title: The C-terminus of the B-chain of human insulin-like peptide 5 is critical for cognate RXFP4 receptor activity.
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.
authors.affiliation: Howard Florey Research Laboratories, Florey Institute for Neuroscience and Mental Health, University of Melbourne, Melbourne, VIC, Australia.|School of Chemistry, Bio21 Institute, University of Melbourne, Melbourne, VIC, Australia.
authors.forename: Nitin A
authors.initials: NA
authors.lastname: Patil
authors.affiliation: Howard Florey Research Laboratories, Florey Institute for Neuroscience and Mental Health, University of Melbourne, Melbourne, VIC, Australia.|Department of Biochemistry and Molecular Biology, University of Melbourne, Melbourne, VIC, Australia.
authors.forename: Ross A D
authors.initials: RAD
authors.lastname: Bathgate
authors.affiliation: Monash Institute of Pharmaceutical Sciences, Parkville, VIC, Australia.
authors.forename: Martina
authors.initials: M
authors.lastname: Kocan
authors.affiliation: Monash Institute of Pharmaceutical Sciences, Parkville, VIC, Australia.
authors.forename: Sheng Yu
authors.initials: SY
authors.lastname: Ang
authors.affiliation: Howard Florey Research Laboratories, Florey Institute for Neuroscience and Mental Health, University of Melbourne, Melbourne, VIC, Australia.
authors.forename: Julien
authors.initials: J
authors.lastname: Tailhades
authors.affiliation: School of Chemistry, Bio21 Institute, University of Melbourne, Melbourne, VIC, Australia.
authors.forename: Frances
authors.initials: F
authors.lastname: Separovic
authors.affiliation: Monash Institute of Pharmaceutical Sciences, Parkville, VIC, Australia.
authors.forename: Roger
authors.initials: R
authors.lastname: Summers
authors.affiliation: Takeda California Inc, San Diego, USA.
authors.forename: Johannes
authors.initials: J
authors.lastname: Grosse
authors.affiliation: Department of Pharmacology, University of Melbourne, Melbourne, VIC, Australia.
authors.forename: Richard A
authors.initials: RA
authors.lastname: Hughes
authors.affiliation: Howard Florey Research Laboratories, Florey Institute for Neuroscience and Mental Health, University of Melbourne, Melbourne, VIC, Australia. john.wade@florey.edu.au.|School of Chemistry, Bio21 Institute, University of Melbourne, Melbourne, VIC, Australia. john.wade@florey.edu.au.
authors.forename: John D
authors.initials: JD
authors.lastname: Wade
authors.affiliation: Howard Florey Research Laboratories, Florey Institute for Neuroscience and Mental Health, University of Melbourne, Melbourne, VIC, Australia. akhter.hossain@unimelb.edu.au.|School of Chemistry, Bio21 Institute, University of Melbourne, Melbourne, VIC, Australia. akhter.hossain@unimelb.edu.au.
authors.forename: Mohammed Akhter
authors.identifier: http://orcid.org/0000-0002-9961-0006
authors.initials: MA
authors.lastname: Hossain
mesh_terms: D000577:Amides / Q000138:chemical synthesis / Q000737:chemistry* / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D016466:CHO Cells; D003412:Cricetulus; D000242:Cyclic AMP / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D007328:Insulin / Q000138:chemical synthesis / Q000737:chemistry* / Q000494:pharmacology; D007700:Kinetics; D051379:Mice; D019950:Mitogen-Activated Protein Kinase 1 / Q000235:genetics / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000235:genetics / Q000378:metabolism; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry* / Q000494:pharmacology; D011485:Protein Binding; D000072417:Protein Domains; D011506:Proteins / Q000138:chemical synthesis / Q000737:chemistry* / Q000494:pharmacology; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry / Q000378:metabolism*; D018000:Receptors, Peptide / Q000737:chemistry / Q000378:metabolism*; D060327:Solid-Phase Synthesis Techniques; D055672:Static Electricity; D013329:Structure-Activity Relationship
chemical_list: D000577:Amides; D007328:Insulin; C084797:Leydig insulin-like protein; D010455:Peptides; D011506:Proteins; C480239:RXFP4 protein, human; D043562:Receptors, G-Protein-Coupled; D018000:Receptors, Peptide; D000242:Cyclic AMP; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3
keywords: INSL5; Insulin-like peptide 5; Peptide; Relaxin