Conventional approaches to identify secreted factors that regulate homeostasis are limited in their abilities to identify the tissues/cells of origin and destination. We established a platform to identify secreted protein trafficking between organs using an engineered biotin ligase (BirA*G3) that biotinylates, promiscuously, proteins in a subcellular compartment of one tissue. Subsequently, biotinylated proteins are affinity-enriched and identified from distal organs using quantitative mass spectrometry. Applying this approach in Drosophila, we identify 51 muscle-secreted proteins from heads and 269 fat body-secreted proteins from legs/muscles, including CG2145 (human ortholog ENDOU) that binds directly to muscles and promotes activity. In addition, in mice, we identify 291 serum proteins secreted from conditional BirA*G3 embryo stem cell-derived teratomas, including low-abundance proteins with hormonal properties. Our findings indicate that the communication network of secreted proteins is vast. This approach has broad potential across different model systems to identify cell-specific secretomes and mediators of interorgan communication in health or disease.
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.
Glycoprotein hormone receptors mediate a diverse range of physiological functions in vertebrate and invertebrate organisms. The heterodimeric glycoprotein hormone GPA2/GPB5 and its receptor LGR1, constitute a recently discovered invertebrate neuroendocrine signaling system that remains to be functionally characterized. We previously reported that LGR1 is expressed in the testes of adult Aedes aegypti mosquitoes, where its immunoreactivity is particularly regionalized. Here, we show that LGR1 immunoreactivity is associated with the centriole adjunct of spermatids and is observed transiently during spermatogenesis in mosquitoes, where it may act to mediate the regulation of flagellar development. RNA interference to downregulate LGR1 expression was accomplished by feeding mosquito larvae with bacteria that produced LGR1-specific dsRNA, which led to defects in spermatozoa, characterized with shortened flagella. LGR1 knockdown mosquitoes also retained ∼60% less spermatozoa in reproductive organs and demonstrated reduced fertility compared to controls. To date, the endocrine regulation of spermatogenesis in mosquitoes remains an understudied research area. The distribution of LGR1 and detrimental effects of its knockdown on spermatogenesis in A. aegypti indicates that this heterodimeric glycoprotein hormone signaling system contributes significantly to the regulation of male reproductive biology in this important disease-vector.
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