ESTIMATION OF THE KINETIC CONSTANTS FOR BINDING OF EPINEPHRINE TO BETA-ADRENERGIC RECEPTORS OF THE S49 CELL

The dissociation constant (K(d)) for the binding of epinephrine to beta-adrenergic receptors of the S49 cell is 2-mu-M, which is the ratio of the rate constants for dissociation (k(off)) and association (k(on)), K(d) = k(off)/k(on). Although the K(d) is known by direct measurement, the individual rate constants k(on) and k(off) are unknown since they are both too large to be measured by conventional experimental methods. We present here an analysis in which a minimum value for these constants is calculated. The analysis uses a "transiently private receptor" model for coupling of receptors to G protein/adenylate cyclase that is based on the limits prescribed by the known empirical relationship between the beta-adrenergic receptor occupancy by epinephrine (characterized by K(d)) and their coupling to adenylate cyclase (characterized by EC50 = 10 nM) as a function of epinephrine concentration. The model makes only the assumption (based on previous evidence) that a receptor cannot activate more than one cyclase during the course of one cycle of binding and unbinding of an epinephrine molecule. In such a model, and with such a large separation between the K(d) and the EC50, the rate of G protein/adenylate cyclase activation by epinephrine-bound receptors can be related to the frequency of receptor binding at low concentrations of epinephrine, from which minimum values for the rate constant for association can be derived. This gives the estimates k(on) > 10(8)/M/min and k(off) > 280/min at 37-degrees. The on-rate constant is comparable to the on-rate constants that have been measured for other beta-adrenergic receptor ligands. This result supports the transiently private receptor coupling model as an explanation for the previously observed contribution of the frequency of epinephrine binding to the rate of activation of adenylate cyclase in the S49 cell.