Identification of the domains in RXFP4 (GPCR142) responsible for the high affinity binding and agonistic activity of INSL5 at RXFP4 compared to RXFP3 (GPCR135).
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.
Zhu
Jessica
J
Johnson & Johnson Pharmaceutical Research and Development, L.L.C., 3210 Merryfield Row, San Diego, CA 92121, USA.
Kuei
Chester
C
Sutton
Steven
S
Kamme
Fredrik
F
Yu
Jingxue
J
Bonaventure
Pascal
P
Atack
John
J
Lovenberg
Timothy W
TW
Liu
Changlu
C
D001665:Binding Sites; D006801:Humans; D007328:Insulin / Q000378:metabolism / Q000494:pharmacology*; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000378:metabolism / Q000494:pharmacology*; D043562:Receptors, G-Protein-Coupled / Q000819:agonists / Q000737:chemistry* / Q000378:metabolism; D018000:Receptors, Peptide / Q000819:agonists / Q000737:chemistry* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D012065:Relaxin / Q000378:metabolism / Q000494:pharmacology
D007328:Insulin; C084797:Leydig insulin-like protein; D011506:Proteins; C480238:RLN3 protein, human; C410092:RXFP3 protein, human; C480239:RXFP4 protein, human; D043562:Receptors, G-Protein-Coupled; D018000:Receptors, Peptide; D011993:Recombinant Fusion Proteins; D012065:Relaxin