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Ipsen 5i is a Novel Potent Pharmacoperone for Intracellularly Retained Melanocortin-4 Receptor Mutants.

Tao YX, Huang H - Front Endocrinol (Lausanne) (2014)

Bottom Line: Ipsen 5i increased cell surface expression of three mutants (S58C, G98R, and F261S) but did not affect signaling.Ipsen 5i had no effect on mutant MC4Rs with other defects (Δ88-92, D90N, I102S) or no defect (N274S).It also did not affect trafficking of a misrouted MC3R mutant (I335S).

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy, Physiology and Pharmacology, College of Veterinary Medicine, Auburn University , Auburn, AL , USA.

ABSTRACT
Inactivating mutations of the melanocortin-4 receptor (MC4R) cause early-onset severe obesity in humans. Comprehensive functional studies show that most of the inactivating mutants of the MC4R are retained intracellularly. In the present study, we investigated whether a small molecule inverse agonist of the MC4R, Ipsen 5i, could act as a pharmacoperone and correct the cell surface expression and function of intracellularly retained mutant MC4Rs using multiple cell lines, including HEK293 and two neuronal cell lines. We showed that Ipsen 5i rescued the cell surface expression of all 11 intracellularly retained mutant MC4Rs studied herein in at least one cell line. Ipsen 5i functionally rescued seven mutants in all cell lines used. One mutant (Y157S) was functionally rescued in HEK293 cells but not in the two neuronal cell lines. Ipsen 5i increased cell surface expression of three mutants (S58C, G98R, and F261S) but did not affect signaling. Ipsen 5i had no effect on mutant MC4Rs with other defects (Δ88-92, D90N, I102S) or no defect (N274S). It also did not affect trafficking of a misrouted MC3R mutant (I335S). Cell impermeable peptide ligands of the MC4R or cell permeable small molecule ligand of δ opioid receptor could not rescue misrouted mutant MC4R. In summary, we demonstrated that Ipsen 5i was a novel potent pharmacoperone of the MC4R, correcting trafficking and signaling of a significant portion (73%) of intracellularly retained mutants. Additional studies are needed to demonstrate its in vivo efficacy.

No MeSH data available.


Related in: MedlinePlus

Ipsen 5i rescued the cell surface expression of intracellularly retained mutant MC4Rs in HEK293 cells as visualized by confocal microscopy. HEK293 cells stably expressing WT or mutant MC4Rs were treated with 10−6 M Ipsen 5i for 24 h and then stained with Alexa Fluor 488-conjugated antibody. Cells were visualized using a Nikon A1 confocal microscope. Results are representative of three independent experiments.
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Figure 2: Ipsen 5i rescued the cell surface expression of intracellularly retained mutant MC4Rs in HEK293 cells as visualized by confocal microscopy. HEK293 cells stably expressing WT or mutant MC4Rs were treated with 10−6 M Ipsen 5i for 24 h and then stained with Alexa Fluor 488-conjugated antibody. Cells were visualized using a Nikon A1 confocal microscope. Results are representative of three independent experiments.

Mentions: To investigate whether Ipsen 5i acted as a pharmacoperone rescuing the cell surface expression of mutant MC4Rs, we studied 11 mutants (S58C, N62S, I69R, P78L, C84R, G98R, Y157S, W174C, P260Q, F261S, and C271Y) that have reduced or absent cell surface expression (16, 28, 30) (Figure 1). We first visualized the cell surface expression of the 11 mutants stably expressed in HEK293 cells treated with 10−6 M Ipsen 5i using a confocal microscope. As shown in Figure 2, with 10−6 M Ipsen 5i treatment for 24 h, the immunostaining of 10 mutants (S58C, N62S, P78L, C84R, G98R, Y157S, W174C, P260Q, F261S, and C271Y) was dramatically enhanced compared with that of the DMSO-treated control cells whereas that of one mutant (I69R) was not obviously changed.


Ipsen 5i is a Novel Potent Pharmacoperone for Intracellularly Retained Melanocortin-4 Receptor Mutants.

Tao YX, Huang H - Front Endocrinol (Lausanne) (2014)

Ipsen 5i rescued the cell surface expression of intracellularly retained mutant MC4Rs in HEK293 cells as visualized by confocal microscopy. HEK293 cells stably expressing WT or mutant MC4Rs were treated with 10−6 M Ipsen 5i for 24 h and then stained with Alexa Fluor 488-conjugated antibody. Cells were visualized using a Nikon A1 confocal microscope. Results are representative of three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4120685&req=5

Figure 2: Ipsen 5i rescued the cell surface expression of intracellularly retained mutant MC4Rs in HEK293 cells as visualized by confocal microscopy. HEK293 cells stably expressing WT or mutant MC4Rs were treated with 10−6 M Ipsen 5i for 24 h and then stained with Alexa Fluor 488-conjugated antibody. Cells were visualized using a Nikon A1 confocal microscope. Results are representative of three independent experiments.
Mentions: To investigate whether Ipsen 5i acted as a pharmacoperone rescuing the cell surface expression of mutant MC4Rs, we studied 11 mutants (S58C, N62S, I69R, P78L, C84R, G98R, Y157S, W174C, P260Q, F261S, and C271Y) that have reduced or absent cell surface expression (16, 28, 30) (Figure 1). We first visualized the cell surface expression of the 11 mutants stably expressed in HEK293 cells treated with 10−6 M Ipsen 5i using a confocal microscope. As shown in Figure 2, with 10−6 M Ipsen 5i treatment for 24 h, the immunostaining of 10 mutants (S58C, N62S, P78L, C84R, G98R, Y157S, W174C, P260Q, F261S, and C271Y) was dramatically enhanced compared with that of the DMSO-treated control cells whereas that of one mutant (I69R) was not obviously changed.

Bottom Line: Ipsen 5i increased cell surface expression of three mutants (S58C, G98R, and F261S) but did not affect signaling.Ipsen 5i had no effect on mutant MC4Rs with other defects (Δ88-92, D90N, I102S) or no defect (N274S).It also did not affect trafficking of a misrouted MC3R mutant (I335S).

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy, Physiology and Pharmacology, College of Veterinary Medicine, Auburn University , Auburn, AL , USA.

ABSTRACT
Inactivating mutations of the melanocortin-4 receptor (MC4R) cause early-onset severe obesity in humans. Comprehensive functional studies show that most of the inactivating mutants of the MC4R are retained intracellularly. In the present study, we investigated whether a small molecule inverse agonist of the MC4R, Ipsen 5i, could act as a pharmacoperone and correct the cell surface expression and function of intracellularly retained mutant MC4Rs using multiple cell lines, including HEK293 and two neuronal cell lines. We showed that Ipsen 5i rescued the cell surface expression of all 11 intracellularly retained mutant MC4Rs studied herein in at least one cell line. Ipsen 5i functionally rescued seven mutants in all cell lines used. One mutant (Y157S) was functionally rescued in HEK293 cells but not in the two neuronal cell lines. Ipsen 5i increased cell surface expression of three mutants (S58C, G98R, and F261S) but did not affect signaling. Ipsen 5i had no effect on mutant MC4Rs with other defects (Δ88-92, D90N, I102S) or no defect (N274S). It also did not affect trafficking of a misrouted MC3R mutant (I335S). Cell impermeable peptide ligands of the MC4R or cell permeable small molecule ligand of δ opioid receptor could not rescue misrouted mutant MC4R. In summary, we demonstrated that Ipsen 5i was a novel potent pharmacoperone of the MC4R, correcting trafficking and signaling of a significant portion (73%) of intracellularly retained mutants. Additional studies are needed to demonstrate its in vivo efficacy.

No MeSH data available.


Related in: MedlinePlus