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

Heather G Marco,Jozef Vanden Broeck,Gerd Gäde,Heleen Verlinden

doi:10.1038/s41598-017-06805-9

Heather G Marco, Jozef Vanden Broeck + Show 2 more

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https://doi.org/10.1038/s41598-017-06805-9

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Journal: Scientific Reports	Publication Date: Jul 31, 2017
Citations: 26	License type: open-access

Affiliation: University of Cape Town, KU Leuven

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.

Highlights

The common water flea Daphnia pulex (Class: Branchiopoda, Leydig 1860) is a planktonic filter-feeding crustacean that inhabits freshwater bodies
red pigment concentrating hormone (RPCH) is structurally similar to an insect neuropeptide, the adipokinetic hormone (AKH), which is produced in cerebral neurohaemal organs called the corpora cardiaca, and which has an effect on intermediate metabolism in insects[15]
Several sequence predictions have been made for the preprohormone sequence of the red pigment-concentrating hormone in D. pulex (Dappu-RPCH) and it’s G protein-coupled receptor (GPCR) (Dappu-RPCHR)

Summary

Introduction

The common water flea Daphnia pulex (Class: Branchiopoda, Leydig 1860) is a planktonic filter-feeding crustacean that inhabits freshwater bodies. The entire genome of D. pulex is sequenced and represents the first crustacean genome available for data mining[7], making it very interesting for comparative bioinformatic analyses, especially with a focus on peptide hormones[8, 9] This class of hormones, peptides originating from neuroendocrine centres, such as the X-organ – sinus gland complex, has been well-studied for decades in various infraorders of decapod crustaceans. Despite the paucity of information on crustacean RPCHR sequences, the RPCH signalling cascade has been investigated in several decapod crustacean species on a physiological and biochemical level[30] From one such recent investigation with a G-protein antagonist, it was experimentally deduced that Panbo-RPCH lowers cAMP levels in ovarian chromatophores of the freshwater shrimp, Macrobrachium olfersi[31]. In insects on the other hand, the AKHs seem to increase intracellular Ca2+ and cAMP levels[32]

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