Multiple affinity states of opiate receptor in neuroblastoma x glioma NG108-15 hybrid cells. Opiate agonist association rate is a function of receptor occupancy

The existence of multiple affinity states for the opiate receptor in neuroblastoma x glioma NG108-15 hybrid cells has been demonstrated by competition binding studies with tritiated diprenorphine and [D-Ala²,D-Leu⁵]enkephalin (DADLE). In the presence of 10 mM Mg²⁺, all receptors exist in a high affinity state with K(d) = 1.88 ± 0.16 nM. Addition of 10 μM guanyl-5'-yl imidodiphosphate (Gpp(NH)p) decreased the affinity of DADLE to K(d) = 8.08 ± 0.93 nM. However, in the presence of 100 mM Na⁺, which is required for opiate inhibition of adenylate cyclase activity, analysis of competition binding data revealed three sites: the first, consisting of 17.5% of total receptor population has a K(d) = 0.38 ± 0.18 nM; the second, 50.6% of the population, has a K(d) = 6.8 ± 2.2 nM; and the third, 31.9% of the population, has a K(d) of 410 ± 110 nM. Thus, in the presence of sodium, a high affinity complex between receptor (R), GTP binding component (N(i)), and ligand (L) was formed which was different from that formed in the absence of sodium. These multiple affinity states of receptor in the hybrid cells are agonist-specific, and the percentage of total opiate receptor in high affinity state is relatively constant in various concentrations of Na⁺. Multiple affinity states of opiate receptor can be demonstrated further by Scatchard analysis of saturation binding studies with [³H]DADLE. In the presence of Mg²⁺, or Gpp(NH)P, analysis of [³H]DADLE binding demonstrates that opiate receptor can exist in a single affinity state, with apparent K(d) values of [³H]DADLE in 10 mM Mg²⁺ = 1.75 ± 0.28 nM and in 10 μM Gpp(NH)p = 0.85 ± 0.12 nM. There is a reduction of B(max) value from 0.19 ± 0.02 nM in the presence of Mg²⁺ to 0.14 ± 0.03 nM in the presence of Gpp(NH)p. In the presence of 100 mM Na⁺, Scatchard analysis of saturation binding of [³H]DADLE reveals nonlinear plots; two-site analysis of the curves yields K(d) = 0.43 ± 0.09 and 7.9 ± 3.2 nM. These K(d) values are analogous to that obtained with competition binding studies. Again, this conversion of single site binding Scatchard plots to multiple sites binding plots in the presence of Na⁺ is restricted to ³H-agonist binding only. [³H]Diprenorphine binding is not affected by varying the conditions of binding. The apparent increase in ³H-agonist binding with concomitant decrease in the B(max) values of high affinity sites suggests that agonist association rate is a function of receptor occupancy, which is supported by dissociation and association rates studies. Hence, the formation of the ligand-receptor complex with GTP binding component and the dissociation of the complex during opiate regulation of adenylate cyclase activity results in a decrease in opiate affinity with respect to receptor occupancy.