Cloning and characterization of the pheromone biosynthesis activating neuropeptide receptor from the silkmoth, Bombyx mori. Significance of the carboxyl terminus in receptor internalization.
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.
Hull
J Joe
JJ
Molecular Entomology Laboratory, RIKEN (The Institute of Physical and Chemical Research), Hirosawa 2-1, Wako, Saitama 351-0198, Japan.
Ohnishi
Atsushi
A
Moto
Ken'ichi
K
Kawasaki
Yu
Y
Kurata
Ryuichiro
R
Suzuki
Masataka G
MG
Matsumoto
Shogo
S
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D012831:Bombyx; D002118:Calcium / Q000737:chemistry / Q000378:metabolism; D002460:Cell Line; D003001:Cloning, Molecular; D017931:DNA Primers / Q000737:chemistry; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D015723:Gene Library; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000378:metabolism; D007313:Insecta; D008024:Ligands; D008565:Membrane Proteins / Q000737:chemistry; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D009479:Neuropeptides / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D017434:Protein Structure, Tertiary; D018013:Receptors, Neuropeptide / Q000737:chemistry* / Q000235:genetics*; D017981:Receptors, Neurotransmitter / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D013696:Temperature; D013997:Time Factors
D017931:DNA Primers; D018076:DNA, Complementary; D008024:Ligands; D008565:Membrane Proteins; D009479:Neuropeptides; D018013:Receptors, Neuropeptide; D017981:Receptors, Neurotransmitter; C082444:neuromedin U receptor; C079709:pheromone biosynthesis activating neuropeptide, Helicoverpa zea; D049452:Green Fluorescent Proteins; D002118:Calcium