A prolactin-dependent immune cell line (Nb2) expresses a mutant form of prolactin receptor.
The Nb2 cell line is a pre-T rat lymphoma that is dependent on prolactin (PRL) for mitogenesis. Two forms of PRL receptor (PRL-R), which differ in the length of their cytoplasmic domains have been identified in different tissues and species. In the present study we have cloned the cDNA and characterized the mitogenic form of PRL-R in Nb2 cells. Polymerase chain reaction amplification of first strand cDNA prepared from Nb2-11C (PRL-dependent) and Nb2-Sp (PRL-independent) cell lines was performed using oligonucleotide primers specific for the binding domain, the short form of the PRL-R, and the cytoplasmic domain of the long form of the PRL-R. These studies indicate that both cell lines express a novel form of PRL-R. A cDNA was isolated from an Nb2-Sp cDNA library, which contains 1446 base pairs identical to the nucleotide sequence of the long form of the rat PRL-R. However, the cDNA sequence is missing 594 base pairs in the cytoplasmic domain compared with the long form of the PRL-R. The cDNA encodes a protein of 393 amino acids, lacking 198 amino acids in the cytoplasmic domain. Scatchard analysis of 125I-labeled ovine prolactin (oPRL) binding to microsomes prepared from transiently transfected COS-7 cells with either PRL-R long form cDNA or Nb2 PRL-R cDNA indicates that the long form of PRL-R binds oPRL with high affinity (K alpha = 8.8 x 10(9) M-1), while the Nb2 PRL-R showed a 3.3-fold increased affinity for PRL (K alpha = 29.1 x 10(9) M-1). In addition, immunoblot analysis of these microsomes using 125I-labeled monoclonal antibody (U6) to the PRL-R demonstrates a Mr of approximately 82,000 for the long form and approximately 62,000 for the Nb2 form of PRL-R. Polymerase chain reaction amplification of genomic DNA prepared from PRL-dependent and -independent cell lines suggests that this form of PRL-R results from a deletion in the PRL-R gene. The identification of a modified long form of PRL-R in the Nb2 cell line should help localize domains of the PRL-R involved in signal transduction and further the investigation of prolactin's role in immune cell proliferation.
Ali
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Laboratory of Molecular Endocrinology, McGill University, Royal Victoria Hospital, Montreal, Quebec, Canada.
Pellegrini
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Kelly
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D015153:Blotting, Western; D002460:Cell Line; D004247:DNA / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction; D011388:Prolactin / Q000378:metabolism*; D012313:RNA / Q000235:genetics; D051381:Rats; D011981:Receptors, Prolactin / Q000235:genetics*
D011981:Receptors, Prolactin; D012313:RNA; D011388:Prolactin; D004247:DNA