title: Characterization and regulation of prolactin receptors in MA-10 Leydig cells.
abstract: The aim of this study is to further characterize the prolactin receptors (PRL-R) previously reported in the murine Leydig tumor MA-10 cell line, as well as to study their homologous and heterologous regulation. Two forms of PRL-R, a high and a low molecular weight form, were revealed by studies of covalent crosslinking of 125I-human GH to cultured MA-10 cells or cell membranes and immunoprecipitation of the solubilized PRL-R complexes with polyclonal anti PRL-R antibody, followed by SDS-PAGE and autoradiography. The long form had a molecular weight of 101 kDa and was predominant when the study was performed in the presence of protease inhibitors. The short form, with a molecular weight of 39 kDa, appeared, at least in part, to be a proteolytic product of the longer form. The same size forms of PRL-R were detected by crosslinking studies in the parental C57BL/6 mouse testicular Leydig cells, indicating the physiological relevance of the MA-10 cell model to the study of Leydig cell PRL-R. Homologous down-regulation of PRL-R was demonstrated in cultured MA-10 cells exposed for 24 h to increasing concentrations of PRL. In contrast, heterologous, 3 5-fold up-regulation of PRL-R was induced by various cAMP-elevating agents, including 8-bromo-cAMP (10(-4) -10(-3) M), dibutyryl cAMP (3 x 10(-3) M) and cholera toxin (1-10 ng/ml), although not by hCG (up to 100 ng/ml). This up-regulatory effect was apparently the result of a change in affinity, since cholera toxin caused a 2.4-fold increase in PRL-R affinity, with no change in the number of binding sites. In summary, these studies provide further evidence that MA-10 Leydig cells can serve as a physiologically relevant model for the study of PRL and PRL-R interactions, both at the functional level, as shown in our previous study, and at the structural and regulatory levels as shown in the current study.
authors.affiliation: Department of Pharmacology, The Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa.
authors.forename: E
authors.initials: E
authors.lastname: Weiss-Messer
authors.forename: R
authors.initials: R
authors.lastname: Ber
authors.forename: T
authors.initials: T
authors.lastname: Amit
authors.forename: R J
authors.initials: RJ
authors.lastname: Barkey
mesh_terms: D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000818:Animals; D003994:Bucladesine / Q000494:pharmacology; D002772:Cholera Toxin / Q000494:pharmacology; D000242:Cyclic AMP / Q000819:agonists; D007985:Leydig Cells / Q000378:metabolism*; D008297:Male; D051379:Mice; D011388:Prolactin / Q000378:metabolism* / Q000494:pharmacology; D011981:Receptors, Prolactin / Q000819:agonists / Q000737:chemistry / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D015854:Up-Regulation
chemical_list: D011981:Receptors, Prolactin; D015124:8-Bromo Cyclic Adenosine Monophosphate; D003994:Bucladesine; D011388:Prolactin; D002772:Cholera Toxin; D000242:Cyclic AMP