Properties of prostaglandin F 2α receptors in bovine corpus luteum cell membranes

Abstract

The specific binding of 3H-labeled prostaglandin (PG) F 2α to bovine corpus luteum cell membranes was a rapid ( K 1 = 1.1 × 10 4 M −1 s −1) and reversible ( K −1 = 3.3 × 10 −4 s −1) process at 22°C. The specific binding was also a saturable process exhibiting two classes of receptors with apparent dissociation constants ( K d s) of 1.6 × 10 −9 M and 2.4 × 10 −8 M. The heterogenous nature of [ 3H]PGF 2α binding does not appear to be due to negative cooperativity but merely to represent the existence of two independent groups of receptor sites with discrete affinities. Free energy changes of +11.9 and + 10.3 Kcal/mol were calculated from the K d s of high and low affinity receptors, respectively. The binding of [ 3H]PGF 2α to the membranes was not accompanied by any detectable changes in receptor-bound or free [ 3H] PGF 2α. Addition of increasing amounts of unlabeled PGF 2α resulted in a dose-dependent inhibition of [ 3H]PGF 2α binding to the membranes, with complete inhibition occurring at 10 −6 M. Other unlabeled PGs such as PGF 1α, PGE 2 (5-fold), PGE 1 (120-fold), PGA 1 and PGB 1 (about 10,000-fold) were less effective when compared to unlabeled PGF 2α in inhibiting [ 3H]PGF 2α binding to the membranes. The metabolites of PGF 2α, 15-keto-PGF 2α and 13,14-dihydro-15-keto-PGF 2α had 100-fold less affinity for PGF 2α receptors. 15(S)15-Methyl-PGF 2α, an analogue of PGF 2α, had a fairly high affinity but lower than its parent molecule. Various unsaturated fatty acids, indomethacin and 7-oxa-13-prostynoic acid had 3,000- to 10,000-fold less affinities for PGF 2α, receptors. Incubation of membranes with various enzymes revealed that PGF 2α receptor molecules are protein in nature which require membrane lipids and specific phospholipids for binding function. Among the various phospholipids used, sphingomyelin was found to be very effective in restoring the loss of [ 3H]PGF 2α binding in phospholipase C-treated membranes. N- Ethylmaleimide, but not other SH group alkylating agents inhibited binding. The binding was also inhibited by tetranitromethane, dinitrofluorobenzene and acetic anhydride. This suggested that tyrosyl, histidyl, tryptophan and amino (any one or all of them) but not SH groups were involved in binding interaction.