Liu Changlu, Kuei Chester, Sutton Steven, Chen Jingcai, Bonaventure Pascal, Wu Jiejun, Nepomuceno Diane, Kamme Fredrik, Tran Da-Thao, Zhu Jessica, Wilkinson Tracey, Bathgate Ross, Eriste Elo, Sillard Rannar, Lovenberg Timothy W
Johnson & Johnson Pharmaceutical Research and Development, San Diego, California 92121, USA.
J Biol Chem. 2005 Jan 7;280(1):292-300. doi: 10.1074/jbc.M409916200. Epub 2004 Nov 2.
Insulin-like peptide 5 (INSL5) is a peptide that belongs to the relaxin/insulin family, and its receptor has not been identified. In this report, we demonstrate that INSL5 is a specific agonist for GPCR142. Human INSL5 displaces the binding of (125)I-relaxin-3 to GPCR142 with a high affinity (K(i) = 1.5 nM). In a saturation binding assay, (125)I-INSL5 binds GPCR142 with a K(d) value of 2.5 nM. In functional guanosine (gamma-thio)-triphosphate binding and cAMP accumulation assays, INSL5 potently activates GPCR142 with EC(50) values of 1.3 and 1.2 nM, respectively. In addition, INSL5 stimulates Ca(2+) mobilization in HEK293 cells expressing GPCR142 and G alpha(16). Overall, INSL5 behaves as an agonist for GPCR142 similar to relaxin-3. However, unlike relaxin-3, which is also a potent agonist for GPCR135 and LGR7, INSL5 does not activate either GPCR135 or LGR7. INSL5 inhibits (125)I-relaxin-3 binding to GPCR135 with a low potency (K(i) = 500 nM). A functional assay shows that INSL5 (1 microm) is a weak antagonist for GPCR135. In addition, INSL5 (up to 1 microm) shows no affinity or activity at LGR7 or LGR8 either in a binding assay or a bio-functional assay. Previously, we have demonstrated that GPCR142 mRNA is expressed in peripheral tissues, particularly in the colon. Here we show that INSL5 mRNA is expressed in many peripheral tissues, similar to GPCR142. The high affinity interaction between INSL5 and GPCR142 coupled with their co-evolution and partially overlapping tissue expression patterns strongly suggest that INSL5 is an endogenous ligand for GPCR142.
胰岛素样肽5（INSL5）是一种属于松弛素/胰岛素家族的肽，其受体尚未被鉴定出来。在本报告中，我们证明INSL5是GPCR142的特异性激动剂。人INSL5以高亲和力（K(i)=1.5 nM）取代(125)I-松弛素-3与GPCR142的结合。在饱和结合试验中，(125)I-INSL5以2.5 nM的K(d)值结合GPCR142。在功能性鸟苷（γ-硫代）-三磷酸结合和环磷酸腺苷积累试验中，INSL5分别以1.3和1.2 nM的EC(50)值有效激活GPCR142。此外，INSL5刺激表达GPCR142和Gα(16)的HEK293细胞中的Ca(2+)动员。总体而言，INSL5表现为与松弛素-3类似的GPCR142激动剂。然而，与也是GPCR135和LGR7的强效激动剂的松弛素-3不同，INSL5不激活GPCR135或LGR7。INSL5以低效力（K(i)=500 nM）抑制(125)I-松弛素-3与GPCR135的结合。功能试验表明，1 μM的INSL5是GPCR135的弱拮抗剂。此外，在结合试验或生物功能试验中，高达1 μM的INSL5在LGR7或LGR8上均无亲和力或活性。此前，我们已证明GPCR142 mRNA在外周组织中表达，特别是在结肠中。在此我们表明，INSL5 mRNA与GPCR142类似，在许多外周组织中表达。INSL5与GPCR142之间的高亲和力相互作用及其共同进化和部分重叠的组织表达模式强烈表明，INSL5是GPCR142的内源性配体。
