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SUMMARY The Kell blood group locus (KEL) is tightly linked to the prolactin-inducible protein locus (PIP) with z=9.12 at o= 0.00 for combined paternal and maternal meioses. In view of the regional localization of PIP to 7q32-q36 (Myal et al. 1989a), a similar assignment for KEL is favoured.

The mechanism of action of prolactin in target cells and the role of prolactin in human breast cancer are poorly understood phenomena. The present study examines the effect of human prolactin (hPRL) on the synthesis of unique proteins by a human breast cancer cell line, T-47D, in serum-free medium containing bovine serum albumin. [35S]Methionine-labeled proteins were analysed by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis and fluorography. Treatment of cells with hPRL (1-1000 ng/ml) and hydrocortisone (1 microgram/ml) for 36 h or longer resulted in the synthesis and secretion of three proteins having molecular weights of 11,000, 14,000, and 16,000. Neither hPRL nor hydrocortisone alone induced these proteins. Of several other peptide hormones tested, only human growth hormone, a hormone structurally and functionally similar to hPRL, could replace hPRL in causing protein induction. These three proteins were, therefore, referred to as prolactin-inducible proteins (PIP). Each of the three PIPs was purified to homogeneity by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and specific antibodies were generated to them in rabbits. By immunoprecipitation and immunoblotting (Western blot) of proteins secreted by T-47D cells, it was demonstrated that the three PIPs were immunologically identical to one another. In addition, the 16-kDa and 14-kDa proteins (PIP-16 and PIP-14), and not the 11-kDa protein (PIP-11), incorporated [3H]glycosamine. Furthermore, 2-deoxyglucose (2 mM) and tunicamycin (0.5 micrograms/ml), two compounds known to inhibit glycosylation, blocked the production of PIP-16 and PIP-14, with a concomitant increase in the accumulation of PIP-11. These results indicate PIP-16 and PIP-14 are glycosylated variants of PIP-11. Finally, in vitro translation of poly(A)+ messenger RNA followed by immunoprecipitation revealed a 12.5-kDa protein, possibly the precursor form of PIPs. In addition, T-47D cells treated with hPRL plus hydrocortisone contained 10-fold more mRNA for PIPs than control cells, suggesting that the hormones' action is at the level of gene expression. Our finding represents a first demonstration of prolactin regulation of gene expression in human target cells. The human breast cancer cells, T-47D, appear to be an excellent model to afford future studies on the molecular action of prolactin and on the possible role of prolactin in human breast cancer.

Antisera against a partially purified prolactin-receptor preparation derived from pregnant-rabbit mammary glands were generated in guinea pigs. On double immuno-diffusion, each antiserum produced a single precipitin line with the prolactin receptors. The anti-receptor sera also specifically inhibited the binding of 125I-labelled sheep prolactin to membrane particles as well as to highly purified prolactin receptors derived from the rabbit mammary glands. The same antisera, however, had no effect on the binding of 125I-labelled insulin to the same membranes. These antisera did not bind or destroy prolactin. Moreover, the binding of 125I-LABELLED PROLACTIN TO MEMBRANE PARTICLES DErived from different tissues from a number of species was also inhibited by the antisera, thus suggesting that the immunological determinants of the prolactin receptors are similar in various tissues derived from different species. The factors in the antisera that were responsible for inhibiting the binding of 125I-labelled prolactin to its receptors were found to be associated with the gamma-globulin fraction. In addition, 131I-labelled gamma-globulins derived from one antiserum were shown to bind to membrane particles derived from mammary glands, and an increase in binding of gamma-globulin was accompanied by a decrease in binding of prolactin. Kinetic analyses of inhibition of 125I-labelled prolactin binding by antisera by using the methods of Lineweaver & Burk [J. Am. Chem. Soc. (1934) 56, 658-666] and Dixon [Biochem. J. (1953) 55, 170-171], revealed that the mechanism is a hyperbolic competitive inhibition. The demonstration of hormone-receptor-antibody complexes further favours this mechanism. The availability of anti-receptor sera should facilitate studies on the functional role as well as other biochemical, immunological and physiological properties of the prolactin receptors.

Two human breast cancer cell lines (T-47D and MCF-7) and one cell line derived from normal human milk (HBL-100) not only specifically bound but also degraded prolactin. Quantitative differences in the ability to bind and degrade prolactin among the cell lines exist, although there was a good correlation between the number of prolactin receptor sites and prolactin degradative activity. Iodo-prolactin as well as native prolactin were degraded. The prolactin molecule was processed to yield at least three small molecular weight peptides which were released into the incubation medium. These peptides neither bound to fresh receptors nor to anti-prolactin antibodies. The protease inhibitor N-alpha-p-tosyl-L-lysine chloromethyl ketone, lysosomotropic agents such as chloroquine and ammonium chloride, and metabolic inhibitors 2,4-dinitrophenol and sodium azide, all abolished prolactin degradation by the breast cancer cells. When prolactin degradation was inhibited, specific binding and the subsequent release of intact 125I-prolactin was still observable, suggesting that hormonal degradation was not a prerequisite to dissociation of prolactin. However, prolactin degradation did account for the accelerated rate of dissociation of prolactin. Studies utilizing inhibitors suggest that the receptor-bound 125I-prolactin was degraded by an energy-dependent internalization process such as pinocytosis; lysosomal enzymes are probably involved in the degradation of prolactin by human breast cancer cells.