Precoupling of alpha-2B adrenergic receptors and G-proteins in transfected PC-12 cell membranes: influence of pertussis toxin and a lysine-directed cross-linker.

The ability of pertussis toxin (PTX) pretreatment to alter the binding of [3H]rauwolscine (RAU) to alpha-2B adrenergic receptors expressed in PC12 cells was examined. PTX caused a 30% increase in the Bmax for [3H]RAU and reduced its KD, whereas in the added presence of Na+ and Gpp(NH)p binding was increased to 75% above the level in untreated membranes. Because all three agents act to reduce receptor/G-protein affinity, the increased binding may reflect extensive precoupling of the alpha-2B receptor. The affinity of the agonist epinephrine in displacing [3H]RAU was normally reduced by both Na+ and Gpp(NH)p; however, in PTX-treated membranes the effect of Gpp(NH)p was eliminated, and Na+ remained effective. The lysine-directed cross-linking reagent ethyleneglycol bis(succinimidyl)succinate (EGS) was utilized in an attempt to cross-link precoupled receptor and G-protein. Maximal [3H]RAU binding was reduced by EGS in a time- and dose-dependent manner, and this action was reversed by prior incubation with Na+ and Gpp(NH)p, suggesting that EGS did indeed cross-link receptor and G-protein. RAU and epinephrine each provided protection against the effect of EGS. The inclusion of Na+ and Gpp(NH)p during [3H]RAU binding studies was able to restore maximal binding in EGS-treated membranes to the same level as untreated membranes. These results indicate that in the absence of Na+ and Gpp(NH)p at least 40% of the total alpha-2B adrenergic receptors in these membranes exist as a precoupled receptor/G-protein complex which fails to bind [3H]RAU.