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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, AI107, BI107) 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–Y10] 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 I107 receptor variants (long and short) or rat neuropeptide S receptor. [Copyright &y& Elsevier]

Abstract: Relaxin-3 is a potent agonist for both G-protein coupled receptors (GPCR) RXFP3 (also known as GPCR135) and RXFP4 (also known as GPCR142) while insulin-like peptides 5 (INSL5) is a selective RXFP4 agonist. INSL5 is also a weak (low affinity) RXFP3 antagonist. RXFP3 and RXFP4 share about 50% homology. We have used gain-of-function (RXFP3 → RXFP4) and loss-of-function (RXFP4 → RXFP3) chimeras to identify the domains critical for the binding and activation induced by INSL5. Replacing extracellular loop (EL) 1 or EL3 of RXFP3 with the corresponding domains from RXFP4 does not change the RXFP3 pharmacological profile. Exchanging the N-terminus and EL2 of RXFP3 with these of RXFP4 results in a chimeric receptor (CR5) with a high affinity for INSL5. However, in contrast to native RXFP4, INSL5 does not elicit an agonist response from CR5. Conversely, replacing the N-terminus and EL2 of RXFP4 with counterparts from RXFP3 (CR15) results in a chimeric receptor for which relaxin-3 and INSL5 are high and low affinity agonists, respectively. Further mutagenesis studies indicate that transmembrane (TM) domains 2, 3 and 5 of RXFP4 are critical determinants of functional receptor activation by INSL5. Replacement of TM2, 3, and 5 of RXFP3 with equivalent domains from RXFP4 results in a chimeric receptor that can be activated by INSL5. These results suggest that the N-terminus and EL2 domains of RXFP3 and RXFP4 are involved in ligand binding while TM2, 3, and 5 are critical for receptor activation. [Copyright &y& Elsevier]