|Lee, Jae - Advance Science Institute, Riken|
|Kawai, Takeshi - University Of Tokyo|
|Kurihara, Masaaki - Advance Science Institute, Riken|
|Tanokura, Masaru - University Of Tokyo|
|Nagata, Koji - University Of Tokyo|
|Nagasawa, Hiromichi - University Of Tokyo|
|Matsumoto, Shogo - Advance Science Institute, Riken|
|Tsuneizumi, K - Advance Science Institute, Riken|
Submitted to: Frontiers in Endocrinology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/1/2012
Publication Date: 4/18/2012
Citation: Lee, J.M., Hull, J.J., Kawai, T., Tsuneizumi, K., Kurihara, M., Tanokura, M., Nagata, K., Nagasawa, H., Matsumoto, S. 2012. Establishment of Sf9 transformants constitutively expressing PBAN receptor variants: application to functional evaluation. Frontiers in Endocrinology. 3(56):1-8.
Interpretive Summary: Multiple PBANR variants were previously found to be expressed in the pheromone glands of diverse moth species. These receptor variants are identical up to the 7th transmembrane domain, at which point the amino acid sequences of the respective receptors diverge. To better understand how these sequence differences affect overall receptor function and regulation, genes encoding the silkmoth (Bombyx mori) PBANR variants were inserted into the genome of cultured insect cells. The resulting transgenic cell lines were found to stably and constitutively express the respective receptors. No deleterious effects on cell morphology or growth were observed as a result of gene insertion. All of the receptor variants bound a red fluorescent analog of PBAN. Cells expressing the short variant (A) were defective with respect to calcium mobilization and ligand-induced internalization. Similar defects were not seen in cells expressing the two long variants (B and C). These findings demonstrate clearly that the intracellular pathways activated by the short variant are functionally distinct from those activated by the long variants. With these established cell lines, it will now be possible to tease apart the specific signaling pathway(s) utilized by the short variant. It is expected that functional characterization of the short variant will provide clues to its in vivo biological role. Similar efforts to unravel the functionality of the short variant in native pheromone glands would be greatly hampered by the underlying complexity of multiple PBANR signaling circuits.
Technical Abstract: To facilitate further evaluation of pheromone biosynthesis activating neuropeptide receptor (PBANR) functionality and regulation, we generated cultured insect cell lines stably expressing a number of fluorescent Bombyx mori PBANR (BommoPBANR) and Pseudaletia separata PBANR (PsesePBANR) variants including BommoPBANR-A, -B, and -C, as well as PsesePBANR-B and -C, all of which were C-terminally fused to EGFP. Cell lines expressing non-chimeric BommoPBANR-B and –C were also generated. Functional evaluation of these stable transformants using confocal laser microscopy revealed that a Rhodamine Red-labeled PBAN derivative (RR-C10PBANR2K) specifically co-localized with all of the respective expressed PBANR variants at the plasma membrane. Near complete internalization of the bound RR-C10PBANR2K 30 min after ligand binding was observed in all cell lines except those expressing the BommoPBANR-A variant, in which the ligand/receptor complex remained at the plasma membrane. Fluorescent Ca2+ imaging further showed that, unlike the BommoPBANR-B or BommoPBANR-C cell lines, RR-C10PBANR2K binding failed to trigger an influx of extracellular Ca2+ in the BommoPBANR-A cell line. These observations suggest that, unlike BommoPBANR-B and -C, BommoPBANR-A is functionally deficient. We also found that, contrary to previous reports, ligand-induced internalization of BommoPBANR-B and BommoPBANR-C in cell lines stably expressing these variants occurred in the absence of extracellular Ca2+.