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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, 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 of 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 human P2Y receptor subtypes expressed in bone cells. This research sheds light on P2Y receptors
Area of Science:
- Molecular Biology
- Cell Biology
- Pharmacology
Background:
- Cellular responses to extracellular nucleotides like ATP and UTP are primarily mediated by G-protein coupled P2Y receptors.
- P2Y receptors are implicated in regulating various physiological processes, including bone metabolism.
- Understanding the specific P2Y receptor subtypes involved in bone cell function is crucial for therapeutic development.
Purpose of the Study:
- To identify novel P2Y receptor subtypes expressed in human bone cells.
- To characterize the expression profile of known P2Y receptor subtypes in human bone and osteoblastic 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 analyze isoforms of the newly identified P2Y6 receptor.
- RT-PCR was performed to assess the expression of various human P2Y receptor subtypes in human bone, osteoblastic cell lines (OHS-4, MG-63), and brain-derived cell lines.
Main Results:
- Degenerate PCR identified human P2Y1 and a novel P2Y6 receptor.
- Three cDNA isoforms of the P2Y6 receptor were identified, including two likely originating from alternative splicing and one pseudogene.
- RT-PCR analysis revealed the expression of all known human P2Y receptor subtypes (P2Y1, P2Y2, P2Y4, P2Y6, P2Y7) in human bone and osteoblastic cell lines.
- A selective expression pattern of P2Y receptor subtypes was observed in brain-derived cell lines, contrasting with bone cells.
Conclusions:
- The P2Y6 receptor is a newly identified member of the human P2Y receptor family expressed in bone cells.
- Human bone and osteoblastic cells express a comprehensive repertoire of known P2Y receptor subtypes.
- Differential expression of P2Y receptors in distinct tissues like bone and brain suggests tissue-specific roles.