title: Identification of relaxin-3/INSL7 as an endogenous ligand for the orphan G-protein-coupled receptor GPCR135.
abstract: GPCR135, publicly known as somatostatin- and angiotensin-like peptide receptor, is expressed in the central nervous system and its cognate ligand(s) has not been identified. We have found that both rat and porcine brain extracts stimulated 35S-labeled guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) incorporation in cells over-expressing GPCR135. Multiple rounds of extraction, purification, followed by N-terminal sequence analysis of the ligand from porcine brain revealed that the ligand is a product of the recently identified gene, relaxin-3 (aka insulin-7 or INSL7). Recombinant human relaxin-3 potently stimulates GTPgammaS binding and inhibits cAMP accumulation in GPCR135 overexpressing cells with EC50 values of 0.25 and 0.35 nM, respectively. 125I-Relaxin-3 binds GPCR135 at high affinity with a Kd value of 0.31 nM. Relaxin-3 is the only member of the insulin/relaxin superfamily that can activate GPCR135. In situ hybridization showed that relaxin-3 mRNA is predominantly expressed in the dorsomedial ventral tegmental nucleus of the brainstem (aka nucleus incertus), as well as in discrete cells in the lateral periaqueductal gray and in the central gray nucleus. GPCR135 is expressed abundantly in the hypothalamus with discrete expression in the paraventricular nucleus of the hypothalamus and supraoptic nucleus, as well as in the cortex, septal nucleus, and preoptical area. Relaxin-3 has previously been shown to bind and activate the LGR7 relaxin receptor. However, we believe that neuroanatomical colocalization of GPCR135 and relaxin-3, coupled with a clear high affinity interaction, suggest that GPCR135 is the receptor for relaxin-3. The identification of relaxin-3 as the ligand for GPCR135 provides the framework for the discovery of a new brainstem/hypothalamus circuitry.
authors.affiliation: Johnson & Johnson Pharmaceutical Research and Development, L.L.C., San Diego, California 92121, USA. cliu9@prdus.jnj.com
authors.forename: Changlu
authors.initials: C
authors.lastname: Liu
authors.forename: Elo
authors.initials: E
authors.lastname: Eriste
authors.forename: Steven
authors.initials: S
authors.lastname: Sutton
authors.forename: Jingcai
authors.initials: J
authors.lastname: Chen
authors.forename: Barbara
authors.initials: B
authors.lastname: Roland
authors.forename: Chester
authors.initials: C
authors.lastname: Kuei
authors.forename: Niven
authors.initials: N
authors.lastname: Farmer
authors.forename: Hans
authors.initials: H
authors.lastname: Jörnvall
authors.forename: Rannar
authors.initials: R
authors.lastname: Sillard
authors.forename: Timothy W
authors.initials: TW
authors.lastname: Lovenberg
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000378:metabolism; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism; D003001:Cloning, Molecular; D006224:Cricetinae; D000242:Cyclic AMP / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006801:Humans; D007328:Insulin / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D008957:Models, Genetic; D008969:Molecular Sequence Data; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology*; D011994:Recombinant Proteins / Q000378:metabolism; D012065:Relaxin / Q000737:chemistry / Q000502:physiology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013552:Swine; D014018:Tissue Distribution; D014466:Ultraviolet Rays
chemical_list: D007328:Insulin; D008024:Ligands; C480238:RLN3 protein, human; D012333:RNA, Messenger; C410092:RXFP3 protein, human; D043562:Receptors, G-Protein-Coupled; D011994:Recombinant Proteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D012065:Relaxin; D000242:Cyclic AMP; D002118:Calcium