cAMP binds with high affinity and specificity to the androgen receptor from murine skeletal muscle

• Published in: Biochemistry (Easton) 1988-11, Vol.27 (23), p.8592-8598
• Main Authors: Ofenloch-Haehnle, Beatus, Eisele, Karl
• Format: Article
• Language: English
• Subjects: Animals; Biological and medical sciences; Cell receptors; Cell structures and functions; Centrifugation, Density Gradient; Chromatography, Affinity; Chromatography, Gel; Cyclic AMP - metabolism; Cytosol - metabolism; Fundamental and applied biological sciences. Psychology; Kinetics; Male; Mice; Mice, Inbred C57BL; Molecular and cellular biology; Muscles - metabolism; Receptors, Androgen - isolation & purification; Receptors, Androgen - metabolism; Testosterone - metabolism
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00423a013

cAMP binding of the androgen receptor (AR) from murine skeletal muscle was studied. Testosterone affinity chromatography yielded androgen receptor with about 4000-fold purification. Determination of the cAMP binding in the affinity eluate, by adsorption of protein-cAMP complexes to cellulose ester filters or removal of unbound cAMP by dextran-coated charcoal, was not possible, as the observed binding was not stable during the assays. Displacement studies suggest that this is due to a very fast dissociation kinetics of the binding. The problem could be solved by assaying the components of affinity eluate immobilized to a testosterone affinity resin that stabilizes the cAMP-protein complexes. The cAMP binding found in the affinity eluate shows an upward concave Scatchard plot and is compatible with a model containing two independent binding sites with dissociation constants of 7 and 58 nM. However, a larger number of binding sites or negative cooperativity cannot be excluded. Sixteen cAMP binding sites were observed per testosterone binding site. The binding affinity of cAMP exceeds that of cGMP 200-fold, that of cCMP 2000-fold, and that of AMP and 2',3'-cAMP more than 10,000-fold. Results indicate that cAMP is bound by the AR, although it only represents about 1% of the total protein in the affinity eluate: (i) Specific testosterone and cAMP binding of affinity eluate was copurified by affinity chromatography, density gradient centrifugation, and gel filtration. The ratio of cAMP to testosterone binding in each peak was about 16:1, identical with that found in the total affinity eluate.