Immunohistochemical mapping and transcript expression of the GPA2/GPB5 receptor in tissues of the adult mosquito, Aedes aegypti.

ABSTRACT

GPA2/GPB5 is a glycoprotein hormone found in most bilateral metazoans including the mosquito, Aedes aegypti. To elucidate physiological roles and functions of GPA2/GPB5, we aim to identify prospective target tissues by examining the tissue- and sex-specific expression profile of its receptor, the leucine-rich repeat-containing G protein-coupled receptor 1 (LGR1) in the adult mosquito. Western analyses using a heterologous system with CHO-K1 cells, transiently expressing A. aegypti LGR1, yielded a 112-kDa monomeric band and high-molecular weight multimers, which associated with membrane-protein fractions. Moreover, immunoblot analyses on protein isolated from HEK 293 T cells stably expressing a fusion construct of A. aegypti LGR1-EGFP (LGR1 105 kDa+EGFP 27 kDa) yielded a band with a measured molecular weight of 139 kDa that also associated with membrane-protein fractions and upon deglycosylation, migrated as a lower molecular weight band of 132 kDa. Immunocytochemical analysis of HEK 293 T cells stably expressing this fusion construct confirmed EGFP fluorescence and LGR1-like immunoreactivity colocalized primarily to the plasma membrane. Immunohistochemical mapping in adult mosquitoes revealed LGR1-like immunoreactivity is widespread in the alimentary canal. Importantly, LGR1-like immunoreactivity localizes specifically to basolateral regions of epithelia and, in some regions, appeared as punctate intracellular staining, which together indicates a potential role in feeding and/or hydromineral balance. LGR1 transcript expression was also detected in gut regions that exhibited strong LGR1-like immunoreactivity. Interestingly, LGR1 transcript expression and strong LGR1-like immunoreactivity was also identified in reproductive tissues including the testes and ovaries, which together suggests a potential role linked to spermatogenesis and oogenesis in male and female mosquitoes, respectively.