Mutagenesis studies of neuropeptide S identify a suitable peptide tracer for neuropeptide S receptor binding studies and peptides selectively activating the I 107 variant of human neuropeptide S receptor

Abstract

Neuropeptide S and its receptor represent a novel neurotransmitter system mainly expressed in the brain. A single nucleotide polymorphism in the first extracellular loop (I107) increases the potency of neuropeptide S and has been identified for both the human neuropeptide S receptor short (A) and long (B) C-terminal forms. Preliminary human genetic studies link this polymorphism to asthma, panic disorders and altered sleep behavior. No polymorphism or splice variants have been reported for the rat neuropeptide S receptor, however it carries an isoleucine at position 107. To identify a suitable tracer for neuropeptide S receptor binding and investigate the role of specific amino acids within neuropeptide S we carried out mutagenesis of the peptide and assessed the ability of the mutations to stimulate calcium release in HEK293 cells expressing human neuropeptide S receptor variants (A, B, AI 107, BI 107) and rat neuropeptide S receptor. Replacement of threonine at position 8 by arginine and methionine at position 10 by tyrosine resulted in a mutant peptide slightly more potent on all neuropeptide S receptor variants compared to neuropeptide S and more importantly the iodinated mutant peptide was found to be a suitable tracer for binding studies with improved signal to noise ratio and stability compared to [ 125I–Y 10] neuropeptide S. Replacement of serine at position 1 of neuropeptide S peptide by arginine resulted in a complete loss of potency for the neuropeptide S receptor (long and short form) but not for the I 107 receptor variants (long and short) or rat neuropeptide S receptor.