Abstract
By screening patients with severe early onset obesity for mutations within the melanocortin 4 receptor (MC4R) gene, we have identified a missense mutation (C271R) that occurs homozygous in two siblings with obesity. In-depth functional characterization of C271R revealed a right-shifted concentration response curve due to lower affinity to natural and synthetic MC4R agonists and a reduced cell surface expression. Cys-271 is located in the third extracellular loop. Here, we provide evidence that Cys-271 forms an intra-loop disulfide bond with Cys-277. Unexpectedly, we found that loss of receptor function is not only caused by the disruption of this disulfide bridge. Our data strongly support a new mechanism in which the receptor malfunction in the C271R mutant is induced by formation of a functionally disastrous disulfide bridge between Cys-277 and a third Cys residue at position 279. Mutational and chemical disruption of this improper disulfide bond was able to restore normal receptor potency. By demonstrating that a loss of a disulfide bond-participating Cys residue can favor a functionally disastrous disulfide bond, we now add a new mechanism of how Cys residues can be involved in G-protein-coupled receptor malfunction.
Highlights
Introduction of an additionalCys residue at five different positions into EL3 had no significant impact on functional properties of the WT receptor
By screening patients with severe early onset obesity for mutations within the melanocortin 4 receptor (MC4R) gene, we have identified a missense mutation (C271R) that occurs homozygous in two siblings with obesity
Loss and Gain of Cys Residues Are Frequently Found as Molecular Basis of G-protein-coupled receptors (GPCR) Inactivation—Mutational screening revealed a frequency of 3–5% mutations in the MC4R of severely early onset obese children [6, 7, 15,16,17,18,19]
Summary
Mutation Detection and Construction of Wild-type and Mutant Receptors—Genomic DNA was prepared from peripheral white blood cells using a commercial kit (Blood amp kit, Qiagen, Hilden, Germany). The wild-type MC4R was amplified from a healthy control and cloned in the pcDps expression vector. PCR fragments containing the various mutations were cloned into the EcoRI/Bsu36I or EcoRI/ SpeI sites of the MC4R-pcDps vector. The correctness of all of the PCR-derived sequences were proven by automatic sequencing. Cell Culture, Transfection, and Functional Characterization—Cells were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 g/ml streptomycin at 37 °C in a humidified 5% CO2 incubator. COS-7 cells were seeded into 12-well plates (2 ϫ 105 cells/well) for investigation of agonist-stimulated cAMP accumulation and transiently transfected with 0.5 g of DNA/well using the LipofectAMINE Plus method according to the manufacturer’s protocol (Invitrogen).
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have