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Functional expression of rat M5 muscarinic acetylcholine receptor in yeast
H J Huang1, C F Liao, B C Yang
1Graduate Institute of Botany, National Taiwan University, Taipei, Republic of China.
Abstract:
We have produced the rat M5 muscarinic acetylcholine receptor, an integral membrane protein, in the yeast Saccharomyces cerevisiae. This was achieved by placing an M5 gene in the yeast vector under the control of the yeast alpha-factor promoter and leader sequence. Northern blotting revealed the presence of M5 transcripts in yeast transformed with the M5 plasmid constructs. Crude extract prepared from the transformant yeasts showed saturable binding of the muscarinic antagonist [3H]-N-methyl scopolamine ([3H]NMS) with a kd of 22.77 nM and Bmax of 134.76 fmole per mg protein. Results deduced from saturation binding assay of intact cell demonstrated clearly that the M5 receptor was translocated across the membrane of the endoplasmic reticulum using the secretion signal on alpha-leader sequence and its binding site was still functional. Yeast expressing M5 receptor did not exhibit cell-cycle arrest in the presence of carbachol, a acetylcholine agonist, indicating that the recombinant M5 receptor could not couple directly to the endogenous yeast pheromone signaling G-protein.
Insights
Researchers successfully produced the rat M5 muscarinic acetylcholine receptor in yeast. The recombinant receptor, expressed in Saccharomyces cerevisiae, maintained functional binding capabilities.
Area of Science:
- Molecular biology
- Biochemistry
- Yeast genetics
Background:
- Muscarinic acetylcholine receptors (mAChRs) are crucial for neurotransmission.
- Heterologous expression of integral membrane proteins like mAChRs in yeast presents significant challenges.
- Understanding the functional expression of M5 mAChR in a non-mammalian system is key for drug discovery.
Purpose of the Study:
- To express the rat M5 muscarinic acetylcholine receptor (M5 mAChR) in the yeast Saccharomyces cerevisiae.
- To verify the functional integrity and localization of the expressed M5 mAChR.
- To investigate the potential for G-protein coupling of the recombinant M5 mAChR in yeast.
Main Methods:
- Gene cloning and vector construction using the yeast alpha-factor promoter and leader sequence.
- Northern blotting to confirm M5 transcript presence in transformed yeast.
- Saturation binding assays using [3H]-N-methyl scopolamine ([3H]NMS) on crude extracts and intact cells.
Main Results:
- Successful expression of M5 mAChR transcripts and proteins in Saccharomyces cerevisiae.
- Demonstrated saturable binding of [3H]NMS to the recombinant M5 mAChR with specific binding affinity (Kd) and capacity (Bmax).
- Evidence of M5 mAChR translocation to the endoplasmic reticulum membrane and functional binding site in intact yeast cells.
Conclusions:
- The yeast Saccharomyces cerevisiae can be utilized as a system for producing functional rat M5 muscarinic acetylcholine receptors.
- The recombinant M5 mAChR retains its ligand-binding properties after expression and translocation in yeast.
- The expressed M5 mAChR does not appear to directly couple with endogenous yeast G-proteins, suggesting system-specific limitations or requirements.