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Cooperative Binding of Insulin-Like Peptide 3 to a Dimeric Relaxin Family Peptide Receptor 2

Receptor Systems Biology Laboratory (A.M.S., T.M.E., P.D.M., J.N.), Hagedorn Research Institute, DK-2820 Gentofte, Denmark; Institute of Anatomy, Histology, and Embryology (M.V.), Veterinary Faculty, University of Ljubljana, Sl-1000 Ljubljana, Slovenia; 7TM Pharma A/S (A.H.), DK-2970 Hørsholm, Denmark; Chemical API Supply Isotopes (J.B.K.), Novo Nordisk, DK-2760 Måløv, Denmark; and Howard Florey Institute of Experimental Physiology and Medicine (J.D.W., R.A.D.B.), University of Melbourne, Victoria 3010, Australia

Address all correspondence and requests for reprints to: Jane Nøhr, Ph.D, Receptor Systems Biology Laboratory, Hagedorn Research Institute, Niels Steensens Vej 6, DK-2820 Gentofte, Denmark. E-mail: jnql{at}novonordisk.com.

Insulin-like peptide 3 (INSL3) binds to a G protein-coupled receptor (GPCR) called relaxin family peptide receptor 2 (RXFP2). RXFP2 belongs to the leucine-rich repeat-containing subgroup (LGR) of class A GPCRs. Negative cooperativity has recently been demonstrated in other members of the LGR subgroup. In this work, the kinetics of INSL3 binding to HEK293 cells stably transfected with RXFP2 (HEK293-RXFP2) have been investigated in detail to study whether negative cooperativity occurs and whether this receptor functions as a dimer. Our results show that negative cooperativity is present and that INSL3-RXFP2 binding shows both similarities and differences with insulin binding to the insulin receptor. A dose-response curve for the negative cooperativity of INSL3 binding had a reverse bell shape reminiscent of that seen for the negative cooperativity of insulin binding to its receptor. This suggests that binding of INSL3 may happen in a trans rather than in a cis way in a receptor dimer. Bioluminescence resonance energy transfer (BRET²) experiments confirmed that RXFP2 forms constitutive homodimers. Heterodimerization between RXFP2 and RXFP1 was also observed.

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