Ligand screening system using fusion proteins of G protein coupled receptors with G protein α subunits

Transcription

1 2 Ligand screening system using fusion proteins of G protein coupled receptors with G protein α subunits G protein coupled receptors A key player of signaling transduction. Cell membranes are packed with membrane proteins that occupy ~50% of their volume. Authors: Hinako Suga, Tatsuya Haga Source: Neurochem. Int. 51:140 64, Review Speaker: Hsu Yuan Fu Advisor: Chii Shen Yang, Ph. D. Sep. 28, Why signaling? How important the membrane it is! How does the life come from?

2 5 6 G protein coupled receptors G protein coupled receptors A key player of signaling transduction. Cell membranes are packed with membrane proteins that occupy ~50% of their volume. A large protein family of transmembrane receptors. Be found only in eukaryotes, including yeast, plants, and animals. The Nobel Prize in Physiology or "G proteins and the role of these proteins in signal transduction in cells". The Nobel Prize in Physiology or Medicine

3 9 10 G protein signaling system G protein signaling system: signaling cycle Resting state (ground state) Ligand binding Activated state (signaling state) Recovery state Ex: s protein coupled receptor The Nobel Prize in Physiology or Medicine G proteins GPCR,, Gβγ The genes of human GPCRs Two significant feature of GPCR genes Intronless Encode 7 hydrophobic region in amino acid sequence G A F B E C D Pi Pi Gγ Gβ Large scale analysis of gene structure in rhodopsin like GPCRs: evidence for widespread loss of an ancient intron, Gene 338 (2004), pp

4 13 14 The proteins of human GPCRs 7 transmembrane spanning α helices An extracellular N terminus An intracellular C terminus Three interhelical loops on each side of the membrane. NH3 + ECL1 ECL2 A B C D E F G ICL1 ICL2 ICL3 G ECL3 COO - A F B E C D Cell exterior Cell interior Oligomerization of human GPCRs Homodimer Rhodopsin form para crystalline arrays of parallel dimers in mouse disc membranes. Curr. Opin. Struct. Biol. 16 (2006), Heterodimer Opioid GPCR (κ & δ ) heterodimerization modulates receptor function. Nature 399 (1999), Intermolecular "cross talk" The α 2C adrenergic (α2) and m3 muscarinic receptor Proc. Natl. Acad. Sci. USA 90 (1993), Oligomerization of human GPCRs G proteins GPCRs may exist as functionally active oligomers in cultured cells. GPCR,, Gβγ It has not been settled yet, however, If GPCRs are generally present as homo or heterooligomers in situ. If the oligomerization is generally prerequisite for their function. G A F B E C D Pi Pi Gγ Gβ

5 17 18 subunit & Gβγ subunits GPCR desensitization Three phase of desensitization Uncoupling of the GPCR from G proteins Internalization (sequestration) of the GPCR into intracellular compartments Down regulation of the GPCR (irreversible breakdown of the GPCR) 49% 38% 28% What was the situation here? Screen System Orphan GPCRs: deorphaning Endogenous ligands have not been identified for more than 100 of GPCRs. Deorphaning may lead to discovery of novel hormones, neurotransmitters, or other factors. Drug Screening The GPCR family present a wide range of opportunities as therapeutic targets in these areas as demonstrated by the fact that 50 60% of approved drugs elicit their therapeutic effect by selectively addressing members of the GPCR family. Website: GE healthcare Annu. Rev. Pharmacol. Toxicol. 44 (2004), 43 66

6 21 22 Technologies proposed now Example: Tango GPCR Assay System (Invitrogen( Invitrogen) Ligands for q or s coupled receptors can be screened relatively easily by expressing these receptors in cultured cells and measuring agonist induced increase in intracellular Ca 2+ or camp, respectively. Activation of i coupled receptors which inhibited camp formation is difficult to be measured efficiently. Various other techniques have been proposed, such as the use of melanophore cells, cytosensor, [ 35 S]GTPγS binding, yeast mating pathway, fluorescently tagged proteins, and reporter based assays Difficulty of deorphan to orphan GPCRs and drug screening 1. Expression active GPCRs in cultured cell. Helper (partner) proteins Hetero oligomer formation 2. Interact partner species of G proteins can not be identify from the sequence data. s coupled receptors are highly homologous. i and q coupled receptors can not be distinguished using the sequence data. For example, urotensin II receptor is most homologous to somatostatin receptors (coupled to i family G proteins), but has turned out to couple with q family G proteins. 3. A satisfactory method is not available for screening of ligands for i coupled receptors, in spite of the fact that they constitute the largest group and the demand for such a method is high. 23 subunit q/i chimera The first few N ter. & last few C ter. amino acids of GPCR recognition site q/i chimera is activated by the i coupled receptor and activates phospholipase C pathway as the q G16 & G15 in hematopoietic cells They are promiscuously activated by different kinds of GPCRs, which are intrinsically coupled to s, i, or q family G proteins, and to activate phospholipase Cβ 24

7 Why GPCR fusion proteins? 25 Characterization of fusion proteins between GPCRs and 26 The fusion proteins have been used for studies of the interaction between GPCRs and by taking advantage of their robust interaction under 1:1 stoichiometry. Constructions: 5 gpcr tag (or gfp) gpa 3 Cell exterior A B C D E F G A B C D E F G Cell interior GFP Characterization of fusion proteins between GPCRs and 27 Characterization of fusion proteins between GPCRs and 28 Constructions: 5 gpcr tag (or gfp) gpa 3 Activity: agonist stimulation of activity: [ 35 S]GTPγS binding & GTP hydrolysis downstream signaling: adenylyl cyclase activity (Δ[cAMP]), Δ[Ca 2+ ] Example The β2 adrenoceptor s fusion proteins in S49 cyccells or insect Sf9 cells agonist stimulated Δ[cAMP]. Example The β2 adrenoceptor s fusion proteins in S49 cyc cells or insect Sf9 cells agonist stimulated Δ[cAMP]. The 5 HT1A receptor i1 fusion protein in HEK293 cells agonist stimulated Δ[cAMP]. The α 1B adrenoceptor 11 fusion protein or GPR40 q fusion protein in EF88 fibroblast cells Δ[Ca 2+ ]. The α 2A adrenoceptor G15, α 2B adrenoceptor G15, 5 HT1A receptor G15, M2 receptor G16, CX3CR1 G16 and P2Y12 receptor G16 fusion proteins in CHO cells Δ[Ca 2+ ]. Bertin et al., 1994, Seifert et al., 1998, Kellett et al., 1999, Stevens et al., 2001, Stoddart et al., 2007, Pauwels et al., 2000, Pauwels et al., 2001, Wurch et al., 2003, Suga et al., 2004, Nonaka et al., 2005, Martin et al., 2002, Hirasawa et al., 2005, Vorobiov et al., 2000, Medici et al., 1997

8 Considering the physilogical function of GPCR fusion protein 29 Example: downstream signaling of GPCR fusion protein Downstream signaling of GPCR fusion protein The activated may not necessarily be capable of activating the downstream effectors. 2. Effects of Gβγ subunits to GPCR fusion protein The presence of Gβγ promotes coupling between GPCR and. Fusion proteins have been shown to interact directly with Gβγ complexes using a variety of approaches. Adenosine A1 receptor GFP Cys351Gly i1 in CHO K1 cells Muscarinic acetylcholine receptor M2 subtype z in Xenopus oocytes 5 HT1A receptor Cys351Ile i1 in HEK293 cells [ 35 S]GTPγS binding endogenous v Fusion v Downstream signaling adenylyl cyclase and MAP kinase responses endogenous G proteins endogenous Gβγ released from endogenous i activate G protein activated K + channel agonist induced inhibition of adenylyl cyclase activity tethered i1, but not by endogenous i family G proteins However 31 As a tool for ligand screening: some successful story 32 Coexpression of the Gβγ with β2 adrenoceptor s in Sf9 membranes had no significant effect on the fraction of β2 adrenoceptors with high affinity for agonist and slightly increased the basal GTPase activity. Detection of endogenous ligand Example: A model study using nociceptin receptor i2 fusion protein. Deorphan by using combination of chemical compounds and lipid library Example: Identification of 5 oxo eicosatetraenoic acid (5 oxo ETE) as the ligand of hgpcr48 using the receptor fusion protein. Eur. J. Biochem. 255 (1998),

9 33 34 Summary G protein signaling system play important roles in sensing molecules outside the cell and activating inside signal transduction pathways, and, finally, cellular responses. Orphan GPCRs may be involved in explaining many unknown signaling pathway. GPCRs are involved in many diseases, and are also the target of around half of all modern medicinal drugs. Summary Efficacious ligand screening platform is not only for deorphaning but also for further drugdiscovery Thank you for your attention!!