Characterization and Applications of Monoclonal Antibodies to the Prolactin Receptor*

Abstract

Monoclonal antibodies (mAbs) were produced in BALB/c mice immunized with partially purified PRL receptors from rat liver. Two mAbs (T1 and T6) were able to completely inhibit [125I]ovine PRL ([125I]oPRL) binding to solubilized rat liver PRL receptors, while two other mAbs (U5 and U6) showed only a small effect on PRL binding, but were able to precipitate hormone-receptor complexes. Scatchard analysis of [125I]oPRL binding to rat liver microsomes in the presence of mAbs resulted in a decrease in the number of sites without changing the affinity of PRL binding by T1 and T6, whereas U5 and U6 altered neither parameter. [125I]mAb binding to rat liver microsomes was performed in the presence of various concentrations of unlabeled mAbs or oPRL to examine the interaction between mAbs. Competition of binding to the receptor was observed, respectively, between T1 and T6, U5 and U6, and U5 and E21 (a mAb to the rat liver PRL receptor previously produced). Both [125I]T1 and [125I]T6 binding were inhibited by oPRL, although not completely (80% inhibition at the higher concentrations). When [125I]T1 binding was analyzed by Scatchard analysis, two classes of binding sites to rat liver microsomes were found, of which only the number of higher affinity sites was affected by the presence of oPRL, in incubation. Similar results were observed for [125I]T6 binding. [125I]mAb binding to microsomes from other tissues and species was examined. All five mAbs were able to bind to microsomes from rat tissues (liver, ovary, adrenal, prostate, and Nb2 lymphoma cells), similar to the level of [125I]oPRL binding in these tissues. The binding characteristics of [125I]T6 or [125I]U5 were essentially identical in all rat tissues examined. Although T1, U6 and E21 showed strong species specificity, there was significant binding of T6 to rabbit liver and mammary gland and of U5 to rabbit and pig mammary gland and mouse liver. Competition curves of [125I]U5 binding were parallel for rat, rabbit, and mouse tissues, while [125I]T6 binding was able to distinguish PRL receptors in rabbit mammary gland from those in rat tissues. The use of 125I-labeled mAb in immunoblot analysis of the PRL receptor resulted in a marked increase in sensitivity. All mAbs detected microsomal PRL receptors in rat liver with mol wt of 84,000, 42,000 and 40,000. As little as 4 fmol reception can be identified using this approach. Microsomal PRL receptors from rat ovary, prostate, and Nb2 cells and purified receptors from pig and rabbit mammary gland were subjected to immunoblot analysis. Autoradiograms showed several mol wt bands for each tissue: 84,000, 51,000 and 42,000 for ovary; 84,000 and 42,000 for prostate; 64,000, 52,000 and 42,000 or Nb2 cells; 66,000 and 36,000 for pig mammary gland; and 77,000, 55,000, 45,000 and 36,000 for rabbit mammary gland. The results demonstrate that 1) T1 and T6 are directed against two separate epitopes of the rat liver PRL receptor, one the hormone-binding site and the other to which PRL is unable to bind; 2) the antigenic domains of U5 and U6 are distinct from the hormone-binding site; 3) the immunogenic domains of the PRL receptor, possibly including the hormone-binding site, are highly conserved within the same species; 4) there are some immunogenic domains of the receptor conserved between species; and 5) immunoblot analysis using [125I]mAbs is a powerful tool to detect and analyse PRL receptors in various tissues.