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.

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Acknowledgements

This research was funded by institutional new investigator start-up funds and a Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grant to J.P.P. The authors are indebted to Carmela Curcuruto and Andreea Matei for technical assistance with protein electrophoresis and mosquito dissections, respectively. In addition, the authors would like to thank Professor Andrew Donini (Biology, York University) for providing the Alexa 488-phalloidin.

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Correspondence to Jean-Paul V. Paluzzi.

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Rocco, D.A., Kim, D.H. & Paluzzi, JP.V. Immunohistochemical mapping and transcript expression of the GPA2/GPB5 receptor in tissues of the adult mosquito, Aedes aegypti . Cell Tissue Res 369, 313–330 (2017). https://doi.org/10.1007/s00441-017-2610-3

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Keywords

  • GPA2/GPB5
  • Glycoprotein hormone
  • LGR1
  • Leucine-rich repeat-containing G protein-coupled receptor 1
  • Aedes aegypti
  • Mosquito