CLONING, SEQUENCING AND FUNCTIONAL-ANALYSIS OF A TRUNCATED CDNA-ENCODING RED DEER PROLACTIN RECEPTOR - AN ALTERNATIVE TYROSINE RESIDUE MEDIATES BETA-CASEIN PROMOTER ACTIVATION

This study reports the isolation and in vitro characterisation of a tr uncated cDNA encoding the red deer long form prolactin receptor. The c DNA sequence predicts a protein of 557 amino acids which differs from the rat sequence by a 3' truncation of the cytoplasmic domain located 34 residues before the stop codon. The deer sequence shares the region s of homology which are important for maintenance of structural and fu nctional integrity, high affinity binding and signal transduction. How ever, the truncated deer receptor lacks the most C-terminal tyrosine r esidue in the intracellular domain which is believed to be essential f or activation of the beta-casein promoter. Transfection studies of the cervine cDNA into human 293 fibroblast cells confirmed the expression of a receptor that has high affinity binding to ovine prolactin (K-a = 0.65 x 10(9)M(-1) and B-max = 548.6 fmol/mg protein). Co-transfectio n of CHO cells with expression vector encoding the cervine prolactin r eceptor cDNA along with a fusion gene containing the promoter region o f beta-casein followed by beta-luciferase coding sequence led to 8.13 +/- 0.13-fold induction of luciferase enzyme activity in the presence of 400 ng/ml ovine prolactin. This was comparable to fold induction ob served with the wild type long form rat prolactin receptor (6.37 +/- 0 .48); macaque growth hormone receptor was without effect. Western blot analysis demonstrated tyrosine phosphorylation of the cervine recepto r and the associated kinase Jak2. following stimulation with prolactin . This confirms that the cervine cDNA although truncated is fully func tional and that Jak2 and an alternative tyrosine residue in the intrac ellular domain are involved in the signalling pathway leading to activ ation of the beta-casein promoter. Northern blot analysis provides evi dence that the prolactin receptor in the liver is encoded by transcrip ts of approximately 2.5 and 3.5 kb. Comparison of Northern blots of di fferent deer species suggests that the receptor is conserved amongst t he Cervidae. Northern blot analysis of red deer testis suggests that t his species expresses a second form of the receptor, encoded by a tran script of 1.7 kb, which may correspond to a smaller receptor form or a binding protein.