Characterisation and pharmacological analysis of a crustacean G protein-coupled receptor: the red pigment-concentrating hormone receptor of Daphnia pulex

Abstract

This is the first pharmacological characterisation of a neuropeptide G protein-coupled receptor (GPCR) in a crustacean. We cloned the ORF of the red pigment-concentrating hormone from a German strain of Daphnia pulex (Dappu-RPCH), as well as that of the cognate receptor (Dappu-RPCHR). Dappu-RPCHR has the hallmarks of the rhodopsin superfamily of GPCRs, and is more similar to insect adipokinetic hormone (AKH) receptor sequences than to receptor sequences for AKH/corazonin-like peptide or corazonin. We provide experimental evidence that Dappu-RPCH specifically activates the receptor (EC50 value of 65 pM) in a mammalian cell-based bioluminescence assay. We further characterised the properties of the ligands for the Dappu-RPCHR by investigating the activities of a variety of naturally-occurring peptides (insect AKH and crustacean RPCH peptides). The insect AKHs had lower EC50 values than the crustacean RPCHs. In addition, we tested a series of Dappu-RPCH analogues, where one residue at a time is systematically replaced by an alanine to learn about the relative importance of the termini and side chains for activation. Mainly amino acids in positions 1 to 4 and 8 of Dappu-RPCH appear responsible for effective activation of Dappu-RPCHR. The substitution of Phe4 in Dappu-RPCH had the most damaging effect on its agonistic activity.

Structural diversity of adipokinetic hormones in the hyperdiverse coleopteran Cucujiformia

“…Harax‐AKH in the Coccinelloidea and Trica‐AKH in the Tenebrionoidea (see Table ). Interestingly, the latter 2 peptides share the C‐terminal amidated tetrapeptide completely (STGW amide) or mostly (STDW amide) with the ancestral Schgr‐AKH‐II; a substitution at position 7 has previously not shown much difference in AKH receptor activation in vitro (Caers et al, ; Marco, Verlinden, Vanden Broeck, & Gäde, ; Marchal et al, ) or in vivo (Gäde & Hayes, ; Marco & Gäde, ).…”

Transcriptomic analysis of crustacean neuropeptide signaling during the moult cycle in the green shore crab, Carcinus maenas

“…Whilst there are studies which have sought to identify crustacean neuropeptide GPCRs based on sequence similarity to known neuropeptide GPCRs (e.g. [ 29 , 32 , 51 ]), until recently no crustacean neuropeptide GPCR had been de-orphanised [ 30 , 31 ]. For this reason predicted receptors were identified as a result of searching using search terms for Tribolium casteneum and Drosophila receptors, for which functional de-orphanisation has been achieved.…”

“…Through NGS and transcriptome mining within crustaceans, orthologs of insect neuropeptides have been documented [ 27 ], but functional roles for these peptides remain lacking. Similarly, whilst the receptors for numerous neuropeptides have been functionally demonstrated for insects, only a few G protein-coupled receptors (GPCRs) have been de-orphanised among crustaceans [ 30 , 31 ] and so identification of their GPCRs lags considerably behind. Indeed, only recently have efforts been focussed on revealing the structural identity of putative GPCRs in a crustacean, the spiny lobster Sagmariasus verreauxi [ 32 ].…”

Insights on Molecular Mechanisms of Ovarian Development in Decapod Crustacea: Focus on Vitellogenesis-Stimulating Factors and Pathways

“…Buckley et al ( 106 ) proposed the identity of a putative RPCH-like receptor to be a G-protein coupled receptor based on in silico mining of transcriptomic data, with this receptor being expressed during the metamorphic molt of the spiny lobster, Sagmariasus verreauxi . Marco et al ( 107 ) pharmacologically characterized the red pigment-concentrating hormone receptor (RPCHR) from the water flea, Daphnia pulex , and revealed it to be similar in terms of sequence to insect adipokinetic hormone (AKH) receptor. This was the first report of a deorphanized neuropeptide G protein-coupled receptor (GPCR) in crustaceans.…”