Functional Elucidation and Characterization of GPA2/GPB5 and Its Receptor LGR1 in the Mosquito, Aedes Aegypti
Rocco, David Angelo
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Glycoprotein hormones govern key aspects of physiology like reproduction, growth and development as well as metabolism in vertebrates. In 2002, a novel glycoprotein hormone formed by the heterodimeric interactions of GPA2 and GPB5 (GPA2/GPB5) subunits was discovered. Unlike other glycoprotein hormones, GPA2/GPB5 homologs are present in most vertebrate and invertebrate genomes, however its exact function remains elusive. The present set of studies aimed to elucidate the function of GPA2/GPB5 and its receptor LGR1 in the disease vector mosquito, Aedes aegypti. First, the tissue-specific expression and cellular localization of LGR1 was characterized to identify potential targets for GPA2/GPB5 in adult mosquitoes. LGR1 transcript and immunoreactivity was detected in gut epithelia, which supported a pre-established ionoregulatory / osmoregulatory role in the alimentary canal of adult mosquitoes. Interestingly, strong and regionalized immunoreactivity was detected in the female ovaries and male testes suggesting GPA2/GPB5 signalling may function to regulate reproduction in A. aegypti. In the male testes, the subcellular expression of LGR1 during spermatogenesis was characterized further, and results found LGR1 immunoreactivity specifically localized to the plasma membrane of the centriole adjunct, which contains proteins important for flagellar formation in developing spermatids. Using RNA interference, downregulation of LGR1 led to sperm defects, such as shortened flagella. Moreover, newly emerged males were characterized with significantly less spermatozoa and rendered less fertile. The GPA2 and GPB5 subunits were found to be co-expressed in the abdominal ganglia of the central nervous system in both male and female adult mosquitoes. Mammalian cell lines were then employed to produce recombinant A. aegypti GPA2 and GPB5 proteins and study subunit interactions, ligand-receptor and interactions and determine downstream signalling events upon LGR1 activation. Results demonstrated A. aegypti GPA2/GPB5 are capable of subunit homodimerization in vitro. However, only tethered A. aegypti GPA2/GPB5, rather than individual subunits alone, were required to activate LGR1, which interestingly, appears to involve coupling to an inhibitory G protein. Together, these studies have demonstrated that signalling involving the glycoprotein hormone system is imperative for male reproductive-related processes in adult mosquitoes, a largely understudied research area, and presents novel insight to the function of its homologs in other animals.