Abstract
In most Lepidoptera, pheromone biosynthesis is regulated by a neuropeptide termed pheromone biosynthesis activating neuropeptide (PBAN). Although much is known about the cellular targets of PBAN, identification and functional characterization of the PBAN receptor (PBANR) has proven to be elusive. Given the sequence similarity between the active C-terminal regions of PBAN and neuromedin U, it was hypothesized that their respective receptors might also be similar in structure (Park, Y., Kim, Y. J., and Adams, M. E. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 11423-11428). Consequently, utilizing primers constructed from the conserved regions of insect neuromedin U receptor homologues, a full-length 2780-nucleotide clone encoding a 46-kDa G protein-coupled receptor was amplified from a Bombyx mori pheromone gland cDNA library. Tissue distribution analyses revealed that the receptor transcript is specific to the pheromone gland where it undergoes significant up-regulation in the day preceding eclosion. When transiently expressed in Sf9 cells, the B. mori PBANR responds to PBAN by mobilizing extracellular calcium in a dose-dependent manner. Confocal microscopic studies demonstrated the specificity of enhanced green fluorescent protein-tagged B. mori PBANR for PBAN and showed that PBAN induces internalization of the PBANR.PBAN complex. The rapid onset of internalization is mediated by a 67-amino acid C-terminal extension absent in the cloned Helicoverpa zea PBANR, which suggests that receptor internalization in that species likely utilizes a different mechanism. From these results, we have concluded that the cloned receptor gene encodes the B. mori PBANR and that it is both structurally and functionally distinct from the H. zea PBANR.
Highlights
In most Lepidoptera, pheromone biosynthesis is regulated by a neuropeptide termed pheromone biosynthesis activating neuropeptide (PBAN)
The protein, which we have designated as B. mori PBAN receptor (PBANR), contains several features characteristic of the rhodopsin-like family of G protein-coupled receptor (GPCR) [32] such as a number of highly conserved residues implicated in structural stabilization of the receptor and/or in promoting G protein-coupling (Arg137), the presence of N-linked glycosylation sites (Asn18 and Asn21) in the extracellular N terminus, a potential disulfide bridge (Cys112-Cys193) connecting the first two extracellular loops, as well as numerous potential phosphorylation sites throughout the intracellular loops and the C terminus
An ionic interaction involving the highly conserved Arg in the D/ERY motif located at the C-terminal end of TM3 (Arg137 in B. mori PBANR) and a Glu at the C terminus of TM6 has been proposed to stabilize the inactive state of some GPCRs [33, 34]
Summary
In B. mori, a similar rise in the levels of the second messenger is not required [11] for PBANinduced reduction of the fatty-acyl group [12], i.e. the final step in the B. mori pheromone biosynthetic pathway These findings could be an indication that the PBAN receptors (PBANRs) of these two insect species are functionally different. Based on the sequence similarities between the C-terminal pentapeptide motifs necessary for biological activity in NmU, FRPRNamide [16], and PBAN, FSPRLamide, it had been postulated that the respective receptors may likewise exhibit regions of similarity In support of this hypothesis, Hewes and Taghert [17] had earlier reported the presence of multiple GPCRs in the Drosophila genome homologous with the NmU/arginine vasopressin family of receptors specific for peptides containing the C-terminal PRXamide motif. We provide the first evidence that PBAN induces rapid internalization of the B. mori PBANR and that the C-terminal extension is essential for that internalization
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