title: Radioligand assays for oestradiol and progesterone conjugated to protein reveal evidence for a common membrane binding site in the medial preoptic area-anterior hypothalamus and differential modulation by cholera toxin and GTPgammaS.
abstract: In this study membrane oestradiol (E) binding sites in the medial preoptic area-anterior hypothalamus (MPOA-AH) of ovariectomized (OVX) rats were characterized using standard radioligand binding techniques employing E conjugated to bovine serum albumin (BSA) at position 6 and radiolabeled with 125I (E-6-[125I-BSA]). In previous studies binding of a radioactive conjugate of progesterone (P) and BSA (P-3-[125I-BSA]) was examined using the same membrane preparation. E-6-[125I-BSA] binding was linear across a tissue concentration range of 0.005-0.02 mg protein/0.1 ml of membrane suspension. An association T1/2 of 9.5 min and a dissociation T1/2 of 52.1 min for E-6-[125I-BSA] were derived from kinetic experiments. Competition binding experiments revealed high (Ki=0.63+/-(0.50 nM) and low (Ki=161.5(96.5 nM) affinity binding sites for E-6-[125I-BSA], demonstrating different binding parameters than shown in our previous work for P-3-[125I-BSA] binding. Further studies on MPOA-AH membranes treated with cholera toxin (CTX) and GTPgammaS suggested that E-6-BSA binding sites are associated with G proteins. E-6-[125I-BSA] binding demonstrated both high-and low-affinity sites. GTPgammaS added to the assay reduced both E-6-[125I-BSA] and P-3-[125I-BSA] binding suggesting that G proteins are associated with both binding sites. Extensive analysis of both E-6-[125I-BSA] and P-3-[125I-BSA] binding sites demonstrated a reciprocal relationship such that high-affinity E-6-[125I-BSA] binding sites exhibit low affinity for P-3-[125I-BSA] and low-affinity E-6-[125I-BSA] binding sites exhibit high affinity for P-3-[125I-BSA]. Preincubating membranes with CTX or GTPgammaS reduced high-affinity E-6-[125I-BSA] binding and enhanced high-affinity P-3-[125I-BSA] binding. These results suggest that, in the MPOA-AH, membrane steroid binding sites exist in two interconvertible conformations that preferentially bind either E-6-BSA or P-3-BSA, depending on their association with a G protein. Additional studies with free steroids revealed that: (1) oestrogens (17beta-oestradiol, diethylstilbestrol) as well as synthetic oestrogen antagonists tamoxifen and ICI 182 780 displaced P-3-[125I-BSA] further suggesting a relationship between membrane binding sites for E and P-3-[125I-BSA] binding sites; and (2) treatment of OVX rats with E decreased displacement by P-3-BSA and increased displacement by ICI 182,780 and tamoxifen suggesting these antagonists affect membrane P-3-[125I-BSA] binding sites after in-vivo E treatment. The membrane binding sites for E and P demonstrate interrelationships not demonstrated by their nuclear receptors.
authors.affiliation: Department of Pharmaceutical Sciences, North Dakota State University, Fargo, ND 58105-5055, USA. jcaldwell@plains.nodak.edu
authors.forename: J D
authors.initials: JD
authors.lastname: Caldwell
authors.forename: C H
authors.initials: CH
authors.lastname: Walker
authors.forename: A
authors.initials: A
authors.lastname: Rivkina
authors.forename: C A
authors.initials: CA
authors.lastname: Pedersen
authors.forename: G A
authors.initials: GA
authors.lastname: Mason
mesh_terms: D000818:Animals; D001665:Binding Sites / Q000187:drug effects; D001667:Binding, Competitive / Q000187:drug effects; D002772:Cholera Toxin / Q000494:pharmacology*; D004958:Estradiol / Q000378:metabolism*; D005260:Female; D019204:GTP-Binding Proteins / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology*; D007032:Hypothalamus, Anterior / Q000187:drug effects / Q000378:metabolism; D007700:Kinetics; D008566:Membranes / Q000187:drug effects / Q000378:metabolism; D010052:Ovariectomy; D011301:Preoptic Area / Q000187:drug effects / Q000378:metabolism; D011374:Progesterone / Q000378:metabolism*; D011485:Protein Binding; D011869:Radioligand Assay; D051381:Rats; D017207:Rats, Sprague-Dawley; D012711:Serum Albumin, Radio-Iodinated
chemical_list: D012711:Serum Albumin, Radio-Iodinated; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D011374:Progesterone; D004958:Estradiol; D002772:Cholera Toxin; D019204:GTP-Binding Proteins