Characterization and regulation of prolactin receptors in MA-10 Leydig cells

• Published in: Molecular and cellular endocrinology 1998-08, Vol.143 (1), p.53-64
• Main Authors: Weiss-Messer, Esther, Ber, Rosalie, Amit, Tamar, Barkey, Ronnie J
• Format: Article
• Language: English
• Subjects: 8-Bromo Cyclic Adenosine Monophosphate - pharmacology; Animals; Bucladesine - pharmacology; cAMP; Cholera toxin; Cholera Toxin - pharmacology; Cyclic AMP - agonists; Leydig Cells - metabolism; MA-10 Leydig cells; Male; Mice; PRL receptors; Prolactin - metabolism; Prolactin - pharmacology; Receptors, Prolactin - agonists; Receptors, Prolactin - chemistry; Receptors, Prolactin - metabolism; Regulation of PRL-R; Tumor Cells, Cultured; Up-Regulation
• ISSN: 0303-7207; 1872-8057
• DOI: 10.1016/S0303-7207(98)00134-8

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 (3×10 −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.