«Επταελικοειδείς υποδοχείς που αλληλεπιδρούν µε G- πρωτεΐνες:mo. :Moριακοί

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1 «Επταελικοειδείς υποδοχείς που αλληλεπιδρούν µε G- πρωτεΐνες:mo :Moριακοί στόχοι στη παραγωγή νέων φαρµάκων» Ηρώ Γεωργούση demokritos.gr/georgoussi_cv/htm Εργαστήριο Κυτταρικής Σηµατοδότησης και Μοριακής Φαρµακολογίας, Ινστιτούτο Βιολογίας

2 SIGNALS CAN ENTER CELLS IN MANY WAYS growth factors neurotransmitters tyrosine kinase steroid hormones R R cations nucleus 2 d messengers cell membrane hormones, photons neurotransmitters

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4 G protein signaling

5 GPCR SIGNALING GPCR SIGNALING Hormones,neurotransmit neurotransmit. RECEPTORS G PROTEINS (αβγ) GTP R G GDP EFFECTORS 2d messengers, σσ or other E

6 Diversity of 7-Transmembrane receptors

7 How do G protein-coupled receptors cause G protein activation? RG contact sites GDP is >20A away action at a distance

8 G protein cycle Turnoff P i R α GDP β γ A Receptor activation A R RGS RGS R α β γ GTP α β γ GTP GDP Activation of: Ga effectors Gβγ effectors

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10 Receptor Activation Palczewski et al. Hubbell

11 Heterotrimeric G Proteins α subunit The a Subunits Bind and Hydrolyze GTP Exchange Factors are Heptahelical Receptors GTPase Activating Proteins are RGS Proteins a and βγ Subunit Association-Dissociation Cycle is Regulated by GTP Binding/Hydrolysis Cycle Gβ is a WD repeat Protein Gγ is isoprenylated

12 GTP-Dependent Conformational Switch in Ga GDP-Bound GTP-Bound

13 Structural Basis of GTP-Mediated Subunit Dissociation: Gβγ-Binding Residues GDP-bound Gaβγ GTP-bound Ga

14 Family of G protein a subunits cdna Gαs Gαolf Gαi2 Gαi3 Gαi1 GαoA GαoB Gαt1 Gαt2 Gαz Gαq Gα11 Gα14 Gα15 Gα16 Class Gαs Gαi Gαq Gα12 Gα13 Gα12/13 Amino Acid Identity (%)

15 Specificity of G protein signaling A Neurotransmitters Hormones Chemokines Sensory Signals Local modulators 23 a subunits α Effectors αs Adenylyl Cyclase αi ΑC inhibition αq Phospholipase C αt cgmp Phosphodiesterase α12/13 rho exchange factors >1000 Receptors Gs-coupled Gi-coupled Gq-coupled β-adrenergic, D1 Dopamine A2a Adenosine, 5HT-2, H2 Histamine ACTH, LH, FSH, TSH, MSH, CCK Olfactory, Taste α2-adrenergic, D2 Dopamine M2 Muscarinic, GABAb, 5HT-1a A1 Adenosine, H3 Histamine Opioid mu, kappa, delta, SST IL-8, fmlp, Thrombin Prostaglandin E1, E2 α1-adrenergic, M1 Muscarinic 5HT-1c, Histamine H1 TSH, GnRH CCK, VIP, V1 Vasopressin Bradykinin, Substance P, K Thomboxane, PAF G12, G13 Thrombin, Thromboxane, Endothelin 6 Gβ subunits12 Gγ subunits Gβγ Effectors K, Ca Channels ACII, PLCb2 b-ark, Phosducin PI-3 kinase, src kinase

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17 G protein effectors as αi αq αt GTP a/12,13 α a Effectors Adenylyl Cyclase Adenylyl Cyclase Phospholipase Cβ cgmp Phosphodiesterase RhoGEF γ β βγ Effectors K, Na, Ca Channels ACII, PLCβ modulation b-ark, Phosducin anchor PI-3 kinase Tyr kinases src, Tsk, Btk Second messenger signaling vs membrane-delimited pathways camp, cgmp, Ca 2+ K and Ca channels, lipids

18 Adenylyl Cyclic Cyclases Nucleotide and Metabolism regulation - campof camp metabolism β-adrenergic receptor SIGMA-ALDRICH Some Gs-coupled receptors: ACTH, LH, FSH, TSH, MSH, CCK A2 Adenosine, 5HT-2, H2 Histamine D2 Dopamine, Olfactory, Taste Giα Some Gi-coupled receptors: M2, M4 Muscarinic, GABAb, 5HT-1a a2-adrenergic, D2 Dopamine Opioid mu, kappa, delta, SST IL-8, fmlp, Thrombin Prostaglandin E1, E2 Gsα Important roles in Endocrine Neural Olfactory.. Every physiological system Functional response

19 Gq-coupled Receptors Regulate the Phospholipase C Pathway [Ca] i maintained very low (~50nM). Gq activation causes PLCβ activation via Gaq Gi and Go βγ activation also activates PLCβ Phosphatidyl inositol lipids [Ca] i increase leads to: Activation of Calmodulin and Ca-CAM regulated enzymes; [Ca] oscillations PKC activation by diacylglycerol causes phosphorylation of PKC substrates, and changes in gene expression (phorbol esters are potent tumor promoters)

20 Diversity of G-Protein Coupled Receptor Signal Transduction Pathways Increase rho

21 R1 R2 R3 R1 R2 R3 R1 R2 R3 G 1 G 2 G 3 G 1 G 2 G 3 G E1 E2 E3 E R E R GG G1 G2 G3 E1 E1 E1 E1 E2 E3

22 Ερωτηµατικά που πρέπει να απαντηθούν: α) Κάτω απο ποιές φυσιολογικές συνθήκες ενεργοποιούνται όλοι αυτοί οι τελεστές? β) Ποιοί είναι οι περιοριστικοί παράγοντες που εµποδίζουν όλους αυτούς τους τελεστές να ενεργοποιηθούν την ίδια χρονική στιγµή? γ) Αρκούν µια ή απαιτούνται περισσότερες G πρωτείνες ώστε να αποδώσουν ικανό αριθµόβγυποµονάδων για την ενεργοποίηση όλων αυτών των τελεστών? δ) Ποιά είναι η εξειδίκευση των βγ υποµονάδων µε τους τελεστές? ε) Με ποιό µηχανισµόγίνεταιηενεργοπoίηση των τελεστών? στ) Υπάρχει µηχανισµός turnoff? των βγ υποµονάδων?

23 Use of receptor derived peptides fused or not with GST for G protein coupling & activation adenylyl cyclase inhibition Gβγ involvement Receptor-protein protein I/N Consrtuction of minigenes Site Site directed directed mutagenesis Cellular expression of the 3d loop peptides results in G protein activation, alteration in camp Amino-acids respon- accumulation, blockade of PI sible for activation, hydrolysis and interaction with G protein coupling heterologous receptors effector regulation

24 G protein signaling

25 SIGNALING CIRCUITS LEADING TO CREB PHOSPHORYLATION

26 Hormone binding Receptor activation G protein coupling effector activation 2nd messenger Desensitization G protein uncoupling Phosphorylation (sec, min) Sequestration (min) Down regulation (min, hrs)

27 Molecular mechanisms of desensitization and downregulation 1. Phosphorylation 2. Arrestin binding } 3. Recruitment of clathrin 4. Endocytosis } desensitization (uncoupling from signaling) } downregulation (decreased # receptors on cell surface)

28 Trafficking itineraries of GPCRs lysosome

29 NH2 AC Gαi/oi/o Gβ γ COOH GPCRs can form protein assemblies which should be viewed as dynamic complexes that involve interactions between receptor-receptor receptor, receptor-g G protein and receptor interacting proteins all of which contribute to the intricate and finally tuned process of downstream cell signaling

30 External interactions ligand binding localization 3d intracellular loop signaling cytoskeletal associations C- tail (intracellular interactions) signaling trafficking localization cytoskeletal associations

31 Novel Signaling Paradigms 1. Receptors signal through many more proteins than G proteins 2. GPCRs oligomerize, and take on new pharmacology

32 Role of receptor-receptor I/N in GPCR signaling Signaling beyond G protein Physiological Functional significance? Relevance?

33 Signal transduction cross-talk Cells integrate various signals; the final cellular response is integrated based on all the signals it is receiving. Cell responses are very tissue specific, because of the particular genes each expresses.

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35 Diseases caused by mutations or modifications of G proteins Pathologic organisms: cholera toxin ADP-ribosylates Gsa, blocking GTPase activity pertussis toxin ADP-ribosylates Gia, blocking GTPase activity Mutations in G proteins: Ga subunits: activating -- by blocking GTPase activity adenomas of pituitary and thyroid, adenomas of adrenals and ovaries testotoxicosis, by increased constitutive Mutations in Gsa leading to disease GDP release Absent or inactive G protein a subunit pseudohypoparathyroidism night blindness. Disruption of receptor-mediated activation McCune-Albright syndrome Gβγ subunits: essential hypertension (risk factor) Weinstein et al., 2001

36 Diseases caused by mutations or modifications of receptors Mutations in GPCRs Color Opsins and rhodopsin: Color blindness, Retinitis pigmentosa, Congenital night blindness V2 vasopressin receptor: Nephrogenic diabetes insipidus ACTH receptor: Familial ACTH resistance LH receptor: Familial male precocious puberty TSH receptor: Familial nonautoimmune hypertyroidism Ca2+ receptor: Familial hyperparathyroidism Thromboxane A2 receptor: Congenital bleeding Endothelin B receptor: Hirschsprung disease

37 Marketed drugs targeted at GPCRs Eli Lilly s s : Zyprexa Janssen : Risperdal Schering-Plough Plough s s : Clarinex GlaxoSmithKline s : Zantac, GlaxoSmithKline s : Imigran Novartis : Zelnorm 1997 > million US$

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