Ancestral glycoprotein hormone and its cognate receptor present in primitive chordate ascidian: Molecular identification and functional characterization.

The glycoprotein hormone (GPH) system is fundamentally significant in regulating the physiology of chordates, such as thyroid activity and gonadal function. However, the knowledge of the GPH system in the primitive chordate ascidian species is largely lacking. Here, we reported an ancestral GPH system in the ascidian (Styela clava), which consists of GPH α subunit (Sc-GPA2), GPH β subunit (Sc-GPB5), and the cognate leucine-rich repeat-containing G protein-coupled receptor (Sc-GPHR). Comparative structure analysis revealed that distinct from vertebrate GPH β subunits, Sc-GPB5 was less conserved, showing an atypical N-terminal sequence with a type II transmembrane domain instead of a typical signal peptide. By investigating the presence of recombinant Sc-GPA2 and Sc-GPB5 in cell lysates and culture media of HEK293T cells, we confirmed that these two subunits could be secreted out of the cells via distinct secretory pathways. The deglycosylation experiments demonstrated that N-linked glycosylation only occurred on the conserved cysteine residue (N78) of Sc-GPA2, whereas Sc-GPB5 was non-glycosylated. Although Sc-GPB5 exhibited distinct topology and biochemical properties in contrast to its chordate counterparts, it could still interact with Sc-GPA2 to form a heterodimer. The Sc-GPHR was then confirmed to be activated by tethered Sc-GPA2/GPB5 heterodimer on the Gs-cAMP pathway, suggesting that Sc-GPA2/GPB5 heterodimer-initiated Gs-cAMP signaling pathway is evolutionarily conserved in chordates. Furthermore, in situ hybridization and RT-PCR results revealed the co-expression patterns of Sc-GPA2 and Sc-GPB5 with Sc-GPHR transcripts, respectively in ascidian larvae and adults, highlighting the potential functions of Sc-GPA2/GPB5 heterodimer as an autocrine/paracrine neurohormone in regulating metamorphosis of larvae and physiological functions of adults. Our study systematically investigated the GPA2/GPB5-GPHR system in ascidian for the first time, which offers insights into understanding the function and evolution of the GPH system within the chordate lineage.