RAT PLACENTAL LACTOGEN-I VARIANT (RPL-I-V), PRODUCT OF AN UNIQUE GENE, IS BIOLOGICALLY DIFFERENT FROM RPL-I

The rat placenta expresses two rat placental. lactogen-I (rPL-I) prote ins: the normal rPL-I in the first half of pregnancy and a variant (rP L-I-v) in the second half of pregnancy. They are 70% identical at the amino acid level but arise from different cell types: rPL-I from giant cells and rPL-I-v from cytotrophoblasts. To assess whether rPL-I-v or iginates from alternative splicing of the rPL-I gene or is the product of a separate gene, genomic clones of rPL-I and rPL-I-v were isolated from a lambda EMBL-3 rat genomic library. Restriction enzyme analysis of the 14-kilobase full-length genomic clones of rPL-I and rPL-I-v in dicated that the two genes are distinct. To assess the biological acti vity of the variant protein relative to other members of the rat PL/PR L/GH family, two expression systems were chosen to obtain the purified recombinant protein: 1) a secreted form of rPL-I-v was obtained from Chinese hamster ovary (CHO) cells transfected with rPL-I-v-complementa ry DNA; and 2) a rPL-I-v fusion protein (Bac-rPL-I-v) was obtained fro m Spodoptera frugiperda (Sf9) insect cells that had been infected with a recombinant baculovirus generated from rPL-I-v complementary DNA. A n antibody was generated to the purified Bac-rPL-I-v fusion protein an d used for affinity chromatography to purify recombinant rPL-I-v from the CHO cell media. The mitogenic activity of rPL-I-v was monitored in the Nb2 lymphoma cell bioassay. The relative potency of rPL-I-v compa red with other members of the PL/PRL/GH family follows: ovine PRL 100, rPL-I 200, rPL-II 160, rPRL 40, CHO-rPL-I-v 0.7, and Bac rPL-I-v 0.4. Iodinated CHO-rPL-I-v showed minimal binding to Nb2 lymphoma cells, b ut at a 500-fold protein concentration rPL-I-v was able to displace [I -125]rpL-I from the lymphoma cell PRL receptor. Using recombinant CHO- derived rPL-I-v as standard and antisera against the Bac-rPL-I-v fusio n protein, a RIA was developed for rPL-I-v. In pregnant rats rPL-I-v a ppeared in the serum at day 14, rising to a peak of 2080 +/- 440 ng/ml at day 18, followed by a slight decline. These values reflect the lev els of messenger RNA for rPL-I-v in rat placenta noted previously. In summary, rPL-I-v arises from a gene different from 1 PL-I and the rPL- I protein displays minimal binding and mitogenic activity in the Nb2 l ynphoma cells.