Identification and cyclic AMP-induced modification of the cyclic AMP receptor in Dictyostelium discoideum.
We have recently identified a cell surface cAMP-binding protein by specific photoaffinity labeling of intact Dictyostelium discoideum cells with 8-N3-[32P] cAMP. The major photolabeled protein appears as a doublet (Mr = 40,000-43,000) in sodium dodecyl sulfate-polyacrylamide gel electrophoresis autoradiography. In this study, the doublet is shown to have the characteristics of the cAMP receptor responsible for chemotaxis and cAMP signaling. Both specific photoaffinity labeling of the doublet and binding of 8-N3-[32P]cAMP are saturable (KD = 0.3 microM), the levels of both peak at 5 h, and both are inhibited by cAMP and several cAMP analogs in the same order of potency and with K1 values similar to those measured for inhibition of [3H]cAMP binding. When cAMP-binding activity was partially purified (40-fold) and then photoaffinity labeled, the same bands (Mr = 40,000-43,000) were observed. The relative intensities of the upper and lower bands of the doublet alternated at the same frequency as the spontaneous oscillations in cAMP synthesis. When oscillations were suppressed, the lower band of the doublet predominated. Following addition of cAMP, the relative intensity gradually shifted to the upper band. When cAMP was removed, there was a gradual restoration of the lower band form. We propose that the lower band form of the receptor activates chemotaxis and cAMP signaling and that the upper band form does not. This reversible receptor modification may then be the mechanism of adaptation, the process by which the physiological responses cease to be stimulated by persistent cAMP. Several developmentally regulated genes in D. discoideum have been reported to be induced or suppressed by pulses of cAMP (adaptive regulation) and others by continuous cAMP (nonadaptive regulation). These observations may be explained by the receptor modification reported here if the two forms of the receptor, which bind cAMP with the same affinity, independently influence gene expression.
Klein
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P
Theibert
A
A
Fontana
D
D
Devreotes
P N
PN
D000345:Affinity Labels / Q000378:metabolism; D001386:Azides / Q000378:metabolism; D001667:Binding, Competitive; D002633:Chemotaxis; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology*; D004023:Dictyostelium / Q000254:growth & development / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D010777:Photochemistry; D011953:Receptors, Cyclic AMP / Q000187:drug effects / Q000378:metabolism*
D000345:Affinity Labels; D001386:Azides; D011953:Receptors, Cyclic AMP; C017042:8-azidoadenosine-3',5'-monophosphate; D000242:Cyclic AMP