Expression Profiling, Downstream Signaling and Subunit Interactions of GPA2/GPB5 in the Adult MosquitoAedes aegypti

Abstract

GPA2/GPB5 and its receptor constitute a glycoprotein hormone-signalling system native to the genomes of most vertebrate and invertebrate organisms, including humans and mosquitoes. Unlike the well-studied gonadotropins and thyrotropin, the exact function of GPA2/GPB5 is unclear, and whether it elicits its functions as heterodimers, homodimers or as independent monomers remains unknown. Here, the glycoprotein hormone signalling system was investigated in adult mosquitoes, where GPA2 and GPB5 subunit transcripts co-localized to bilateral pairs of neuroendocrine cells within the first five abdominal ganglia of the central nervous system. Unlike human GPA2/GPB5 that demonstrated strong heterodimerization between subunits, the GPA2/GPB5 subunits in A. aegypti lacked evidence of heterodimerization when heterologously expressed. Interestingly, cross-linking analysis to determine subunit interactions revealed A. aegypti and H. sapiens GPA2 and GPB5 subunits form homodimers, and treatments with independent subunits did not activate A. aegypti LGR1 or H. sapiens TSH receptor, respectively. Since mosquito GPA2/GPB5 heterodimers were not evident by heterologous expression of independent subunits, a tethered construct was generated for expression of the subunits as a single polypeptide chain to improve heterodimer formation. Our findings revealed A. aegypti LGR1 elicited constitutive activity that elevated levels of cAMP as determined by increased cAMP-dependent luminescence. However, upon treatment with recombinant tethered GPA2/GPB5 heterodimers, an inhibitory G protein (Gi) signalling cascade is initiated and forskolin-induced cAMP production is inhibited. These results provide evidence towards the functional deorphanization of LGR1 and, moreover, further support the notion that GPA2/GPB5 heterodimerization is a requirement for glycoprotein hormone receptor activation.