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Functional characterization of the intermediate isoform of the human prolactin receptor
J B Kline1, H Roehrs, C V Clevenger
1Department of Pathology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA.
Abstract:
Prolactin-dependent signaling occurs as the result of ligand-induced dimerization of the prolactin receptor (PRLr). While three PRLr isoforms have been characterized in the rat, studies have suggested the existence of several human isoforms in breast carcinoma species and normal tissues. Reverse transcription polymerase chain reaction was performed on mRNA isolated from the breast carcinoma cell line T47D, revealing two predominant receptor isoforms: the previously described long PRLr and a novel human intermediate PRLr. The nucleotide sequence of the intermediate isoform was found to be identical to the long isoform except for a 573-base pair deletion occurring at a consensus splice site, resulting in a frameshift and truncated intracytoplasmic domain. Scatchard analysis of the intermediate PRLr revealed an affinity for PRL comparable with the long PRLr. While Ba/F3 transfectants expressing the long PRLr proliferated in response to PRL, intermediate PRLr transfectants exhibited modest incorporation of [(3)H]thymidine. Significantly, however, both the long and intermediate PRLr were equivalent in their inhibition of apoptosis of the Ba/F3 transfectants after PRL treatment. The activation of proximal signaling molecules also differed between isoforms. Upon ligand binding, Jak2 and Fyn were activated in CHO-K1 cells transiently transfected with the long PRLr. In contrast, the intermediate PRLr transfectants showed equivalent levels of Jak2 activation but only minimal activation of Fyn. Last, Northern analysis revealed variable tissue expression of intermediate PRLr transcript that differed from that of the long PRLr. Taken together, differences in signaling and tissue expression suggest that the human intermediate PRLr differs from the long PRLr in physiological function.
Insights
A novel human prolactin receptor (PRLr) intermediate isoform was identified, differing from the long isoform by a deletion that truncates its signaling domain. Both isoforms inhibit apoptosis, but signaling molecule activation and tissue expression vary, suggesting distinct physiological roles.
Area of Science:
- Endocrinology
- Molecular Biology
- Cell Biology
Background:
- Prolactin signaling is initiated by prolactin receptor (PRLr) dimerization.
- While rat PRLr isoforms are known, human PRLr isoforms in normal tissues and breast cancer are less understood.
- Previous studies suggested multiple human PRLr isoforms exist.
Purpose of the Study:
- To characterize novel human prolactin receptor (PRLr) isoforms.
- To investigate the functional differences between the long and a newly identified intermediate human PRLr.
- To compare the signaling pathways and tissue expression of these isoforms.
Main Methods:
- Reverse transcription polymerase chain reaction (RT-PCR) was used to identify PRLr isoforms in the T47D breast carcinoma cell line.
- Nucleotide sequencing was performed to determine the structure of the intermediate PRLr.
- Scatchard analysis, cell proliferation assays ([(3)H]thymidine incorporation), apoptosis inhibition assays, and Western blotting (for Jak2 and Fyn activation) were conducted.
- Northern analysis was used to examine tissue expression patterns.
Main Results:
- A novel human intermediate PRLr isoform was identified, characterized by a 573-base pair deletion leading to a truncated intracytoplasmic domain.
- Both long and intermediate PRLr isoforms bound prolactin (PRL) with comparable affinity and were equally effective in inhibiting apoptosis.
- Proliferation responses to PRL differed, with the long isoform promoting proliferation and the intermediate isoform showing modest thymidine incorporation.
- Signaling molecule activation varied: Jak2 activation was equivalent, but Fyn activation was significantly reduced in intermediate PRLr transfectants.
- Northern analysis revealed distinct tissue expression patterns for the intermediate and long PRLr transcripts.
Conclusions:
- The human intermediate PRLr isoform possesses unique structural and functional characteristics compared to the long PRLr.
- Despite shared anti-apoptotic functions, differences in proliferation, signaling molecule activation (Jak2, Fyn), and tissue distribution suggest distinct physiological roles for the two isoforms.
- These findings highlight the complexity of prolactin signaling and the potential for isoform-specific functions in normal physiology and disease.