion channels directly or indirectly Direct G-protein channel interaction was first shown on cardiac muscle Eg: .mAChRs enhances K+ permeability .In neurons.

Free G-protein alpha + Guanosine nucleotide exchange factor Rho protein is activated Activates Rho kinase Controls variety of cellular functions  Important in the pathogenesis of pulmonary hypertension.Rho/Rho kinase system  Activated by certain GPCR’s which couple to the G-proteins of G12/13 type.  Eg:Fasudil 32 .

cytokines .GPCR ligands  End result – cell proliferation  Proving to be a pathogenic pathway for cancer  Many compounds inhibiting this pathway potential anti cancer drugs  Eg: Sorafenib.MAP Kinase system  Activated by: .growth factors . dabrafenib and vemurafenib – Raf kinase Selumetinib. trametinib – MEK inhibitors 33 .

receptor internalisation(endocytosis) Residues in the C-terminal cytoplasmic tail gets phosphorylated Involvement of protein kinase A.protein kinase C and GPCR kinases 34 .DESENSITISATION Involves 2 main processes: .receptor phosphorylation .

35 .

36 .Recent developments in GPCR GPCR dimerisation: GABAb R1 Eg:GABA b GABAb R2 .Beta adrenoreceptor: mutation in the 3rd intracellular loop or overexpression of the receptor.Dopamine 2 -Somatostatin receptors Constitutevely active receptors: . -Histamine(H3) shows constitutive activity in vivo.

Eg: .Neuropeptide CGRP=CRLR(GPCR) + RAMP 1 .Receptor activating modifying proteins(RAMP’s): .Adrenomedullin = CRLR(GPCR) + RAMP 2 37 .family of membrane proteins that associate with GPCR and alter the functional characteristics.

Regulators of G-protein Signalling(RGS)  Family of 20 cellular proteins  Binds specifically to G alpha units  Increase the GTPase activity  Hence exerting an inhibitory effect on G- protein signaling  RAMP’s and RGS:-protein-protein interactions influence the pharmacological behaviour of the receptors in a selective way. 38 .

WHERE IT ACTS??  Anchoring to the lipid bi-layer and thereby targeting the interface  Pepducins for over 15 different GPCR’s have been successfully produced  An anti PAR4 pepducin-extended bleeding time in mice and protected against platelet activation and thrombosis  A CXCR4 agonist pepducin mobilzes hematopoetic stem cells to bone marrow 39 . HOW IT ACTS??  It utilizes the lipid fragments of intracellular GPCR loops to modulate the GPCR action.PEPDUCINS  Cell penetrating peptides  Acts as intracellular modulators of signal transmission from a GPCR to G-proteins.


and efficacy of GPCR-based pepducins in disease models".Refrences 1. N.1159–204. "Mammalian G proteins and their cell type specific functions“:2005.Flower RJ. Ernst OP. 85 (4). "Comprehensive repertoire and phylogenetic analysis of the G protein-coupled receptors in human and mouse". Fredriksson R.Dale MM. Covic L. biodistribution. Hellstrand SH. 5. 4. Gloriam DE. Schwartz TW.p33-42 Wettschureck N. Tchernychev B. Schiöth HB (September 2006). Agarwal A. Annals of the New York Academy of Sciences 1226: 34–49 41 . Kuliopulos A (2011). Kristiansson H. "G protein-coupled receptor modulation with pepducins: moving closer to the clinic". Milligan G. Koukos G. Jacques SL. Methods in Molecular Biology (Clifton.Ritter JM. Bouvier M. Gardella TJ. Sakmar TP. Janz JM. Xu L.  Bjarnadóttir TK. Offermanns S. Genomics 88 (3): 263–73. Tressel SL. "Pharmacology. Rang HP. Hunt SW (May 2011).How drugs act: molecular 2. aspects:7th edition:2008.J. Methods in Molecular Biology 683: 259–75 Dimond P. Covic L. Gerard C. Carlson K. Kuliopulos A. 3.).

8. Cell. Signal. Schwartz TW. Hunt SW (May 2011). Ribas C. 15 (11): 973–81. Sakmar TP. Mayor F (November 2003). "G proteins: transducers of receptorgenerated signals". Carlson K. "The role of beta-arrestins in the termination and transduction of Gprotein-coupled receptor signals". Annu. 42 . Covic L. Agarwal A. Annals of the New York Academy of Sciences 1226: 34–49 9. Rev. Xu L. Gerard C.6. Bouvier M. Janz JM. "Mechanisms of regulation of the expression and function of G protein-coupled receptor kinases". 7. Kuliopulos A. 56: 615–49. Gilman AG (1987). Ernst OP.  Luttrell LM. Cell.  Penela P. 115 (Pt 3): 455–65. J. Gardella TJ. Milligan G. Dimond P. Biochem. Lefkowitz RJ (February 2002). "G protein-coupled receptor modulation with pepducins: moving closer to the clinic". Sci.

43 .