Cyclic AMP receptors and cation fluxes in the turkey erythrocyte
Rudolph, S.A.; Baird, T.M.; Wardell, J.W.
Molecular Pharmacology 21(2): 503-510
1982
ISSN/ISBN: 0026-895X PMID: 7099149 Document Number: 193383
Both membrane-bound and soluble cAMP receptors were found in turkey erythrocytes that may be related to catecholamine-dependent ion fluxes in these cells. To distinguish physiologically relevant cAMP receptors from nonspecific binding sites, the binding of 3H-cAMP to membranes and cytosol prepared from control cells and from (-)-isoproterenol-pretreated cells was measured. Fractions prepared from isoproterenol-treated cells bound less 3H-cAMP than did fractions prepared from untreated cells. The difference in binding is considered specific, since that difference reflects sites that are occupied endogenously in response to isoproterenol. Maximal specific binding was .apprx. 400 fmol/mg of membrane protein for the membrane-bound receptors. Binding was half-maximal at 20 nM 3H-cAMP. Specific binding of 3H-cAMP occurred rapidly at 0.degree. C with a half-time of .apprx. 2 min. cAMP binding sites in the membranes prepared from isoproterenol-treated cells remained occupied for at least 90 min if the membranes were kept at 0.degree. C. If the membranes were heated to 37.degree. C for 20 min prior to measuring 3H-cAMP binding, no difference was observed between membranes prepared from control and isoproterenol-treated cells. The effect of isoproterenol on 3H-cAMP binding was half-maximal at 30 nM, as was the effect of isoproterenol on cation influx. The dose-response curves for the 2 processes were superimposable, suggesting that the cAMP binding sites may be the cAMP receptors responsible for the nucleotide's physiological effect in these cells. The effect of 30 nM (-)-isoproterenol on cAMP receptor occupancy was half-maximal within 1 min, whereas the effect on cation influx required .apprx. 3 min for a half-maximal response, suggesting that some step distal to cAMP receptor occupancy may be rate-limiting. Using the photoaffinity label 32P-8-N3-cAMP, > 95% of the membrane-bound radioactivity and > 90% of the cytosol-bound radioactivity were found in a 48,000 MW protein (by sodium dodecyl sulfate-polyacrylamide gel electrophoresis). This protein is presumably the regulatory subunit of a Type I cAMP-dependent protein kinase.