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Cloning of P2Y6 cDNAs and identification of a pseudogene: comparison of P2Y receptor subtype expression in bone and
R Maier1, A Glatz, J Mosbacher
1Novartis Pharma Inc., Basel, CH-4002, Switzerland.
Abstract:
Cellular responses to ATP/UTP and analogs are mediated by G-protein coupled P2Y receptors and have been proposed to play a role in the regulation of bone metabolism. Using a degenerate PCR approach on MG-63 cell cDNA we found PCR fragments coding for human P2Y1 and a new receptor, P2Y6. cDNA cloning of the P2Y6 receptor identified three cDNA isoforms. Two contained the same contiguous ORFs but differed in their 5' UTRs and may therefore originate by alternative splicing whereas the third represents a pseudogene. Analysis of P2Y receptor subtype expression in human bone and the osteoblastic cell lines OHS-4 and MG-63 by RT-PCR showed that all known human P2Y receptor subtypes (P2Y1, P2Y2, P2Y4, P2Y6, and P2Y7) were expressed. In contrast, analysis of brain-derived cell lines suggests that a selective expression of P2Y receptor subtypes occurs in brain tissue.
Insights
Researchers identified a new P2Y6 receptor and found all known P2Y receptor subtypes expressed in human bone cells. This suggests P2Y receptors play a role in bone metabolism regulation.
Area of Science:
- Molecular Biology
- Cell Biology
- Pharmacology
Background:
- Cellular responses to extracellular nucleotides like ATP and UTP are mediated by G-protein coupled P2Y receptors.
- P2Y receptors are implicated in the regulation of bone metabolism, but their specific roles and expression patterns require further elucidation.
- Understanding P2Y receptor expression is crucial for investigating their involvement in bone physiology and pathology.
Purpose of the Study:
- To identify novel P2Y receptor subtypes involved in cellular responses.
- To characterize the expression profile of various P2Y receptor subtypes in human bone and related cell lines.
- To investigate potential differences in P2Y receptor expression between bone and brain tissues.
Main Methods:
- Degenerate PCR was employed to identify P2Y receptor fragments from MG-63 cell cDNA.
- cDNA cloning was used to isolate and characterize the identified P2Y6 receptor and its isoforms.
- RT-PCR analysis was performed to determine the expression of known human P2Y receptor subtypes in different cell lines and tissues.
Main Results:
- Degenerate PCR identified human P2Y1 and a novel receptor, P2Y6.
- Three cDNA isoforms of the P2Y6 receptor were identified, including two potentially arising from alternative splicing and one pseudogene.
- RT-PCR confirmed the expression of all known human P2Y receptor subtypes (P2Y1, P2Y2, P2Y4, P2Y6, P2Y7) in human bone and osteoblastic cell lines (OHS-4, MG-63).
- Analysis of brain-derived cell lines indicated selective expression of P2Y receptor subtypes in brain tissue.
Conclusions:
- A novel P2Y6 receptor and its isoforms were identified, expanding the known repertoire of P2Y receptors.
- All known human P2Y receptor subtypes are expressed in human bone and osteoblastic cells, suggesting a significant role for these receptors in bone metabolism.
- Differential expression patterns of P2Y receptors in bone versus brain tissues highlight tissue-specific functions.