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.