Interactions of 16alpha,17alpha-Cyclohexane Derivatives of Progesterone with the Progesterone Receptor from Rat Uterus

A. N. Smirnov^1*, E. V. Pokrovskaya¹, V. P. Shevchenko², I. S. Levina³, and A. V. Kamernitzky³

¹Laboratory of Endocrinology, School of Biology, Lomonosov Moscow State University, Moscow, 119899 Russia; fax: (095) 939-4309

²Institute of Molecular Genetics, Russian Academy of Sciences, pl. Kurchatova 45, Moscow, 123182 Russia; fax: (095) 196-0221; E-mail: img@glas.apc.org

³Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Leninskii pr. 47, Moscow, 117913 Russia; fax: (095) 135-5328; E-mail: 1220@syearn2.bitnet

^* To whom correspondence should be addressed.

Received January 5, 1998; Revision received April 2, 1998

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Pregna-D´-pentaranes, 16alpha,17alpha-cyclohexanoprogesterone and 6alpha-methyl-16alpha,17alpha-cyclohexanoprogesterone, were found to specifically interact with the progesterone receptor of soluble fraction from rat uterus. The formation of complexes between ³H-labeled derivatives of these steroids and the protein was complete within 1 to 3 h at 0-4°C. The dissociation of these complexes was a two-phase process, the contribution of the fast dissociating complexes decreasing with increasing preincubation time. The dissociation constant (k_-1) values for progesterone, 16alpha,17alpha-cyclohexanoprogesterone, and 6alpha-methyl-16alpha,17alpha-cyclohexanoprogesterone complexes with the protein after 1 h preincubation were 6.5 ± 0.8, 8.8 ± 5.5, and (16.6 ± 5.6)·10^-4 sec^-1 for the fast phase and 5.1 ± 0.5, 3.5 ± 0.8, and (2.8 ± 0.6)·10^-5 sec^-1 for slow phase, respectively. The equilibrium K_d values were 11.7 ± 2.1, 19.0 ± 2.0, and 66.1 ± 14.6 nM for progesterone, 16alpha,17alpha-cyclohexanoprogesterone, and 6alpha-methyl-16alpha,17alpha-cyclohexanoprogesterone, respectively. The steroids mutually inhibited the binding of their ³H-labeled derivatives to the protein, the inhibition being of competitive type. In the case of [³H]6alpha-methyl-16alpha,17alpha-cyclohexanoprogesterone, the inhibitory efficacy of progesterone declined with an increase of its concentration; this points to possible heterogeneity of binding sites for the ³H-labeled ligand. The comparison of the results with those obtained by us earlier (Biochemistry (Moscow), 1996, 61, 1034-1041) suggests the existence of significant species differences in progesterone receptor structure within or near the region that interacts with the D-ring of a hormone molecule.

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KEY WORDS: progesterone receptor, progesterone analogs, binding specificity, binding kinetics

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