Cooperative binding of insulin-like Peptide 3 to a dimeric relaxin family peptide receptor 2.
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 (BRET(2)) experiments confirmed that RXFP2 forms constitutive homodimers. Heterodimerization between RXFP2 and RXFP1 was also observed.
Receptor Systems Biology Laboratory, Hagedorn Research Institute, Niels Steensens Vej 6, DK-2820 Gentofte, Denmark.
Angela Manegold
AM
Svendsen
Milka
M
Vrecl
Tina M
TM
Ellis
Anders
A
Heding
Jesper Bøggild
JB
Kristensen
John D
JD
Wade
Ross A D
RA
Bathgate
Pierre
P
De Meyts
Jane
J
Nøhr
D002460:Cell Line; D019281:Dimerization; D006801:Humans; D007328:Insulin / Q000378:metabolism*; D008163:Luminescent Measurements; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*; D013696:Temperature; D014162:Transfection
D007328:Insulin; C084797:Leydig insulin-like protein; D011506:Proteins; C472867:RXFP2 protein, human; D043562:Receptors, G-Protein-Coupled