Agonist-Dependent Internalization of the Human Melanocortin-4 Receptors in Human Embryonic Kidney 293 Cells

Gao, Zhenhai; Lei, Dachuan; Welch, Julie L.; Le, Kathy; Lin, Jen‐Kou; Leng, Song; Duhl, David M.

doi:10.1124/jpet.103.055525

Cited by 31 publications

(34 citation statements)

References 30 publications

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“…The ability of the MC4 receptor to undergo receptor internalization in response to agonist has been documented (Gao et al, 2003;Shinyama et al, 2003) and is considered to be an important regulatory mechanism in receptor desensitization. We assessed the ability of the FMC4 receptor to undergo agonist-dependent internalization using FACS analysis.…”

Section: Resultsmentioning

confidence: 99%

“…In addition to activating signaling pathways, ␣-MSH binding to the MC4 receptor activates regulatory pathways, which include receptor internalization (Gao et al, 2003;Shinyama et al, 2003). This is of potential interest because not only is internalization involved in receptor regulation, recent findings have suggested that it may contribute to signaling because receptors can interact with downstream effectors once internalized (Daaka et al, 1998;Holstein et al, 2004).…”

Section: -(1r)-(4-mentioning

confidence: 99%

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Functional Selectivity of Melanocortin 4 Receptor Peptide and Nonpeptide Agonists: Evidence for Ligand-Specific Conformational States

Nickolls¹,

Fleck²,

Hoare³

et al. 2005

J Pharmacol Exp Ther

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Agonists of the melanocortin 4 (MC4) receptor have potential pharmaceutical benefit in the treatment of obesity and sexual dysfunction. In this study, we have compared the ability of a number of peptide and nonpeptide agonists to activate a FLAG-tagged human MC4 (FMC4) receptor, as measured by both cAMP accumulation and calcium mobilization using a fluorometric imaging plate reader (FLIPR). In addition, we have analyzed the ability of these agonists to cause receptor internalization, as measured by fluorescence-activated cell sorting analysis. The endogenous agonist ␣-melanocortin-stimulating hormone (␣-MSH) increased cAMP accumulation, calcium mobilization, and receptor internalization in a dose-dependent manner in human embryonic kidney 293 cells expressing the FMC4 receptor. The activity of the other agonists varied considerably in these assays, and overall, the potency and intrinsic activity of the agonists in the cAMP accumulation assays did not correlate with their potency or intrinsic activity in either the FLIPR or receptor internalization assays. Agonists could be clearly separated into two functional classes based on their structure. Peptide agonists ␤-MSH, des-acetyl-␣-MSH, and [Nle 4 , D-Phe 7 ]-␣-melanocortin-stimulating hormone exhibited 80 to 112% of the maximal ␣-MSH response in cAMP accumulation and 62 to 96% in FLIPR assays and were able to cause 75 to 118% of receptor internalization induced by ␣-MSH. Conversely, although the nonpeptide agonists exhibited 73 to 149% of the ␣-MSH response in the cAMP accumulation assays, they were significantly impaired in the FLIPR (7-40%) and receptor internalization (Ϫ5-38%) assays. These findings demonstrate an important difference in activation and internalization of the MC4 receptor by nonpeptide versus peptide agonists and provides evidence of agonist-specific conformational states.The melanocortin 4 receptor (MC4R) is a member of the seven transmembrane receptor family that initiate signal transduction through activation of heterotrimeric G proteins. There are four additional members of the melanocortin receptor subfamily, distinguishable by their distribution and physiology. Melanocortin receptors are all activated by one or more pro-opiomelanocortin-derived peptides, which include ␣-, ␤-, and ␥-melanocortin-stimulating hormone (MSH). In addition, the melanocortin receptors are regulated by the antagonists/inverse agonists, AGRP and agouti (Lu et al., 1994;Ollmann et al., 1997).The melanocortin receptors all signal through G␣ s , which stimulates the production of cAMP by adenylate cyclase. The MC3 receptor also functions through phospholipase C to increase intracellular calcium (Konda et al., 1994). Likewise, the MC4 receptor mediates an increase in intracellular calcium, but reportedly through a cholera toxin-sensitive pathway rather than phospholipase C (Mountjoy et al., 2001).

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Section: Resultsmentioning

confidence: 99%

Section: -(1r)-(4-mentioning

confidence: 99%

Functional Selectivity of Melanocortin 4 Receptor Peptide and Nonpeptide Agonists: Evidence for Ligand-Specific Conformational States

Nickolls¹,

Fleck²,

Hoare³

et al. 2005

J Pharmacol Exp Ther

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“…The intracellular traffic of MC4R along the endosomal route appears to play an important role in the function of the receptor. In this respect, prolonged exposure to ␣-MSH promotes its localization to lysosomes (15). Moreover, trafficking of intracellular MC4R back to the cell surface rather than to lysosomes underlies the ability of mutations of attractin and Mahogunin Ring Finger-1 (Mgrn1) to rectify the obesity in mice overexpressing agouti-signaling protein, an antagonist of MC4R (18).…”

Section: Mc4rmentioning

confidence: 99%

“…Constitutive Internalization of ␤ 2 AR-Attachment of hemagglutinin (HA) and GFP tags to the N and C termini of MC4R, respectively, does not change the ability of the receptor to bind to ␣-MSH and signal (14,15). By using both HA-MC4R and HA-MC4R-GFP, we have found that the receptor is internalized at the same rate in the absence and presence of ␣-MSH (17).…”

Section: Constitutive Internalization Of Mc4r Occurs Faster Thanmentioning

confidence: 99%

“…The data presented in this study indicate that modifications at Thr-312 and Ser-329 of MC4R, most likely phosphorylation, function to retain the receptor in the endosomal compartment. It has been reported that exposure to ␣-MSH and to agouti-signaling protein directs MC4R to lysosomes (15,18). Moreover, mutations in two factors that rectify obesity in a mouse overexpressing agouti-signaling protein, attractin, and MGRN1, block agouti-signaling protein-dependent traffic of MC4R to lysosomes (18).…”

Section: Internalization Of Mc4r Is Required To Maintain the Pool Of mentioning

confidence: 99%

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Constitutive Cholesterol-dependent Endocytosis of Melanocortin-4 Receptor (MC4R) Is Essential to Maintain Receptor Responsiveness to α-Melanocyte-stimulating Hormone (α-MSH)

McDaniel

Molden

Mohammad

et al. 2012

Journal of Biological Chemistry

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Background: MC4R is essential for energy homeostasis and cycles continuously. Results: MC4R internalization is blocked by clathrin and cholesterol depletion, reducing receptor response to ␣-MSH, which is partially recovered by mutations at Thr-312/Ser-329. Conclusion: Constitutive internalization of MC4R is cholesterol-dependent and required for receptor function. Significance: These findings provide a potentially novel mechanism by which hypothalamic cell cholesterol content can affect appetite control.

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GPCR Signaling from Intracellular Membranes

Jong¹,

Harmon²,

O’Malley³

2022

GPCRs as Therapeutic Targets

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Agonist-Dependent Internalization of the Human Melanocortin-4 Receptors in Human Embryonic Kidney 293 Cells

Cited by 31 publications

References 30 publications

Functional Selectivity of Melanocortin 4 Receptor Peptide and Nonpeptide Agonists: Evidence for Ligand-Specific Conformational States

Functional Selectivity of Melanocortin 4 Receptor Peptide and Nonpeptide Agonists: Evidence for Ligand-Specific Conformational States

Constitutive Cholesterol-dependent Endocytosis of Melanocortin-4 Receptor (MC4R) Is Essential to Maintain Receptor Responsiveness to α-Melanocyte-stimulating Hormone (α-MSH)

GPCR Signaling from Intracellular Membranes

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