AUTHOR=Kozma Mihika T. , Pérez-Moreno Jorge L. , Gandhi Neha S. , Hernandez Jeppesen Luisanna , Durica David S. , Ventura Tomer , Mykles Donald L. 

TITLE=In silico analysis of crustacean hyperglycemic hormone family G protein-coupled receptor candidates

JOURNAL=Frontiers in Endocrinology

VOLUME=14

YEAR=2024

URL=https://www.frontiersin.org/journals/endocrinology/articles/10.3389/fendo.2023.1322800

DOI=10.3389/fendo.2023.1322800

ISSN=1664-2392

ABSTRACT=<p>Ecdysteroid molting hormone synthesis is directed by a pair of molting glands or Y-organs (YOs), and this synthesis is inhibited by molt-inhibiting hormone (MIH). MIH is a member of the crustacean hyperglycemic hormone (CHH) neuropeptide superfamily, which includes CHH and insect ion transport peptide (ITP). It is hypothesized that the MIH receptor is a Class A (Rhodopsin-like) G protein-coupled receptor (GPCR). The YO of the blackback land crab, <italic>Gecarcinus lateralis</italic>, expresses 49 Class A GPCRs, three of which (Gl-CHHR-A9, -A10, and -A12) were provisionally assigned as CHH-like receptors. CrusTome, a transcriptome database assembled from 189 crustaceans and 12 ecdysozoan outgroups, was used to deorphanize candidate MIH/CHH GPCRs, relying on sequence homology to three functionally characterized ITP receptors (BNGR-A2, BNGR-A24, and BNGR-A34) in the silk moth, <italic>Bombyx mori</italic>. Phylogenetic analysis and multiple sequence alignments across major taxonomic groups revealed extensive expansion and diversification of crustacean A2, A24, and A34 receptors, designated <underline>C</underline>HH <underline>F</underline>amily <underline>R</underline>eceptor <underline>C</underline>andidates (CFRCs). The A2 clade was divided into three subclades; A24 clade was divided into five subclades; and A34 was divided into six subclades. The subclades were distinguished by conserved motifs in extracellular loop (ECL) 2 and ECL3 in the ligand-binding region. Eleven of the 14 subclades occurred in decapod crustaceans. In <italic>G. lateralis</italic>, seven CFRC sequences, designated Gl-CFRC-A2α1, -A24α, -A24β1, -A24β2, -A34α2, -A34β1, and -A34β2, were identified; the three A34 sequences corresponded to Gl-GPCR-A12, -A9, and A10, respectively. ECL2 in all the CFRC sequences had a two-stranded β-sheet structure similar to human Class A GPCRs, whereas the ECL2 of decapod CFRC-A34β1/β2 had an additional two-stranded β-sheet. We hypothesize that this second β-sheet on ECL2 plays a role in MIH/CHH binding and activation, which will be investigated further with functional assays.</p>