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 Go_α 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 α-subunits. Fusion proteins comprising the human A₁-adenosine receptor and Gα_i-1 (A₁/Gα_i-1) or Gα_o (A₁/Gα_o) were expressed in HEK293 cells. Suramin analogues were screened for their ability to differentially affect high affinity binding of the agonist (-)N ⁶-3-[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/Gα_i-1 but modestly enhanced binding of IHPIA to A₁/Gα_o. 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/Gα_i-1 and IHPIA + A₁/Gα_o is comparable and because lowering the affinity of the receptor for the G protein (by introducing point mutations at cys³⁵¹ of Gα_i-1) enhanced the uncoupling effect of NF326. Finally, NF326 did not discriminate between a fusion protein comprising the α_2A-adrenoceptor and Gα_i-1 (α_2A/Gα_i-1) or Gα_o-1 (α_2A/Gα_o-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 Gα_o rather than Gα_i.