Biased inhibition by a suramin analogue of A(1)-adenosine receptor/G protein coupling in fused receptor/G protein tandems: the A(1)-adenosine receptor is predominantly coupled to Go(alpha) in human brain

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 A(1)-adenosine receptor and Galpha(i-1) (A(1)/Galpha(i-1)) or Galpha(o) (A(1)/Galpha(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-[I-125](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 A(1)/Galpha(i-1) but modestly enhanced binding of IHPIA to A(1)/Galpha(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 + A(1)/Galpha(i-1) and IHPIA + A(1)/Galpha(o) is comparable and because lowering the affinity of the receptor for the G protein (by introducing point mutations at cys(351) of Galpha(i-1)) enhanced the uncoupling effect of NF326. Finally, NF326 did not discriminate between a fusion protein comprising the alpha(2A)-adrenoceptor and Galpha(i-1) (alpha(2A)/Galpha(i-1)) or Galpha(o-1) (alpha(2A)/Galpha(o-1)); binding of the agonist [H-3]UK14304 (bromoxidine) to both fusion proteins was inhibited over a comparable concentration range while binding of the antagonist [H-3]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 A(1)-adenosine receptors in human cerebrocortical membranes interacts with Galpha(o) rather than Galpha(i).