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 truncated cDNA encoding the red deer long form prolactin receptor. The cDNA 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 regions of homology which are important for maintenance of structural and functional integrity, high affinity binding and signal transduction. However, the truncated deer receptor lacks the most C-terminal tyrosine residue in the intracellular domain which is believed to be essential for 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 (Ka = 0.65 x 10(9)M(-1) and Bmax = 548.6 fmol/mg protein). Co-transfection of CHO cells with expression vector encoding the cervine prolactin receptor cDNA along with a fusion gene containing the promoter region of 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 observed 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 receptor and the associated kinase Jak2 following stimulation with prolactin. This confirms that the cervine cDNA although truncated is fully functional and that Jak2 and an alternative tyrosine residue in the intracellular domain are involved in the signalling pathway leading to activation of the beta-casein promoter. Northern blot analysis provides evidence that the prolactin receptor in the liver is encoded by transcripts of approximately 2.5 and 3.5 kb. Comparison of Northern blots of different deer species suggests that the receptor is conserved amongst the Cervidae. Northern blot analysis of red deer testis suggests that this species expresses a second form of the receptor, encoded by a transcript of 1.7 kb, which may correspond to a smaller receptor form or a binding protein.
Institute of Zoology, London, UK. h.jabbour@ed-rbu.mrc.ac.uk
H N
HN
Jabbour
L A
LA
Clarke
S
S
Boddy
A
A
Pezet
M
M
Edery
P A
PA
Kelly
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002364:Caseins / Q000096:biosynthesis* / Q000235:genetics*; D002460:Cell Line; D003001:Cloning, Molecular; D006224:Cricetinae; D017931:DNA Primers; D018076:DNA, Complementary; D003670:Deer; D017930:Genes, Reporter; D006801:Humans; D007668:Kidney; D008156:Luciferases / Q000096:biosynthesis; D008297:Male; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D051381:Rats; D011981:Receptors, Prolactin / Q000096:biosynthesis*; D011994:Recombinant Proteins / Q000096:biosynthesis; D017386:Sequence Homology, Amino Acid; D013737:Testis / Q000378:metabolism; D014162:Transfection
D002364:Caseins; D017931:DNA Primers; D018076:DNA, Complementary; D011981:Receptors, Prolactin; D011994:Recombinant Proteins; D008156:Luciferases