title: Biased inhibition by a suramin analogue of A1-adenosine receptor/G protein coupling in fused receptor/G protein tandems: the A1-adenosine receptor is predominantly coupled to Goalpha in human brain.
abstract: The interface between receptors and G proteins can be considered as a drug target. Various classes of low molecular weight inhibitors have been identified that block the ability of receptors to interact with G proteins (e.g. peptides, suramin analogues and amphiphilic cations). Here we have tested if there are compounds that differentially affect the interaction of one receptor with two different (related) G protein alpha-subunits. Fusion proteins comprising the human A1-adenosine receptor and Galphai-1 (A1/Galphai-1) or Galphao (A1/Galphao) were expressed in HEK293 cells. Suramin analogues were screened for their ability to differentially affect high affinity binding of the agonist (-) N6-3-[125I](iodo-4-hydroxyphenylisopropyl) adenosine (IHPIA). One compound [NF326 = 8,8'-(carbonylbis-(imino-3,1-phenylenecarbonylimino))bis-(1-naphthol-3,6-disulfonic acid, disodium salt)] was identified that inhibited high affinity agonist binding to the fusion protein A1/Galphai-1 but modestly enhanced binding of IHPIA to A1/Galphao. This action was specific because NF326 did not affect antagonist binding to either fusion protein. In addition, it was unrelated to a difference in affinity of the receptor for the G protein fusion moiety because the stability of ternary complexes formed by IHPIA + A1/Galphai-1 and IHPIA + A1/Galphao) is comparable and because lowering the affinity of the receptor for the G protein (by introducing point mutations at cys351 of Galphai-1) enhanced the uncoupling effect of NF326. Finally, NF326 did not discriminate between a fusion protein comprising the alpha2A-adrenoceptor and Galphai-1 (alpha2A)/Galphai-1) or Galphao-1 (alpha2A)/Galphao-1); binding of the agonist [3H]UK14304 (bromoxidine) to both fusion proteins was inhibited over a comparable concentration range while binding of the antagonist [3H]yohimbine was unaffected. These observations are consistent with the interpretation that the contact sites that are formed between individual receptors and G proteins differ. These differences suffice to allow for selective disruption by G protein inhibitors of different classes. Using NF326 we show that the bulk of the A1-adenosine receptors in human cerebrocortical membranes interacts with Galphao rather than Galphai.
authors.affiliation: Institute of Pharmacology; University of Vienna, Währinger Strasse 13a, 1090 Vienna, Austria.
authors.forename: Oliver
authors.initials: O
authors.lastname: Kudlacek
authors.forename: Maria
authors.initials: M
authors.lastname: Waldhoer
authors.forename: Matthias U
authors.initials: MU
authors.lastname: Kassack
authors.forename: Peter
authors.initials: P
authors.lastname: Nickel
authors.forename: Jussi I
authors.initials: JI
authors.lastname: Salmi
authors.forename: Michael
authors.initials: M
authors.lastname: Freissmuth
authors.forename: Christian
authors.initials: C
authors.lastname: Nanoff
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D001921:Brain / Q000378:metabolism*; D019556:COS Cells; D004305:Dose-Response Relationship, Drug; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D019204:GTP-Binding Proteins / Q000378:metabolism*; D020962:Heterotrimeric GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D058915:Purinergic P1 Receptor Antagonists; D018047:Receptors, Purinergic P1 / Q000378:metabolism*; D013498:Suramin / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology*; D013552:Swine
chemical_list: D000970:Antineoplastic Agents; D058915:Purinergic P1 Receptor Antagonists; D018047:Receptors, Purinergic P1; D013498:Suramin; D019204:GTP-Binding Proteins; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D020962:Heterotrimeric GTP-Binding Proteins