Exposure of MC4R to agonist in the endoplasmic reticulum stabilizes an active conformation of the receptor that does not desensitize

Abstract

Melanocortin-4 receptor (MC4R) is a G protein-coupled receptor expressed in neurons of the hypothalamus where it regulates food intake. MC4R responds to an agonist, α-melanocyte-stimulating hormone (α-MSH) and to an antagonist/inverse agonist, agoutirelated peptide (AgRP), which are released by upstream neurons. Binding to α-MSH leads to stimulation of receptor activity and suppression of food intake, whereas AgRP has opposite effects. MC4R cycles constantly between the plasma membrane and endosomes and undergoes agonist-mediated desensitization by being routed to lysosomes. MC4R desensitization and increased AgRP expression are thought to decrease the effectiveness of MC4R agonists as an antiobesity treatment. In this study, α-MSH, instead of being delivered extracellularly, is targeted to the endoplasmic reticulum (ER) of neuronal cells and cultured hypothalamic neurons. We find that the ER-targeted agonist associates with MC4R at this location, is transported to the cell surface, induces constant cAMP and AMP kinase signaling at maximal amplitude, abolishes desensitization of the receptor, and promotes both cell-surface expression and constant signaling by an obesity-linked MC4R variant, I316S, that otherwise is retained in the ER. Formation of the MC4R/agonist complex in the ER stabilizes the receptor in an active conformation that at the cell surface is insensitive to antagonism by AgRP and at the endosomes is refractory to routing to the lysosomes. The data indicate that targeting agonists to the ER can stabilize an active conformation of a G protein-coupled receptor that does not become desensitized, suggesting a target for therapy. M elanocortin-4 receptor (MC4R) is a G protein-coupled receptor (GPCR) expressed in the brain where it controls food intake and energy expenditure (1-3). MC4R-knockout mice are obese (4), as are humans with naturally occurring mutations of the receptor (5-8), thus indicating the central importance of MC4R in energy homeostasis. Although MC4R is expressed ubiquitously in the central nervous system (9, 10), regulation of food intake is restored in MC4R-knockout mice when MC4R is expressed in the paraventricular nuclei of the hypothalamus and in the amygdala (11). Multiple anorexigenic hormones from the periphery, including leptin from the adipose tissue, insulin from β-cells of the pancreas, and glucagon-like peptide-1 and cholecystokinin from the gut, are received by pro-opiomelanocortin (POMC) neurons localized in the arcuate nucleus of the hypothalamus. POMC neurons, projecting to the paraventricular nucleus of the hypothalamus, release the anorexigenic hormone α-melanocyte-stimulating hormone (α-MSH), which binds to MC4R expressed by downstream melanocortin neurons. On the other hand, other neurons in the arcuate nucleus that respond to orexigenic hormones from the periphery increase the production of agouti-related protein (AgRP), which is the natural antagonist/inverse agonist of MC4R (12-18). AgRP signaling and AgRP neurons (19-21) appear to be essential to promote feedin...

Obesity‐associated mutant melanocortin‐4 receptors with normal Gαs coupling frequently exhibit other discoverable pharmacological and biochemical defects

“…One of the variants tested in the present study, V50M, did exhibit a defect in glycosylation, reduced responsiveness to α‐ and β‐MSH, and reduced the ability to couple to β‐arrestin, although it is unclear whether the glycosylation defect contributes to the altered signalling properties. Granell et al suggest that glycosylation status may not affect the function of the receptor.…”

The melanocortin pathway and control of appetite-progress and therapeutic implications

“…Changing conformation of newly synthesized MC4R along the secretory pathway may also affect the receptor properties not only to signal, but also to desensitize. In this respect, co-expressing α-MSH together with MC4R in the ER can rescue an obesitylinked variant retained in the ER and stabilize the wildtype receptor in an active conformation that does not route to lysosomes nor desensitizes (Granell et al 2013). These observations indicate that MC4R conformation and ability to signal can be modulated by interactions with agonist in the ER.…”

“…Studies based on undifferentiated cells, neuronal cells and immortalized hypothalamic cells indicate that desensitization of MC4R takes place by a process where, upon prolonged agonist exposure, the receptor routes to the lysosomes, instead of being made available at the cell membrane (Gao et al 2003, Shinyama et al 2003, Mohammad et al 2007, Granell et al 2013. MC4R is internalized at the same rate in the presence or absence of α-MSH agonist (Mohammad et al 2007).…”

“…Agonist exposure induces binding of β-arrestin to the β 2 -adrenergic receptor, receptor internalization and desensitization (Drake et al 2006). In the case of MC4R, desensitization instead takes place because, in the presence of agonist, a population of constitutively recycling receptor is retained in the intracellular localization and routes to lysosomes (McDaniel et al 2012, Granell et al 2013. A factor implicated in MC4R intracellular traffic to lysosomes is Mahogunin Ring Finger-1, a RING domain-containing ubiquitin ligase, which also competes with Gsα to bind to MC4R (Perez-Oliva et al 2009, Overton & Leibel 2011.…”

A Small Molecule Agonist THIQ as a Novel Pharmacoperone for Intracellularly Retained Melanocortin-4 Receptor Mutants

“…Several studies have been performed to correct the localization of misfolded MC4Rs through modulating molecular chaperone such as heat shock cognate protein 70 6, or using chemical chaperones such as 4-phenyl butyric acid 7, or several nonpeptidic MC4R antagonists as pharmacological chaperones (pharmacoperones) 8-11. A more recent study using endoplasmic reticulum-targeted α-MSH showed that it also promotes the cell surface expression of I316S MC4R 12. In these studies, most of the misfolded MC4Rs become functional when corrected to the cell surface.…”