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Kinetics and concentration dependence of reversible cAMP-induced modification of the surface cAMP receptor in
Abstract:
We have previously reported that extracellular cAMP induced a reversible shift, from apparent Mr = 40,000 to 43,000, in the electrophoretic mobility of a polypeptide identified by photoaffinity labeling with [32P]8-N3-cAMP as the cAMP receptor of Dictyostelium (Klein, P., Theibert, A., Fontana, D., and Devreotes, P. (1985) J. Biol. Chem. 260, 1757-1764). In this report, we examine the kinetics and concentration dependence of this stimulus-induced receptor modification. Prior to stimulation, 90% of the receptors migrated as the higher mobility form (Mr = 40,000) and 10% as the lower mobility form (Mr = 43,000). Following 15 min of persistent stimulation with 1 microM cAMP, the per cent of receptors migrating as the lower mobility form rose to 80%. This transition occurred with a half-time of 2.5 min. Removal of the stimulus initiated a return to the basal state which occurred with a half-time of about 6 min at 22 degrees C. No reversal occurred at 0 degrees C. Addition and removal of a 50 nM cAMP stimulus induced transitions with similar kinetics, but the final plateau value reached was only 40% lower mobility form. The stimulus concentration which induced 50% of the maximal transition from higher to lower mobility forms at steady state was 27 nM, similar to the KD for [3H]cAMP binding. Scatchard analysis of [3H]cAMP binding indicated that, although a 20% down-regulation occurs during cAMP stimulation, there is no significant difference in the affinities of the higher and lower mobility forms of the receptor. The unoccupied higher and lower mobility forms of the receptor, designated R and D, are considered to be in rapid equilibrium with liganded forms, designated RL and DL. The rate constants for interconversion of the receptor forms R (Formula: see text) D and RL (Formula: see text) DL were calculated from the kinetic data: k1 = 0.012, k-1 = 0.104, k2 = 0.222, and k-2 = 0.055. The interconversion steps are not at equilibrium, suggesting that an energy expenditure occurs during the receptor modification. The pattern of modulation of the cAMP-induced receptor modification suggests that it may be the biochemical mechanism of adaptation.