Cloning and characterization of a cDNA encoding a highly conserved, putative calcium binding protein, identified by an anti-prolactin receptor antiserum.

Using antisera raised against partially purified rabbit mammary gland prolactin receptor, we have isolated a cDNA from a T-47D human breast cancer cell expression library which encodes the putative calcium-binding protein, calcyclin. Since the protein encoded by this cDNA co-purified with the prolactin receptor, we have tentatively named it a prolactin receptor-associated protein (PRA). Hybrid selection and in vitro translation showed that the protein encoded by this cDNA was approximately 10 kDa, a result confirmed by the amino acid sequence derived after DNA sequencing analysis. The PRA gene product has significant sequence similarity to the S-100 proteins, the cystic fibrosis antigen, and p11 proteins. The PRA/calcyclin cDNA obtained from the T-47D human breast cancer cell library has 37 nucleotides more 5'-untranslated information than that of the calcyclin cDNA from human fibroblasts. Since the transcription start site for the calcyclin gene has been confirmed in fibroblasts, our data suggest that a different transcription start site may be used in the T-47D cells. Isolation and nucleotide sequencing of the rat PRA cDNA showed there was 96% predicted amino acid sequence similarity with the human PRA and confirmed the highly conserved nature of the PRA/calcyclin gene between species. The PRA is expressed in most but not all human breast cancer cell lines, e.g. MCF 7. Northern analyses of RNAs from rat tissue indicated that PRA mRNA levels vary widely. They are low in the liver, brain, testes, and ovary, moderate in skeletal muscle, and high in the lung, kidney, and uterus. The availability of prolactin receptor-positive human breast cancer cell lines, which do (T-47D) and do not (MCF 7) express the PRA/calcyclin gene, will allow the investigation of the role, if any, of PRA in prolactin action.