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The C-terminus of the B-chain of human insulin-like peptide 5 is critical for cognate RXFP4 receptor activity

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Abstract

Insulin-like peptide 5 (INSL5) is an orexigenic peptide hormone belonging to the relaxin family of peptides. It is expressed primarily in the L-cells of the colon and has a postulated key role in regulating food intake. Its G protein-coupled receptor, RXFP4, is a potential drug target for treating obesity and anorexia. We studied the effect of modification of the C-terminus of the A and B-chains of human INSL5 on RXFP4 binding and activation. Three variants of human INSL5 were prepared using solid phase peptide synthesis and subsequent sequential regioselective disulfide bond formation. The peptides were synthesized as C-terminal acids (both A- and B-chains with free C-termini, i.e., the native form), amides (both chains as the C-terminal amide) and one analog with the C-terminus of its A-chain as the amide and the C-terminus of the B-chain as the acid. The results showed that C-terminus of the B-chain is more important than that of the A-chain for RXFP4 binding and activity. Amidation of the A-chain C-terminus does not have any effect on the INSL5 activity. The difference in RXFP4 binding and activation between the three peptides is believed to be due to electrostatic interaction of the free carboxylate of INSL5 with a positively charged residue (s), either situated within the INSL5 molecule itself or in the receptor extracellular loops.

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Abbreviations

INSL5:

Insulin-like peptide 5

mINSL5:

Mouse insulin-like peptide 5

hINSL5:

Human insulin-like peptide 5

IGFI:

Insulin-like growth factor I

IGFII:

Insulin-like growth factor II

INSL3:

Insulin-like peptide 3

INSL4:

Insulin-like peptide 4

INSL6:

Insulin-like peptide 6

RXFP3:

Relaxin-family peptide receptor 3

RXFP4:

Relaxin-family peptide receptor 4

Boc:

tert-Butoxycarbonyl

Acm:

Acetamidomethyl

cAMP:

3′,5′-cyclic adenosine monophosphate

PEG-PS:

Poly(ethylene glycol)-polystyrene

CD:

Circular dichroism

CHO:

Chinese hamster ovary

DMF:

Dimethylformamide

DIEA:

Diisopropylethylamine

Fmoc:

9-fluoroenylmethoxycarbonyl

GPCR:

G-protein coupled receptor

HCTU:

O-(6-chlorobenzotriazol-1-yl)-N,N,N’,N’-tetramethyl uronium hexafluorophosphate

INSLs:

Insulin-like peptides

RXFP:

Relaxin-family peptide

tBu:

tert-Butyl

TIPS:

Triisopropylsilane

TFA:

Trifluoroacetic acid

TFMSA:

Trifluoromethanesulfonic acid

DODT:

3,6-dioxa-1,8-octanedithiol

DPDS:

2,2′-dipyridyldisulfide

BSA:

Bovine serum albumin

HEPES:

4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

ERK:

Extracellular-signal-regulated kinase

DMEM:

Dulbecco’s modified Eagle’s medium

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Acknowledgments

This research was partly funded by NHMRC (Australia) project grants (1023321, 1065481, 1023078) to M. A. H, R. A. D. B and J. D. W and ARC linkage grant (LP120100654) to R. A. H. We are grateful to Tania Ferraro and Sharon Layfield for assistance with biochemical assays. During these studies, M. A. H. was the recipient of Florey Foundation Fellowships. R. A. D. B. is an NHMRC Senior Research Fellow, and J. D. W. is an NHMRC Principal Research Fellow. Studies at the FINMH were supported by the Victorian Government’s Operational Infrastructure Support Program.

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Authors and Affiliations

  1. Howard Florey Research Laboratories, Florey Institute for Neuroscience and Mental Health, University of Melbourne, Melbourne, VIC, Australia

    Nitin A. Patil, Ross A. D. Bathgate, Julien Tailhades, John D. Wade & Mohammed Akhter Hossain

  2. School of Chemistry, Bio21 Institute, University of Melbourne, Melbourne, VIC, Australia

    Nitin A. Patil, Frances Separovic, John D. Wade & Mohammed Akhter Hossain

  3. Department of Biochemistry and Molecular Biology, University of Melbourne, Melbourne, VIC, Australia

    Ross A. D. Bathgate

  4. Monash Institute of Pharmaceutical Sciences, Parkville, VIC, Australia

    Martina Kocan, Sheng Yu Ang & Roger Summers

  5. Takeda California Inc, San Diego, USA

    Johannes Grosse

  6. Department of Pharmacology, University of Melbourne, Melbourne, VIC, Australia

    Richard A. Hughes

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  1. Nitin A. Patil
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  2. Ross A. D. Bathgate
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  3. Martina Kocan
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  4. Sheng Yu Ang
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  6. Frances Separovic
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  7. Roger Summers
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  8. Johannes Grosse
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  9. Richard A. Hughes
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  10. John D. Wade
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  11. Mohammed Akhter Hossain
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Correspondence to John D. Wade or Mohammed Akhter Hossain.

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This article does not contain any studies with human participants performed by any of the authors. This article does not contain any studies with animals performed by any of the authors. This article does not contain any studies with human participants or animals performed by any of the authors.

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Patil, N.A., Bathgate, R.A.D., Kocan, M. et al. The C-terminus of the B-chain of human insulin-like peptide 5 is critical for cognate RXFP4 receptor activity. Amino Acids 48, 987–992 (2016). https://doi.org/10.1007/s00726-015-2144-5

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  • DOI: https://doi.org/10.1007/s00726-015-2144-5

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