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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 function of intracellularly retained mutant MC4Rs in Neuro2a cells. Neuro2a cells transiently expressing WT or mutant MC4Rs were treated with either different concentrations of Ipsen 5i (A), 10−6 M Ipsen 5i (B), or DMSO as control. The results are expressed as % DMSO-treated WT cAMP production after correction of the cAMP production in cells expressing the empty vector. Data points are mean ± SEM of at least three independent experiments. See the legend to Figure 5 for other details.
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Figure 6: Ipsen 5i rescued the function of intracellularly retained mutant MC4Rs in Neuro2a cells. Neuro2a cells transiently expressing WT or mutant MC4Rs were treated with either different concentrations of Ipsen 5i (A), 10−6 M Ipsen 5i (B), or DMSO as control. The results are expressed as % DMSO-treated WT cAMP production after correction of the cAMP production in cells expressing the empty vector. Data points are mean ± SEM of at least three independent experiments. See the legend to Figure 5 for other details.

Mentions: In Neuro2a cells transiently expressing MC4Rs, we also observed an increase in cAMP accumulation at 10−9 M Ipsen 5i and a maximal increase at 10−6 M Ipsen 5i (or at 10−5 M Ipsen 5i for I69R) in Neuro2a cells (Figure 6A). As shown in Figure 6B, with 10−6 M Ipsen 5i treatment in Neuro2a cells, seven mutants (N62S, I69R, P78L, C84R, W174C, P260Q, and C271Y) had significantly increased cAMP accumulation whereas signaling of three mutants (G98R, Y157S, and F261S) was not increased by Ipsen 5i. High concentration of Ipsen 5i did not decrease the cAMP accumulation of WT or mutant MC4Rs as dramatically as seen in HEK293 cells (Figure 6A). Our results obtained from N1E-115 cells were similar with those obtained from Neuro2a cells (Figure 7).


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 function of intracellularly retained mutant MC4Rs in Neuro2a cells. Neuro2a cells transiently expressing WT or mutant MC4Rs were treated with either different concentrations of Ipsen 5i (A), 10−6 M Ipsen 5i (B), or DMSO as control. The results are expressed as % DMSO-treated WT cAMP production after correction of the cAMP production in cells expressing the empty vector. Data points are mean ± SEM of at least three independent experiments. See the legend to Figure 5 for other details.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Ipsen 5i rescued the function of intracellularly retained mutant MC4Rs in Neuro2a cells. Neuro2a cells transiently expressing WT or mutant MC4Rs were treated with either different concentrations of Ipsen 5i (A), 10−6 M Ipsen 5i (B), or DMSO as control. The results are expressed as % DMSO-treated WT cAMP production after correction of the cAMP production in cells expressing the empty vector. Data points are mean ± SEM of at least three independent experiments. See the legend to Figure 5 for other details.
Mentions: In Neuro2a cells transiently expressing MC4Rs, we also observed an increase in cAMP accumulation at 10−9 M Ipsen 5i and a maximal increase at 10−6 M Ipsen 5i (or at 10−5 M Ipsen 5i for I69R) in Neuro2a cells (Figure 6A). As shown in Figure 6B, with 10−6 M Ipsen 5i treatment in Neuro2a cells, seven mutants (N62S, I69R, P78L, C84R, W174C, P260Q, and C271Y) had significantly increased cAMP accumulation whereas signaling of three mutants (G98R, Y157S, and F261S) was not increased by Ipsen 5i. High concentration of Ipsen 5i did not decrease the cAMP accumulation of WT or mutant MC4Rs as dramatically as seen in HEK293 cells (Figure 6A). Our results obtained from N1E-115 cells were similar with those obtained from Neuro2a cells (Figure 7).

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