Ligand–receptor interactions: Detailed evaluation of occupancy‐dependent affinity

The exact nature of the curvilinearity of Scatchard plots derived from hormonal and nonhormonal binding systems has not been definitively resolved. Such plots are compatible with heterogeneous receptors with different but fixed affinities and with negatively interacting binding sites resulting in occupancy‐dependent affinity. In the current study we examined in detail the effect of receptor occupancy by the ligand on receptor affinity under a variety of experimental conditions. We chose the human lymphocyte–leukoagglutinin (LPHA) system, which closely mimics the IM9‐insulin model. Reliable estimates of total binding capacity (728 ng/10 6 cells) essential to our report were calculated from a wide database by the least‐squares model. At occupancies ≥ 0.085, receptors are associated with low and fixed affinity (1.5 × 10 6 M −1 ), whereas at occupancies ⩽ 0.085, affinity is high and fixed (1.8 × 10 8 M −1 ) or high but variable (1 × 10 7 M −1 to 1.5 × 10 6 M −1 ) depending on whether the binding is assumed to be noncooperative or cooperative, respectively. Calculation of receptor–ligand complex dissociation velocity over a wide range of occupancies (0.01–0.40) suggested that occupancy exerts an inversely proportional effect on affinity that is rapid and sustained. Cell activation (DNA synthesis) is initiated at receptor occupancy of ≅ 0.004 and is magnified as ligand binding to high affinity receptors increases up to ≅ 0.07 occupancy (functional sites), beyond which point further binding (to low affinity sites) becomes increasingly ineffective and cytotoxic (redundant sites). These findings suggest that occupancy influences affinity as postulated by the hypothesis of negative cooperativity. Through this effect occupancy may play a significant role in regulating ligand‐induced cell responses.