Sequence and functional characterisation of the marmoset monkey (Callithrix jacchus) prolactin receptor: comparative homology with the human long-form prolactin receptor.
This study demonstrates the cloning and in-vitro characterisation of the marmoset monkey (Callithrix jacchus) prolactin receptor cDNA. The marmoset prolactin receptor cDNA was generated by reverse transcription-polymerase chain reaction using adrenal RNA and primers designed from prolactin receptor conserved regions. Sequence analysis predicts a mature protein of 598 amino acids exclusive of the 24 amino acid signal peptide. The marmoset prolactin receptor cDNA shares 93 and 61% base pair, and 89 and 61% amino acid sequence homologies with the long form human and rat prolactin receptor cDNA, respectively. The marmoset prolactin receptor cDNA sequence retains all the receptor sequences that have been shown previously to be essential for ligand binding, structural integrity and signal transduction. Transfection of human 293 fibroblast cells with the marmoset prolactin receptor cDNA (three independent experiments) confirmed the expression of a receptor that has high binding affinity to human growth hormone (K(a)=3.6+/-0.07 nM(-1) and B(max)=7.55+/-2.06x10(-11) M) and human prolactin (K(a)=3.1+/-0.12 nM(-1) and B(max)=2.87+/-0.66x10(-11) M). Functionality of the receptor was assessed by co-transfection of 293 fibroblast cells with marmoset prolactin receptor cDNA and the Jak2 cDNA, or marmoset prolactin receptor and a Stat5 responsive element linked to the luciferase coding sequence. Incubation of the cells with 18 nM ovine prolactin resulted in rapid phosphorylation of Jak2 as ascertained by Western blotting. In addition, the marmoset prolactin receptor cDNA led to 9.06+/-0.47-fold induction of luciferase gene activity. This was comparable with the induction observed following transfection with the human prolactin receptor cDNA (8.55+/-0. 5-fold). In-vivo prolactin receptor expression in the marmoset monkey was assessed by ribonuclease protection assay and detected in a number of tissues including female reproductive organs. These data confirm the cloning and functionality of the marmoset prolactin receptor cDNA. The marmoset prolactin receptor shares a high sequence homology with the long-form human prolactin receptor, and both receptors bind hormones with comparable affinity and confer a similar intracellular response. The marmoset monkey may provide a useful tool to investigate the role of prolactin in primate reproduction.
Dalrymple
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A
Medical Research Council Human Reproductive Sciences Unit, Centre for Reproductive Biology, 37 Chalmers Street, EH3 9ET, Edinburgh, UK.
Edery
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Jabbour
H N
HN
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001667:Binding, Competitive; D015153:Blotting, Western; D002144:Callithrix / Q000235:genetics*; D002460:Cell Line; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D005260:Female; D017930:Genes, Reporter; D006801:Humans; D053614:Janus Kinase 2; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism; D011518:Proto-Oncogene Proteins; D012313:RNA / Q000378:metabolism; D011981:Receptors, Prolactin / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D017385:Sequence Homology; D015398:Signal Transduction; D015533:Transcriptional Activation; D014162:Transfection
D018076:DNA, Complementary; D011518:Proto-Oncogene Proteins; D011981:Receptors, Prolactin; D012313:RNA; D011505:Protein-Tyrosine Kinases; C507924:JAK2 protein, human; C507927:Jak2 protein, rat; D053614:Janus Kinase 2