Amino Acids

, Volume 46, Issue 5, pp 1393–1402 | Cite as

The highly conserved negatively charged Glu141 and Asp145 of the G-protein-coupled receptor RXFP3 interact with the highly conserved positively charged arginine residues of relaxin-3

  • Wei-Jie Zhang
  • Xin-Yi Wang
  • Yu-Qi Guo
  • Xiao Luo
  • Xue-Juan Gao
  • Xiao-Xia Shao
  • Ya-Li Liu
  • Zeng-Guang Xu
  • Zhan-Yun Guo
Original Article
First Online:
Received:
Accepted:

Abstract

Relaxin-3 is a newly identified insulin/relaxin superfamily peptide that plays a putative role in the regulation of food intake and stress response by activating its cognate G-protein-coupled receptor RXFP3. Relaxin-3 has three highly conserved arginine residues, B12Arg, B16Arg and B26Arg. We speculated that these positively charged arginines may interact with certain negatively charged residues of RXFP3. To test this hypothesis, we first replaced the negatively charged residues in the extracellular domain of RXFP3 with arginine, respectively. Receptor activation assays showed that arginine replacement of Glu141 or Asp145, especially Glu141, significantly decreased the sensitivity of RXFP3 to wild-type relaxin-3. In contrast, arginine replacement of other negatively charged extracellular residues had little effect. Thus, we deduced that Glu141 and Asp145, locating at the extracellular end of the second transmembrane domain, played a critical role in the interaction of RXFP3 with relaxin-3. To identify the ligand residues interacting with the negatively charged EXXXD motif of RXFP3, we replaced the three conserved arginines of relaxin-3 with negatively charged glutamate or aspartate, respectively. The mutant relaxin-3s retained the native structure, but their binding and activation potencies towards wild-type RXFP3 were decreased significantly. The compensatory effects of the mutant relaxin-3s towards mutant RXFP3s suggested two probable interaction pairs during ligand–receptor interaction: Glu141 of RXFP3 interacted with B26Arg of relaxin-3, meanwhile Asp145 of RXFP3 interacted with both B12Arg and B16Arg of relaxin-3. Based on these results, we proposed a relaxin-3/RXFP3 interaction model that shed new light on the interaction mechanism of the relaxin family peptides with their receptors.

Keywords

Insulin superfamily Ligand–receptor interaction Mutagenesis Relaxin-3 RXFP3 

Abbreviations

CD

Circular dichroism

CRE

cAPM response element

GPCR

G-protein-coupled receptor

HEK293T

Human embryonic kidney 293T

INSL5

Insulin-like peptide 5

INSL7

Insulin-like peptide 7

HPLC

High-performance liquid chromatography

TMD

Transmembrane domain

UV

Ultraviolet

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Notes

Acknowledgments

This work was supported by grants from the National Natural Science Foundation of China (31270824, 30970609), the National Basic Research Program of China (973 Program, No. 2010CB912604), and the Fundamental Research Funds for the Central Universities. We thank Promega Corporation for providing the plasmids encoding nanoluciferase.

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

726_2014_1705_MOESM1_ESM.doc (2.8 mb)
Supplementary material 1 (DOC 2903 kb)

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Copyright information

© Springer-Verlag Wien 2014

Authors and Affiliations

  • Wei-Jie Zhang
    • 1
    • 2
  • Xin-Yi Wang
    • 1
  • Yu-Qi Guo
    • 1
  • Xiao Luo
    • 1
  • Xue-Juan Gao
    • 1
  • Xiao-Xia Shao
    • 1
  • Ya-Li Liu
    • 2
  • Zeng-Guang Xu
    • 2
  • Zhan-Yun Guo
    • 1
    • 2
  1. 1.Institute of Protein Research, College of Life Sciences and TechnologyTongji UniversityShanghaiChina
  2. 2.Central Laboratory, Shanghai East HospitalTongji University School of MedicineShanghaiChina

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