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Detection of specific protein bands with melatonin-like immunoreactivity in different cell lines and human brain
D K Lahiri1, D Davis, J I Nurnberger
1Department of Psychiatry, Indiana University School of Medicine, Indianapolis 46202-4887, USA.
Insights
Researchers identified specific proteins in human brain and cell extracts that bind to melatonin. These melatonin-binding proteins, particularly abundant in PC12 cells, may regulate free melatonin levels and circadian rhythms.
Area of Science:
- Neuroendocrinology
- Molecular Biology
- Chronobiology
Background:
- Melatonin, a pineal hormone, is crucial for regulating mammalian circadian rhythms.
- Understanding melatonin's mechanism of action requires identifying its interacting proteins.
Purpose of the Study:
- To investigate melatonin-like immunoreactivity (MLI) in human brain and cell extracts.
- To identify specific proteins that bind to melatonin.
Main Methods:
- Western immunoblots using an anti-melatonin antibody.
- Analysis of cell extracts from human brain and various cell lines, including PC12 cells.
- Purification of the anti-melatonin antibody.
Main Results:
- Detected high-molecular-mass protein bands (85-135 kDa) that specifically reacted with the anti-melatonin antibody.
- These protein bands were present in human brain and diverse cell line extracts.
- The 135-kDa protein band showed the highest immunoreactive signal in the PC12 neuroendocrine cell line.
Conclusions:
- Melatonin appears to bind to specific proteins in cellular and brain extracts.
- These melatonin-binding proteins are maximally synthesized in PC12 cells.
- The findings suggest the existence of novel proteins with melatonin-like epitopes or melatonin-binding proteins regulating free melatonin availability.
Abstract:
The pineal hormone melatonin regulates various neural and endocrine processes involved in mammalian circadian rhythms. To understand how melatonin mediates these functions, we investigated melatonin-like immunoreactivity (MLI) in cell extracts and human brain. In Western immunoblots, we detected high-molecular-mass protein bands (85-135 kDa) that specifically reacted with the anti-melatonin antibody. The specific protein bands were present in cell extracts from the human brain and cell lines of different origins. The immunoreactive signal of the 135-kDa protein band was highest in a neuroendocrine PC12 cell line, which was 10-fold higher than the signal observed in any cell extracts used. The commercial antibody employed in the Western blots was further purified against serum proteins and thyroglobulins. We have previously reported that the antibody against melatonin recognizes MLI as detected by a sensitive RIA. In the present report we have detected the putative melatonin-specific binding proteins, which could contribute to the MLI. Our results suggest that melatonin binds with specific proteins in different cellular and brain extracts, the protein(s) being maximally synthesized in PC12 cells. These results may indicate a group of yet unknown proteins sharing a melatonin-like epitope or the presence of melatonin-binding protein(s) that regulate availability of free melatonin, or both.
