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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 HEK293 cells. HEK293 cells stably 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 for 24 h. Cells were washed twice and stimulated with 10−6 M NDP-MSH for 1 h. Intracellular cAMP samples were collected and cAMP concentrations were measured using RIA. The results are expressed as % DMSO-treated WT cAMP production. Data points are shown as mean ± SEM of at least two or three independent experiments. *Significantly different from the DMSO-treated control group, p < 0.05.
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Figure 5: Ipsen 5i rescued the function of intracellularly retained mutant MC4Rs in HEK293 cells. HEK293 cells stably 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 for 24 h. Cells were washed twice and stimulated with 10−6 M NDP-MSH for 1 h. Intracellular cAMP samples were collected and cAMP concentrations were measured using RIA. The results are expressed as % DMSO-treated WT cAMP production. Data points are shown as mean ± SEM of at least two or three independent experiments. *Significantly different from the DMSO-treated control group, p < 0.05.

Mentions: We next investigated whether Ipsen 5i-rescued mutant MC4Rs were functional in generating cAMP at the cell surface. HEK293 cells stably expressing WT or mutant MC4Rs were incubated with different concentrations of Ipsen 5i for 24 h, and then stimulated with 10−6 M NDP-MSH. The intracellular cAMP accumulation was measured. As shown in Figure 5, the cAMP accumulation of WT MC4R was decreased by approximately 30% with 10−6 Ipsen 5i treatment and by 80% with 10−5 M Ipsen 5i treatment. We observed an increase in cAMP accumulation at 10−9 M Ipsen 5i for C84R and W174C and a maximal increase at a concentration between 10−8 and 10−6 M for N62S, P78L, C84R, Y15YS, W174C, P260Q, and C271Y. Unlike most of the mutants that decreased cAMP accumulation at 10−5 M Ipsen 5i, I69R had a maximal cAMP accumulation at that concentration. The signaling of S58C and G98R was not increased by Ipsen 5i.


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 HEK293 cells. HEK293 cells stably 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 for 24 h. Cells were washed twice and stimulated with 10−6 M NDP-MSH for 1 h. Intracellular cAMP samples were collected and cAMP concentrations were measured using RIA. The results are expressed as % DMSO-treated WT cAMP production. Data points are shown as mean ± SEM of at least two or three independent experiments. *Significantly different from the DMSO-treated control group, p < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Ipsen 5i rescued the function of intracellularly retained mutant MC4Rs in HEK293 cells. HEK293 cells stably 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 for 24 h. Cells were washed twice and stimulated with 10−6 M NDP-MSH for 1 h. Intracellular cAMP samples were collected and cAMP concentrations were measured using RIA. The results are expressed as % DMSO-treated WT cAMP production. Data points are shown as mean ± SEM of at least two or three independent experiments. *Significantly different from the DMSO-treated control group, p < 0.05.
Mentions: We next investigated whether Ipsen 5i-rescued mutant MC4Rs were functional in generating cAMP at the cell surface. HEK293 cells stably expressing WT or mutant MC4Rs were incubated with different concentrations of Ipsen 5i for 24 h, and then stimulated with 10−6 M NDP-MSH. The intracellular cAMP accumulation was measured. As shown in Figure 5, the cAMP accumulation of WT MC4R was decreased by approximately 30% with 10−6 Ipsen 5i treatment and by 80% with 10−5 M Ipsen 5i treatment. We observed an increase in cAMP accumulation at 10−9 M Ipsen 5i for C84R and W174C and a maximal increase at a concentration between 10−8 and 10−6 M for N62S, P78L, C84R, Y15YS, W174C, P260Q, and C271Y. Unlike most of the mutants that decreased cAMP accumulation at 10−5 M Ipsen 5i, I69R had a maximal cAMP accumulation at that concentration. The signaling of S58C and G98R was not increased by Ipsen 5i.

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