Regulatory mechanisms underlying pheromone biosynthesis activating neuropeptide (PBAN)-induced internalization of the Bombyx mori PBAN receptor.
Internalization of the Bombyx mori pheromone biosynthesis activating neuropeptide receptor (PBANR) has been attributed to the presence of a 67 amino acid C-terminal extension absent in PBANRs from Helicoverpa. To identify the structural motif(s) responsible for internalization, a series of truncation mutants fused with enhanced green fluorescent protein were constructed and transiently expressed in insect Sf9 cells. Confocal microscopy analyses revealed that truncation at Gly357 severely inhibited internalization while truncation at Gln367 did not, indicating that the PBANR internalization motif resides between Gly357-Gln367. Alanine substitution studies suggest that Tyr360 and Leu363 may constitute a YXXL endosomal targeting motif that facilitates endocytosis, however, this motif does not appear to be the primary determinant; an indication that multiple sites are involved. Furthermore, we determined that internalization of the PBANR proceeds via a clathrin-dependent pathway, is dependent on the influx of extracellular calcium, and likely does not involve a G protein-coupled receptor kinase.
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
Matsumoto
Shogo
S
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D012831:Bombyx / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D004705:Endocytosis / Q000502:physiology*; D005786:Gene Expression Regulation / Q000502:physiology*; D008969:Molecular Sequence Data; D009479:Neuropeptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010675:Pheromones / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D018013:Receptors, Neuropeptide / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*; D013329:Structure-Activity Relationship; D014018:Tissue Distribution
D009479:Neuropeptides; D010675:Pheromones; D018013:Receptors, Neuropeptide; D011994:Recombinant Proteins; C079709:pheromone biosynthesis activating neuropeptide, Helicoverpa zea