The cyclic
adenosine 3':5'-monophosphate (cAMP)-
binding proteins of dysplastic (control) and neoplastic human breast tissue cytosols were investigated after photoaffinity labeling with 8-azido-cyclic
adenosine 3':5'-[32P]monophosphate (8-N3-cAMP) by
sodium dodecyl sulfate/
polyacrylamide gel electrophoresis. Four main
binding proteins, all specific for cAMP, were identified, with molecular weights of 52,000, 49,000, 39,000, and 37,000. According to their molecular weights, elution on
diethylaminoethyl cellulose, and in vitro phosphorylation, the Mr 49,000 and 52,000 species correspond to the regulatory subunits (R-I, R-II) of
cAMP-dependent protein kinases types I and II. The smaller
cAMP receptors (Mr 39,000 and 37,000) are proteolytic fragments of the intact R-
proteins. Dissociation constants (Kd) with
8-N3-cAMP of 0.8 nM for R-I, and 0.12 microM for R-II were obtained; the proteolytic fragments exhibited Kd's similar to that of R-I. No difference in the
8-N3-cAMP affinities and labeling efficiencies was found between control and neoplastic tissues. Although the average incorporation of
8-N3-cAMP was 0.29 +/- 0.02 (S.E.) pmol/mg
protein for control and 0.45 +/- 0.06 pmol/mg
protein for neoplastic breast tissue cytosol, this difference does not reflect different cellular concentrations of
cAMP receptors since the content of
blood protein components is lower in
tumor tissue. However,
tumor cytosols exhibited an increased content of proteolytic R-fragments, and the ratio of intact
cAMP receptors versus proteolyzed R-
proteins was significantly (p less than 0.01) higher in control (8.3 +/- 0.9) than in
tumor (3.0 +/- 0.5) tissue. The average R-I/R-II ratio was greater than 1 in each case, but no significant difference was observed between control and neoplastic tissue. Inverse relationships were obtained, especially between proteolyzed R-fragments and
estrogen receptors, when the contents and ratios of cAMP-
binding proteins were correlated with the contents of
estrogen and
progesterone receptors in
tumor tissue by a Spearman rank correlation coefficient r = -0.55 (significance of difference from zero being p less than 0.01).