Hamster Melatonin Receptors: Cloning and Binding Characterization of MT1 and Attempt to Clone MT2

• Published in: International journal of molecular sciences 2018-07, Vol.19 (7), p.1957
• Main Authors: Gautier, Célia, Dufour, Emilie, Dupré, Clémence, Lizzo, Giulia, Caignard, Sarah, Riest-Fery, Isabelle, Brasseur, Chantal, Legros, Céline, Delagrange, Philippe, Nosjean, Olivier, Simonneaux, Valérie, Boutin, Jean, Guenin, Sophie-Pénélope
• Format: Article
• Language: English
• Subjects: Amino acids; Bats; Binding; Cloning; Conserved sequence; Crystallization; Hamsters; Melatonin; Melatonin receptors; Proteins; Sheep; Stop codon
• ISSN: 1422-0067; 1661-6596; 1422-0067
• DOI: 10.3390/ijms19071957

For many years, it was of interest to identify the sequences encoding the two melatonin receptors (MT1 and MT2) from various species. After publishing the basic molecular characterization of the human, rat, mouse, sheep, and platypus MT1, MT2, or Mel1c receptors, we began cloning the genes from other animals, such as birds, bats, and vipers. The goal was to advance the receptor crystallization, which could greatly contribute the understanding of the sequence/stability relationship. European hamster MT1 receptor was cloned for the first time from this gender, was expressed in stable form in cells, and its binding characterized with a sample of 19 melatonin ligands. Siberian hamster (Phodopus sungorus) expresses a non-functional MT2. We observed that unlike this hamster, the European hamster (Cricetus cricetus) does not have a stop codon in the MT2 sequence. Thus, we undertook the tedious task of cloning the MT2 receptor. We partially succeeded, sequencing the complete exon 2 and a fragment of exon 1 (from putative amino acids 12 to 38 and 77 to 323), after several years of efforts. In order to show that the protein parts we cloned were capable to sustain some binding capacities, we designed a chimeric MT2 receptor using a consensus sequence to replace the unknown amino acids, based on other small rodent MT2 sequences. This chimeric construct could bind melatonin in the nanomolar range. This work is meant to be the basis for attempts from other laboratories of the community to determine the complete natural sequence of the European hamster MT2 receptor. The present work is the first to show that, among the hamsters, if the Siberian is a natural knockout for MT2, the European one is not.