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Title: Re-evaluation of the PBAN receptor molecule: characterization of PBANR variants expressed in the pheromone glands of moths

Author
item LEE, JAE - Advance Science Institute, Riken
item Hull, Joe
item KAWAI, TAKESHI - University Of Tokyo
item GOTO, CHIE - National Agricultural Research Center - Japan
item KURIHARA, MASAAKI - Advance Science Institute, Riken
item TANOKURA, YU - University Of Tokyo
item NAGATA, KOJI - University Of Tokyo
item NAGASAWA, HIROMICHI - University Of Tokyo
item MATSUMOTO, SHOGO - Advance Science Institute, Riken

Submitted to: Frontiers in Endocrinology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/9/2012
Publication Date: 1/24/2012
Citation: Lee, J.M., Hull, J.J., Kawai, T., Goto, C., Kurihara, M., Tanokura, M., Nagata, K., Nagasawa, H., Matsumoto, S. 2012. Re-evaluation of the PBAN receptor molecule: characterization of PBANR variants expressed in the pheromone glands of moths. Frontiers in Endocrinology. 3(6):1-12.

Interpretive Summary: While it is accepted that sex pheromone production in most moths is initiated following pheromone biosynthesis activating neuropeptide receptor (PBANR) activation, questions persist regarding the molecular nature of the PBANR utilized. Initial identification of PBANRs in Helicoverpa zea and Bombyx mori revealed the presence of two PBANR “subtypes” - one in B. mori that has a relatively long C-terminus that is essential for receptor feedback regulation (ie ligand-induced internalization), and a shorter one in H. zea PBANR that lacks features crucial for internalization. The functional significance of the two receptor subtypes was initially ascribed to differences in signal transduction cascades activated (cAMP dependent vs cAMP independent). Recently, though, multiple PBANRs, two of which have C-terminal tails similar to B. mori PBANR, have been reported to be concurrently expressed in the larval CNS of Heliothis virescens. In this study, we report the concurrent expression of four PBANR variants (As, A, B, and C) in pheromone glands of three moth species. The variants are distinguishable only by the length and composition of their respective C-terminal tails with the PBANR-A variant analogous to the H. zea PBANR and the PBANR-C variant analogous to the B. mori PBANR. When expressed as fluorescent chimeras in insect cells, only the long C-terminal variants (PBANR-B and PBANR-C) localized exclusively to the cell surface and underwent ligand-induced internalization. Furthermore, RT-PCR analyses showed that PBANR-C is the predominant transcript expressed in the pheromone gland. Based on these results, we conclude that PBANR-C is the principal receptor molecule involved in PBAN signaling regardless of moth species and suggest that previous “preferential” amplification of PBANR-A like receptors from other species was a PCR artifact that resulted from the high GC content of the C-terminal coding sequence in the B and C variants.

Technical Abstract: Sex pheromone production in most moths is initiated following pheromone biosynthesis activating neuropeptide receptor (PBANR) activation. PBANR was initially cloned from pheromone glands (PGs) of Helicoverpa zea and Bombyx mori. The B. mori PBANR is characterized by a relatively long C-terminus that is essential for ligand-induced internalization, whereas the H. zea PBANR has a shorter C-terminus that lacks features present in the B. mori PBANR critical for internalization. Recently, multiple PBANRs were reported to be concurrently expressed in the larval CNS of Heliothis virescens. In the current study, we sought to identify all of the PBANRs expressed in the pheromone glands (PGs) of three moths and to ascertain their potential functional relevance. Four PBANR variants (As, A, B, and C) were cloned from the PGs of all three species with PBANR-C the most highly expressed. The variants, distinguishable only by the length and composition of their respective C-terminal tails, arise from alternative splicing. Transient expression of fluorescent PBANR chimeras in insect cells revealed that PBANR-B and PBANR-C localized exclusively to the cell surface while PBANR-As and PBANR-A exhibited varying degrees of cytosolic localization. Similarly, only the PBANR-B and PBANR-C variants underwent ligand-induced internalization. Taken together, our results suggest that PBANR-C is the principal receptor molecule involved in PBAN signaling regardless of moth species. The high GC content of the C-terminal coding sequence in the B and C variants, which makes amplification using conventional polymerases difficult, likely accounts for previous “preferential” amplification of PBANR-A like receptors from other species.