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Functional characterization and pharmacological rescue of melanocortin-4 receptor mutations identified from obese patients.

Fan ZC, Tao YX - J. Cell. Mol. Med. (2009)

Bottom Line: To identify potential therapeutic approaches for patients with intracellularly retained MC4R mutants, we tested the effect of an MC4R inverse agonist, ML00253764, on C84R and W174C.The rescued mutants are functional with increased cAMP production in response to agonist stimulation.In conclusion, of 10 mutants we studied, 6 had decreased cell surface expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy, Physiology and Pharmacology, College of Veterinary Medicine, Auburn University, Auburn, AL 36849-5519, USA.

ABSTRACT
As the most common monogenic form of human obesity, about 130 naturally occurring melanocortin-4 receptor (MC4R) gene mutations have been identified. In this study, we reported detailed functional characterization of 10 novel human MC4R (hMC4R) mutants including R7C, C84R, S127L, S136F, W174C, A219V, P230L, F261S, I317V and L325F. Flow cytometry experiments showed that six mutants, including R7C, C84R, S127L, W174C, P230L and F261S, have decreased cell surface expression. The other four mutants are expressed at similar levels as the wild-type hMC4R. Binding assays showed that the mutants have similar binding affinities for the agonist and endogenous antagonist agouti-related protein. Signalling assays showed that S136F is defective in signalling. Multiple mutagenesis showed that S136 of hMC4R is required for the normal function of the receptor. To identify potential therapeutic approaches for patients with intracellularly retained MC4R mutants, we tested the effect of an MC4R inverse agonist, ML00253764, on C84R and W174C. We showed that ML00253764 could function as a pharmacological chaperone rescuing the mutant MC4Rs to the cell surface. The rescued mutants are functional with increased cAMP production in response to agonist stimulation. In conclusion, of 10 mutants we studied, 6 had decreased cell surface expression. Pharmacological chaperone is a potential approach for treating obesity caused by MC4R mutations that result in intracellular retention.

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Basal activities of the WT and mutant hMC4Rs. The basal activities of WT and mutant hMC4Rs were assessed by measuring intracellular cAMP levels in HEK293T cells transiently expressing WT or mutant hMC4Rs without hormone stimulation. The results are expressed as percentage of basal cAMP level of WT hMC4R-expressing HEK293T cells. Shown are mean ± S.E.M. of three or more experiments. The basal cAMP level in the WT hMC4R was 181.9 ± 76.9 pmol/106 cells (mean ± S.E.M. of six experiments). Star (⋆) indicates significant difference from WT hMC4R (P < 0.05).
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fig05: Basal activities of the WT and mutant hMC4Rs. The basal activities of WT and mutant hMC4Rs were assessed by measuring intracellular cAMP levels in HEK293T cells transiently expressing WT or mutant hMC4Rs without hormone stimulation. The results are expressed as percentage of basal cAMP level of WT hMC4R-expressing HEK293T cells. Shown are mean ± S.E.M. of three or more experiments. The basal cAMP level in the WT hMC4R was 181.9 ± 76.9 pmol/106 cells (mean ± S.E.M. of six experiments). Star (⋆) indicates significant difference from WT hMC4R (P < 0.05).

Mentions: Previous studies suggested that loss of basal signalling might be one defect of the receptor in MC4R mutants identified from obese patients [47]. Therefore, we also determined the constitutive activities of the mutants in this study. As shown in Fig. 5, when compared with the WT hMC4R, S127L and P230L had significantly increased constitutive activities, whereas W174C and A219V had significantly decreased constitutive activities. The other six mutants had similar constitutive activities as the WT hMC4R (Fig. 5).


Functional characterization and pharmacological rescue of melanocortin-4 receptor mutations identified from obese patients.

Fan ZC, Tao YX - J. Cell. Mol. Med. (2009)

Basal activities of the WT and mutant hMC4Rs. The basal activities of WT and mutant hMC4Rs were assessed by measuring intracellular cAMP levels in HEK293T cells transiently expressing WT or mutant hMC4Rs without hormone stimulation. The results are expressed as percentage of basal cAMP level of WT hMC4R-expressing HEK293T cells. Shown are mean ± S.E.M. of three or more experiments. The basal cAMP level in the WT hMC4R was 181.9 ± 76.9 pmol/106 cells (mean ± S.E.M. of six experiments). Star (⋆) indicates significant difference from WT hMC4R (P < 0.05).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4516484&req=5

fig05: Basal activities of the WT and mutant hMC4Rs. The basal activities of WT and mutant hMC4Rs were assessed by measuring intracellular cAMP levels in HEK293T cells transiently expressing WT or mutant hMC4Rs without hormone stimulation. The results are expressed as percentage of basal cAMP level of WT hMC4R-expressing HEK293T cells. Shown are mean ± S.E.M. of three or more experiments. The basal cAMP level in the WT hMC4R was 181.9 ± 76.9 pmol/106 cells (mean ± S.E.M. of six experiments). Star (⋆) indicates significant difference from WT hMC4R (P < 0.05).
Mentions: Previous studies suggested that loss of basal signalling might be one defect of the receptor in MC4R mutants identified from obese patients [47]. Therefore, we also determined the constitutive activities of the mutants in this study. As shown in Fig. 5, when compared with the WT hMC4R, S127L and P230L had significantly increased constitutive activities, whereas W174C and A219V had significantly decreased constitutive activities. The other six mutants had similar constitutive activities as the WT hMC4R (Fig. 5).

Bottom Line: To identify potential therapeutic approaches for patients with intracellularly retained MC4R mutants, we tested the effect of an MC4R inverse agonist, ML00253764, on C84R and W174C.The rescued mutants are functional with increased cAMP production in response to agonist stimulation.In conclusion, of 10 mutants we studied, 6 had decreased cell surface expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy, Physiology and Pharmacology, College of Veterinary Medicine, Auburn University, Auburn, AL 36849-5519, USA.

ABSTRACT
As the most common monogenic form of human obesity, about 130 naturally occurring melanocortin-4 receptor (MC4R) gene mutations have been identified. In this study, we reported detailed functional characterization of 10 novel human MC4R (hMC4R) mutants including R7C, C84R, S127L, S136F, W174C, A219V, P230L, F261S, I317V and L325F. Flow cytometry experiments showed that six mutants, including R7C, C84R, S127L, W174C, P230L and F261S, have decreased cell surface expression. The other four mutants are expressed at similar levels as the wild-type hMC4R. Binding assays showed that the mutants have similar binding affinities for the agonist and endogenous antagonist agouti-related protein. Signalling assays showed that S136F is defective in signalling. Multiple mutagenesis showed that S136 of hMC4R is required for the normal function of the receptor. To identify potential therapeutic approaches for patients with intracellularly retained MC4R mutants, we tested the effect of an MC4R inverse agonist, ML00253764, on C84R and W174C. We showed that ML00253764 could function as a pharmacological chaperone rescuing the mutant MC4Rs to the cell surface. The rescued mutants are functional with increased cAMP production in response to agonist stimulation. In conclusion, of 10 mutants we studied, 6 had decreased cell surface expression. Pharmacological chaperone is a potential approach for treating obesity caused by MC4R mutations that result in intracellular retention.

Show MeSH
Related in: MedlinePlus