Abstract
In Dictyostelium discoideum cells, extracellular cAMP induces the rapid (within 2 s) activation of guanylate cyclase, which is followed by complete desensitization after about 10 s. cAMP binding to these cells is heterogeneous, showing a subclass of fast dissociating sites coupled to adenylate cyclase (A-sites) and a subclass of slowly dissociating sites coupled to guanylate cyclase (B-sites). The kinetics of the B-sites were further investigated on a seconds time scale. Statistical analysis of the association of [3H]cAMP to the B-sites and dissociation of the complex revealed that the receptor can exist in three states which interconvert according to the following scheme. (formula; see text). cAMP binds to the BF-state (off-rate 2.5 s) which rapidly (t1/2 = 3 s) converts to the BS-state (off-rate 15 s) and subsequently (without a detectable delay) into the BSS-state (off-rate 150 s). In membranes, both the BS- and BSS-states are converted to the BF-state by GTP and GDP, suggesting the involvement of a G-protein. Densensitized cells show a 80% reduction of the formation of the BSS-state, but no reduction of the BF- or BS-state. These data are combined into a model in which the transitions of the B-sites are mediated by a G-protein; activation of the G-protein and guanylate cyclase is associated with the transition of the BS- to the BSS-state of the receptor, whereas desensitization is associated with the inhibition of this transition.
Highlights
From the$Cell Biology and Morphgenesis Unit,Zoological Laboratory, and the qDepartment of Physiology and Physiological Physics, University of Leiden, 2300 RA Leiden and the §Laboratory of Biochemistry, University of Amsterdam, 1000 BH Amsterdam, The Nether1and.s
Prolonged stimulation of D. discoideum cells with constant cAMP concentrations induces desensitization by at least two mechanisms: (i) down-regulation of cAMP binding activity after a long incubation (5-30 min) with high cAMP concentrations (1-100 pM) (after removal of CAMP,cells resensitize with a half-life of about 60 min [6, 7]); (ii) a rapid desensitization of the CAMP-mediated activation of the cyclases by nanomolar cAMP concentrations [8, 9] without a loss of CAMP-bindingsites (after removal of CAMP,cells resensitize with a half-life of 1-2 min for the guanylate cyclase [9] and
We observed a class of fast dissociating sites (A-sitews ith tM= 1-2 s) that are guanine nucleotide-sensitive and that are presumably responsible for the activation of adenylate cyclase by extracellular CAMP
Summary
Material~-[2,8-~H]cAMP(1.5 TBq/mmol) was obtainedfrom The Radiochemical Centre, Amersham, United Kingdom. Binding of cAMP to all siteswas measured by centrifugation of 95 pl of the incubation mixture through silicon oil (AR 200AR 20 = 1:2). Cells were incubated with 10 nM [3H]cAMPas described above and were centrifuged through silicon oil at the moment of binding equilibrium, a t 10s, or a t 2 min after the onset of dissociation. It should be noted that cAMP does accelerate the release of bound [3H]cAMPwhen cells (or membranes prepared from them) were used which were starved insuspension for an unphysiologicallylong period (7-9 h). The release of bound [3H]cAMPas a function of time was fitted to sums-of-exponentials models To this end, the mean (2:) of the replicas at time ti was used as the single value, and thesample variance [3]of the replicas was divided by their number ni as weights. The degrees of freedom of F are ( p z - p1, N - p z )
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