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Hepatic prolactin receptors in the rat: characterization using monoclonal antireceptor antibodies.

Monoclonal antibodies (mAbs) to the rat hepatic PRL receptor were produced and used for characterization of the receptor. A microsomal fraction from female rat liver was solubilized, purified 300- to 500-fold by affinity chromatography, and injected into mice. Two hybridoma clones (E21 and E29) were established, and immunoglobulin G fraction was obtained. Both E21 and E29 at 200 micrograms/ml could inhibit [125I]ovine PRL (oPRL) binding to microsomes from rat liver by 40% and 95%, respectively. E29 also inhibited PRL binding to solubilized receptors, whereas E21 stimulated PRL binding by about 50%. The action of E21 was markedly attenuated when [125I]human GH (hGH) was used as tracer in both microsomal (inhibition) and solubilized (stimulation) receptors. Specificity studies using microsomes from other tissues showed that both mAbs were specific to rat tissues (mammary gland, ovary, prostate, testis, and adrenal) and did not cross-react with tissues from other species (rabbit, mouse, human, pig, and cat) examined. Immunoprecipitation of PRL receptors with mAbs were assessed using 125I-labeled or [125I]oPRL-labeled PRL receptors. Both E21 and E29 were capable of immunoprecipitating a 44,000 mol wt band, the migration of which on a sodium dodecyl sulfate-electrophoresis gel was not affected by the absence or presence of a reducing agent. Only E21 was able to precipitate [125I]oPRL-receptor complexes. Binding studies of 125I-labeled mAb to microsomal receptors showed that oPRL could inhibit 90% of specific E29 binding, whereas inhibition of E21 binding was only 30%. Immunoblotting of PRL receptors confirmed the finding of immunoprecipitation; a band with a similar mol wt was identified with E21, although two closely located bands could be distinguished. There was no reaction in the presence of a reducing agent. These studies demonstrate that E21 recognizes a region distinct from the lactogen-binding site, while E29 binds to a closely related but not completely coincident domain; those regions recognized by both antibodies are specific to PRL receptors in the rat but not to those in other species; from immunoprecipitation and immunoblotting studies, the mol wt of the PRL receptor (or its subunit) is estimated to be 42-46K, similar to that reported for the rabbit mammary gland; this receptor molecule does not appear to bind to other receptor molecules through disulfide linkages; and hGH appears to recognize the PRL receptor-binding site in a somewhat different manner from that of oPRL.