Negative cooperativity in H2 relaxin binding to a dimeric relaxin family peptide receptor 1.
H2 relaxin, a member of the insulin superfamily, binds to the G-protein-coupled receptor RXFP1 (relaxin family peptide 1), a receptor that belongs to the leucine-rich repeat (LRR)-containing subgroup (LGRs) of class A GPCRs. We recently demonstrated negative cooperativity in INSL3 binding to RXFP2 and showed that this subgroup of GPCRs functions as constitutive dimers. In this work, we investigated whether the binding of H2 relaxin to RXFP1 also shows negative cooperativity, and whether this receptor functions as a dimer using BRET(2). Both binding and dissociation were temperature dependent, and the pH optimum for binding was pH 7.0. Our results showed that RXFP1 is a constitutive dimer with negative cooperativity in ligand binding, that dimerization occurs through the 7TM domain, and that the ectodomain has a stabilizing effect on this interaction. Dimerization and negative cooperativity appear to be general properties of LGRs involved in reproduction as well as other GPCRs.
Svendsen
Angela Manegold
AM
Receptor Systems Biology Laboratory, Hagedorn Research Institute, Gentofte, Denmark.
Zalesko
Anna
A
Kønig
Julie
J
Vrecl
Milka
M
Heding
Anders
A
Kristensen
Jesper Bøggild
JB
Wade
John D
JD
Bathgate
Ross A D
RA
De Meyts
Pierre
P
Nøhr
Jane
J
D001667:Binding, Competitive / Q000502:physiology*; D002478:Cells, Cultured; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007457:Iodine Radioisotopes / Q000493:pharmacokinetics; D009994:Osmolar Concentration; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs / Q000502:physiology; D055503:Protein Multimerization; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism* / Q000502:physiology; D018000:Receptors, Peptide / Q000378:metabolism* / Q000502:physiology; D012065:Relaxin / Q000737:chemistry / Q000378:metabolism* / Q000493:pharmacokinetics / Q000502:physiology; D013379:Substrate Specificity; D013696:Temperature
D007457:Iodine Radioisotopes; C095068:RLN2 protein, human; C417543:RXFP1 protein, human; D043562:Receptors, G-Protein-Coupled; D018000:Receptors, Peptide; D012065:Relaxin