SPECIES-DIFFERENCES IN A(1) ADENOSINE RECEPTOR G PROTEIN COUPLING - IDENTIFICATION OF A MEMBRANE-PROTEIN THAT STABILIZES THE ASSOCIATION OFTHE RECEPTOR/G PROTEIN COMPLEX/

Reconstitution experiments with purified components reproduce the basi c characteristics of receptor/G protein coupling, i.e., GTP-sensitive high affinity agonist binding and receptor-promoted GTP binding. Howev er, the interaction of agonists with the A, adenosine receptor in rat and bovine but not human brain membranes deviates from the ternary com plex model since the agonist/receptor/G protein complex cannot be diss ociated by high concentrations (greater than or equal to 100 mu M) of the hydrolysis-resistant analogue GTP gamma S. The reason for this phe nomenon referred to as a ''tight coupling mode'' has remained enigmati c. We show that it is attributable to a distinct membrane protein, whi ch we labeled the coupling cofactor. Extraction of the protein from ra t brain membranes with the detergent 3-(cholamidopropyl)diamethylammon io]-1-propanamium increased the potency of GTP gamma S by 1000-fold. A fter extraction, the potency was comparable to that in human brain mem brane. Detergent extracts from rat brain membranes were used to resolv e the component from solubilized receptors and G protein alpha and bet a gamma subunits by sequential DEAE-Sephacel chromatography and Supero se gel filtration (molecular weight of similar to 150 kDa in (cholamid opropyl)diamethylammonio]-1-propanamium). Coupling cofactor restored g uanine nucleotide refractoriness in a concentration-dependent manner t o both detergent-extracted rat brain membranes and, albeit with lower affinity, human brain membranes. However, in human brain extracts, cof actor activity was detectable on reconstitution with rat acceptor memb ranes, indicating an intrinsic difference between rat and human recept ors in their ability to interact with the cofactor. With high amounts of coupling cofactor present, GTP gamma S no longer decreased but rath er increased agonist affinity. Readdition of partially purified coupli ng cofactor to acceptor membranes reduced the rate of A(1) adenosine r eceptor-mediated G protein turnover. These observations show that the component identified traps the ternary agonist/receptor/G protein comp lex in a stable conformation, impedes signaling of the A(1) adenosine receptor, and thereby regulates the level of signal amplification.