Properties of prostaglandin F2alpha receptors in bovine corpus luteum cell membranes.
The specific binding of 3H-labeled prostaglandin (PG) F2alpha to bovine corpus luteum cell membranes was a rapid (K1=1.1 X 10(4) M(-1)S(-1) and reversible (K(-1)=3.3 X 10(-4) S(-1)) process at 22 degrees C. The specific binding was also a saturable process exhibiting two classes of receptors with apparent dissociation constants (Kds) of 1.6 X 10(-9) M and 2.4 X 10(-8) M. The heterogenous nature of [3H]PGF2alpha binding does not appear to be due to negative cooperatively 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 Kds of high and low affinity receptors, respectively. The binding of [3H]PGF2alpha to the membranes was not accompanied by any detectable changes in receptor-bound or free [3H]PGF2alpha. Addition of increasing amounts of unlabeled PGF2alpha resulted in a dose-dependent inhibition of [3H]PGF2alpha binding to the membranes, with complete inhibition occurring at 10(-6) M. Other unlabeled PGs such as PGF1alpha, PGE2 (5-fold), PGE1 (120-fold), PGA1 and PGB1 (about 10,000-fold) were less effective when compared to unlabeled PGF2alpha in inhibiting [3H] PGF2alpha binding to the membranes. The metabolites of PGF2alpha, 15-keto-PGF2alpha and 13,14-dihydro-15-keto-PGF2alpha had 100-fold less affinity for PGF2alpha receptors. 15(S)15-Methyl-PGF2alpha, an analogue of PGF2alpha, 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 PGF2alpha receptors. Incubation of membranes with various enzymes revealed that PGF2alpha 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]PGF2alpha 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.
D000818:Animals; D001667:Binding, Competitive; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D003338:Corpus Luteum / Q000378:metabolism*; D005260:Female; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008565:Membrane Proteins / Q000378:metabolism; D010740:Phospholipases; D010743:Phospholipids / Q000494:pharmacology; D011460:Prostaglandins F / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000378:metabolism*; D011982:Receptors, Prostaglandin / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013696:Temperature
D008565:Membrane Proteins; D010743:Phospholipids; D011460:Prostaglandins F; D011956:Receptors, Cell Surface; D011982:Receptors, Prostaglandin; D010740:Phospholipases