Molecular cloning and expression studies of a prolactin receptor in goldfish (Carassius auratus)

A full-length cDNA clone, of a size of 4.6 kb, for the goldfish prolactin r eceptor has been isolated. This cDNA clone encodes a protein of 600 amino a cids homologous to prolactin receptors of other species. A Kyte-Doolittle h ydropathy analysis of the receptor indicates that the translated protein co nsists of a signal peptide of 22 amino acids, an extracellular domain of 22 8 amino acids, a single transmembrane domain of 24 amino acids, and an intr acellular domain of 346 amino acids. Several characteristic landmarks of pr olactin receptor could be identified in this clone. These include the four conserved cysteine residues and the WS motif within the extracellular domai n, and the box: I and box 2 regions of the intracellular domain. Among all the prolactin receptor sequences known to date, this clone bears the closes t resemblance to the tilapia prolactin receptor, although homology between these two fish prolactin receptors is rather low. There are only 57.4% of n ucleotide and 48.3% of amino acid sequence identities between these two fis h receptors. This receptor cDNA was transfected into CHO-K1 cells for funct ional analysis. RT-PCR analysis with a pair of gene specific primers indica te that the receptor was transcribed in the transfected cells. Using a cell proliferation assay based on the reduction of the tetrazolium salt 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetraz bromide, the receptor transfected CHO-K1 cells can be stimulated to proliferate upon the addition of ovine pr olactin in the culture medium. The tissue distribution of the prolactin rec eptor in goldfish was studied by RT-PCR/Southern analysis and by Northern a nalysis. The results indicated that the receptor is expressed mostly in the kidney, the gill and the intestine of goldfish, corroborating with the osm oregulatory role of prolactin in fish. In addition, an appreciable level of the receptor is also found in the brain and gonads of goldfish. Northern a nalysis showed that there are two transcript sizes, a major 4.6 kb and a mi nor 3.5 kb mRNAs, in the kidney, gill and intestine.