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

Significance

Melanocortin-4 receptor (MC4R) is a cell-surface hormone receptor in the brain that is central to the control of appetite. Upstream pathways sensing energy balance in the organism lead to the secretion of α-melanocyte–stimulating hormone (α-MSH), which stimulates MC4R activity and leads to decreased appetite. Previously, it was found that MC4R becomes resistant to treatment with extracellular agonists by a mechanism that may involve desensitization of the receptor, arguing against their usefulness in treating obesity. In this study, we show that exposing MC4R to α-MSH in the endoplasmic reticulum leads to constant signaling of MC4R without desensitization. Our results indicate that the cellular localization where agonist binding initially takes place affects the conformation and the desensitization properties of MC4R, suggesting a target for therapy.

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, agouti-related 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.

Melanocortin-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 feeding. Exposure of MC4R to α-MSH promotes receptor coupling to the heterotrimeric stimulatory G protein with downstream activation of adenylate cyclase and increased intracellular levels of cAMP. These effects are antagonized by AgRP, which also acts as an inverse agonist by inhibiting constitutive signaling of MC4R.

Following agonist stimulation, MC4R is desensitized by a population of receptors disappearing from the cell surface via a mechanism that includes retention of the receptor in the intracellular compartment and its traffic to lysosomes (22–25). Administration of the potent MC4R agonist melanotan II and of MSH/adrenocorticotropin (ACTH) _4–10 to lean and obese mice causes a decrease in both food intake and body weight (26, 27), thus suggesting that treatment with MC4R agonist could prevent or reverse obesity. However, in mice, prolonged agonist treatment causes tachyphylaxis by a mechanism that may include a compensatory up-regulation of AgRP mRNA levels and desensitization (26, 27). Moreover, overweight humans appear to be resistant to treatment with the MC4R agonists (28). These observations suggest that agonism of MC4R by extracellularly delivered ligands is an ineffective target to treat obesity because the receptor is desensitized and/or because of compensatory up-regulation of AgRP. Intracellular delivery of GPCR agonists and antagonists has been used to rescue conformationally defective receptors along the biosynthetic route and to promote their delivery to the cell surface (29). In this respect, it has been found that many MC4R variants linked to obesity have the tendency to misfold and be retained in the endoplasmic reticulum (ER) (6, 30–32). These mutant receptors can be rescued by pharmacoperones, which are MC4R antagonists that can permeate the membrane, as well as by chemical chaperones (31–35). Here we reasoned that delivery of MC4R agonists, rather than antagonists, to the ER could reduce the binding of MC4R to AgRP and promote the traffic of MC4R variants retained in the ER by changing the conformation of the receptor at this location. We find that binding MC4R to α-MSH in the ER abolishes the receptor response to AgRP and promotes the folding of an obesity-linked variant retained in the ER. In addition, binding MC4R to α-MSH in the ER induces a persistent activity of the receptor at a constantly maximal amplitude because desensitization and routing of the receptor to the lysosomes does not occur. The data indicate that desensitization of a GPCR can be modulated not only by the type of agonist to which the receptor is exposed, as in the case of opioid receptors (36–39), but also by the cell localization where the agonist/GPCR interaction initially takes place. The data presented here suggest that exposure of MC4R to α-MSH in the ER induces a conformation of the receptor different from that stabilized by extracellular α-MSH, so that that the receptor is both active and resistant to desensitization, indicating a potential target to promote GPCR signaling.

Results

α-MSH and γ+α-MSH Targeted to the Secretory Pathway Bind to Exogenous, Tagged MC4R.

Posttranslational processing of POMC generates α-, β-, and γ- (γ-MSH) melanocyte–stimulating hormones and ACTH. These hormones are agonists of MC4R and of other melanocortin receptors (40, 41). To target α-MSH to the ER, we generated a construct in which the signal sequence of POMC directing the hormone precursor to the ER was retained and was followed directly by the sequence of α-MSH with a Myc-tag at the C terminus (ER-α-MSH). The tetrapeptide motif His⁶-Phe⁷-Arg⁸-Trp⁹ of α-MSH is essential for stimulation of MC4R (40, 42–44), and mutation of His⁶ to Trp⁶ within this tetrapeptide reduces potency toward MC4R (40). We generated constructs with α-MSH lacking the tetrapeptide motif (ER–α-MSH–delta) and with His⁶ mutated to Trp⁶ (ER–α-MSH–Trp) (Fig. 1A). Corresponding constructs were generated in which WT and mutated α-MSH are preceded by the N-terminal peptide of POMC, which includes the weak MC4R agonist γ-MSH in addition to α-MSH (ER–γ+α-MSH) (Fig. S1A). We previously generated Neuro2A (N2A) cells, which, in addition to endogenous MC4R, express exogenous HA-MC4R-GFP with the HA tag attached to the N terminus and exposed to the extracellular medium and GFP attached the C terminus and exposed to the cytosol (N2A_MC4R cells) (24). The tags do not change the ability of MC4R to bind to α-MSH or to respond to α-MSH stimulation by increasing intracellular cAMP levels (24, 31). Immunofluorescence microscopy of fixed and permeabilized N2A_MC4R cells transiently expressing ER–α-MSH, ER–α-MSH–Trp, and ER–α-MSH–delta indicated that all these proteins were expressed at similar levels (Fig. S1B). However, ER–α-MSH and ER–α-MSH–Trp accumulated more clearly in a perinuclear compartment than ER–α-MSH–delta, indicating a different cellular distribution (Fig. S1B, arrows). To determine whether ER–α-MSH, ER–α-MSH–Trp, and ER–α-MSH–delta reached the plasma membrane, N2A_MC4R cells were incubated at 4 °C with antibodies against the Myc tag of the ER–α-MSH proteins and antibodies against the HA tag of HA-MC4R-GFP. ER–α-MSH and ER–α-MSH–Trp colocalized with HA-MC4R-GFP at the plasma membrane (Fig. 1B, second and third rows), but ER–α-MSH–delta did not (Fig. 1B, fourth row). Thus, ER–α-MSH and ER–α-MSH–Trp, but not ER–α-MSH–delta, bind to HA-MC4R-GFP at some point along the biosynthetic pathway to be transported to the cell surface. We have shown that MC4R is constitutively endocytosed and recycled back to the plasma membrane (24, 25). Consistent with that process, when live N2A_MC4R cells were incubated at 37 °C with anti-HA antibodies, the antibodies were internalized into a perinuclear endosomal compartment where HA-MC4R-GFP also localizes (Fig. 1C, first row). Also, the anti-Myc antibodies against the Myc tag of the ER–α-MSH and ER–α-MSH–Trp internalized to the same perinuclear compartment (Fig. 1C, second and third rows). However, when ER–α-MSH–delta was expressed, the anti-Myc antibody did not appear in the perinuclear endosomes (Fig. 1C, fourth row). These data indicate that ER–α-MSH and ER–α–MSH–Trp remain bound to HA-MC4R-GFP at the cell surface and through one or more cycles of receptor endocytosis.

Fig. 1.

Intracellular α-MSH (ER–α-MSH) expressed in N2A_MC4R cells binds to tagged MC4R along the biosynthetic pathway and is transported to the cell surface and to endosomes. (A) Schematic diagram of ER–α-MSH constructs used in this work. ER–α-MSH corresponds to the signal peptide of POMC (SP, amino acids 1–26) followed by the fragment of POMC that encodes for α-MSH (amino acids 138–150). (B and C) N2A_MC4R cells stably expressing HA-MC4R-GFP were transfected with empty vector (EV) or ER-α-MSH (WT, Trp, or delta). (B) To determine whether ER–α-MSH is expressed at the cell surface together with HA-MC4R-GFP, cells were incubated with anti-HA antibodies at 4 °C. Total HA-MC4R-GFP (GFP, green fluorescence), HA-MC4R-GFP at the cell surface (anti-HA, Cy3, red fluorescence), and ER–α-MSH at the cell surface (anti-Myc, Cy5, blue fluorescence) are shown. (C) To determine whether ER–α-MSH internalizes together with HA-MC4R-GFP, cells were incubated with anti-HA antibodies at 37 °C. Total HA-MC4R-GFP (GFP, green fluorescence), HA-MC4R-GFP at the cell surface and endosomes (anti-HA, Cy3, red fluorescence), and ER–α-MSH at the cell surface and endosomes (anti-Myc, Cy5, blue fluorescence) are shown. Immunofluorescence and confocal imaging was done as described in SI Methods, and experiments were performed three times. (Scale bar, 5 μm.)

Expression of Intracellular α-MSH in Neuronal Cells Expressing Tagged HA-MC4R-GFP Induces Constant cAMP Signaling.

Because ER–α-MSH appears to reach the plasma membrane/endosomes bound to HA-MC4R-GFP, we reasoned that the intracellular α-MSH might promote signaling of the receptor in the absence of exposure to extracellular α-MSH. MC4R couples to G protein with activation of adenylate cyclase and thereby increases the production of cAMP. To test this possibility, we used N2A cells transiently cotransfected with HA-MC4R-GFP and either empty vector or ER–α-MSH. When the N2A cells transiently expressing HA-MC4R-GFP were exposed for 15 min to α-MSH delivered to the medium, the cAMP level increased by ∼20-fold (Fig. 2, lanes 1 and 2). Conversely, when ER–α-MSH was coexpressed with HA-MC4R-GFP, cAMP already was elevated to the level induced by acute (15-min) exposure to extracellular α-MSH (Fig. 2, lanes 2 and 3). The addition of extracellular α-MSH to these cells did not further increase the level of cAMP being generated (Fig. 2, lanes 3 and 4). The experiment suggests that all the HA-MC4R-GFP appearing at the cell surface is in a complex with ER–α-MSH and signals constantly. Coexpression of HA-MC4R-GFP and ER–α-MSH–delta did not induce any increase of cAMP in the absence of extracellular α-MSH (Fig. 2, lanes 7 and 8). This result is consistent with the expected inability of α-MSH–delta to act as an agonist (40) and with the observation that ER–α-MSH–delta does not appear to bind to HA-MC4R-GFP (Fig. 1). Coexpression of HA-MC4R-GFP and ER–α-MSH–Trp increases intracellular cAMP by ∼10-fold, lower than the level induced by the expression of ER–α-MSH (Fig. 2, lanes 3 and 5); the addition of the extracellular hormone increased signaling further to the level generated by the receptor acutely exposed to extracellular α-MSH (Fig. 2, lanes 6 and 2). This result is consistent with the observation that mutation of His⁶ to Trp⁶ within α-MSH reduces potency toward MC4R (40). These experiments indicate that the expression of intracellular α-MSH leads to cAMP signaling that is constant and that, when the tetrapeptide motif His⁶-Phe⁷-Arg⁸-Trp⁹ of α-MSH is intact, has the same amplitude as the signaling induced by extracellular α-MSH.

Fig. 2.

Coexpression of intracellular α-MSH and tagged MC4R induces constant cAMP signaling at maximal amplitude. N2A cells were transiently cotransfected with HA-MC4R-GFP and with either empty vector (EV) or ER-α-MSH (WT, Trp, or delta). cAMP was measured in the absence (open bars) or in the presence of exogenous 100 nM α-MSH (gray bars). The graph shows a representative experiment in which samples were run in replicates of eight. The experiment was performed three times. Data are expressed as mean ± SD. **P < 0.01, ***P < 0.001; one-way ANOVA; ns, nonsignificant,

When ER–γ+α-MSH (WT, Trp, or delta) was expressed in the N2A_MC4R cells, the cAMP levels were equivalent to those induced by ER–α-MSH (Fig. S2A, lanes 5–7 and 2–4). This result suggests that, only α-MSH, and not γ-MSH in the same protein, functions to induce signaling of MC4R. It is possible that the constant signaling observed with the expression of ER–α-MSH occurs because the hormone is secreted in the medium and induces cAMP signaling by binding to MC4R expressed by the same or another cell. However, when the medium from N2A cells expressing ER–γ+α-MSH was transferred to another population of N2A_MC4R cells, it did not induce an increase of intracellular cAMP (Fig. S2B). Thus, the increase in cAMP depends not on the agonist being released in the medium but rather on the binding of the intracellular agonist to MC4R along the biosynthetic pathway.

Binding of Intracellular α-MSH to Tagged MC4R Occurs in the ER.

The data in Fig. 1 indicate that ER–α-MSH binds to MC4R along the secretory pathway. However, the cellular compartment in which this interaction takes place is unclear. Western blot analysis showed that HA-MC4R-GFP expressed without ER–α-MSH migrated at a different, higher molecular weight (MW) than HA-MC4R-GFP coexpressed with ER–α-MSH, suggesting divergence in posttranslational processing (Fig. 3A). In mammalian cells, N-glycosylation is initiated in the ER and requires the presence and availability of the acceptor sequence Asn-X-Ser/Thr (45, 46). HA-MC4R-GFP expressed either with or without ER–α-MSH and then treated with peptide-N-glycosidase F (PNGase F) migrated as a band with a lower MW than that of the receptor not exposed to glycosidase treatment (Fig. 3B). The shift in MW of MC4R upon PNGase F treatment indicates that the receptor is N-glycosylated. The deglycosylated HA-MC4R-GFP construct migrated equally, whether the receptor was coexpressed with or without ER–α-MSH. These observations suggest that coexpression of MC4R with ER–α-MSH changes the glycosylation pattern of the receptor. MC4R possesses four putative N-glycosylation sites, three at the N terminus (Asn³, Asn¹⁷, and Asn²⁶) and one at the first extracellular loop (Asn¹⁰⁸) (2). HA-MC4R-GFP receptors with mutations at each of these sites migrated faster than the WT receptor on SDS/PAGE (Fig. 3C, lanes 1 and 2–9, respectively). This result indicates that MC4R is N-glycosylated at each of these sites. The abundance of all HA-MC4R-GFP N-glycosylation mutants at the cell surface was similar to that the WT receptor (Fig. S3A). Thus, reduced N-glycosylation of MC4R does not appear to affect the ability of the receptor to mature. HA-MC4R-GFP with mutated Asn³, Asn²⁶, and Asn¹⁰⁸ underwent a further lower shift in MW when coexpressed with ER–α-MSH (Fig. 3C, lanes 3 and 4, 7 and 8, and 9 and 10), indicating that ER–α-MSH inhibits glycosylation at a site different from that blocked by the mutation. Conversely, HA-MC4R-GFP with the Asn¹⁷ mutation had the same MW when expressed without or with ER–α-MSH (Fig. 3C, lanes 5 and 6). Moreover, HA-MC4R-GFP with the Asn¹⁷ mutation had an apparent MW similar to that of the WT receptor coexpressed with ER–α-MSH (Fig. 3C, lanes 2 and 5 and 6, respectively). This result suggests that mutation at Asn¹⁷ and coexpression with ER–α-MSH have the same effect on N-glycosylation of MC4R. Together, these experiments indicate that ER–α-MSH binds to MC4R early during synthesis of MC4R in the ER, before N-glycosylation at Asn¹⁷ takes place. MC4R has intrinsic constitutive activity (17), which can be modulated by mutations at the N-terminal domain of MC4R (47). To determine if ER–α-MSH–mediated inhibition of glycosylation at Asn¹⁷ changed the constitutive activity of the receptor, we measured both the α-MSH–stimulated and constitutive activity of WT HA-MC4R-GFP and HA-MC4R-GFP N17Q. We found that the constitutive and α-MSH–stimulated activity by the WT and N17Q receptors were equivalent (Fig. S3 B and C). This result indicates that the increase in cAMP resulting from coexpression of MC4R with ER–α-MSH is not caused by enhanced constitutive activity of the receptor but rather by signaling after binding to the intracellular ligand.

Fig. 3.

Binding of ER–α-MSH to HA-MC4R-GFP occurs in the ER and changes the posttranslational processing of the receptor. (A) N2A cells were cotransfected with WT HA-MC4R-GFP and with or without ER-α-MSH. Cell lysates were immunoprecipitated and analyzed by Western blot as described in SI Methods. (B) N2A cells were cotransfected, and cell lysates were immunoprecipitated as in A. Immunoprecipitates were incubated with 0 or 20 units of PNGase F as described in SI Methods. Immunoprecipitates were analyzed as in A. (C) Asparagine residues of potential N-glycosylation sites were mutated to glutamine as described in SI Methods. N2A cells were cotransfected with WT or mutated HA-MC4R-GFP and with either empty vector (EV) or ER-α-MSH. Cell lysates were immunoprecipitated and analyzed as in A. The images in A–C show a representative gel from three independent experiments.

Binding of Intracellular α-MSH to Obesity-Linked MC4R Variant I316S Changes the Conformation of the Receptor to Promote Cell Surface Expression and Constant Signaling.

The data presented in Fig. 3 indicate that binding of ER–α-MSH to MC4R occurs in the ER. In this respect, it has been found that delivery of pharmacoperones, which are small lipophilic agonists and antagonists of GPCR that can pass through biological membranes, can change the conformation of these receptors by forming interactions along the biosynthetic pathway so that they can progress to the cell surface to signal (29, 48, 49). Pharmacoperones that are either inverse agonists or antagonists of MC4R have been used to promote progression along the secretory pathway of obesity-linked variants of MC4R that otherwise are retained in the biosynthetic pathway (32–34). We reasoned that if interaction with ER–α-MSH changed the conformation of MC4R I316S in the ER, then it might rescue the mutated receptor to the plasma membrane/endosomes. To determine if such rescue occurred, N2A cells transiently expressing either WT HA-MC4R-GFP or HA-MC4R-GFP I316S were incubated in the presence of anti-HA antibody for 1 h at 37 °C. Although in cells expressing WT HA-MC4R-GFP the staining by the anti-HA antibody virtually coincided with the fluorescence of GFP (Fig. 4A, first row, EV), in cells expressing HA-MC4R-GFP I316S the GFP fluorescence had a predominantly reticular distribution that differed from the staining by the anti-HA antibody (Fig. 4B, first row, EV). This result is consistent with previous findings that MC4R I316S has tendency to be retained in the ER (8, 30, 31, 35, 50). The ratio of red fluorescence (Cy3, recycling receptor) to green fluorescence (GFP, total receptor) is a measure of the fraction of total HA-MC4R-GFP that has exited the biosynthetic pathway to reach the cell surface and recycle (31). The coexpression of ER–α-MSH and ER–α-MSH–Trp increased (by ∼100%) the fraction of mutated (Fig. 4C, lanes 5–7) but not the fraction of WT HA-MC4R-GFP (Fig. 4C, lanes 1–3) able to reach plasma membrane/endosomes (Fig. 4C), whereas the coexpression of ER–α-MSH–delta did not have any effect (Fig. 4C, lanes 5 and 8). Thus, ER–α-MSH and ER–α-MSH–Trp appear to promote the exit of HA-MC4R-GFP I316S from the ER to the cell surface/endosomes. The data indicate that binding of the intracellular agonist to HA-MC4R-GFP I316S changes the conformation of the receptor in the ER so that it no longer is retained by the quality-control machinery.

Fig. 4.

Binding of intracellular α-MSH to HA-MC4R-GFP I316S induces both the exit of the mutated receptor from the ER and constant cAMP signaling. (A and B) N2A cells were cotransfected with either empty vector (EV) or ER-α-MSH (WT, Trp, or delta) and with either WT HA-MC4R-GFP (A) or I316S HA-MC4R-GFP (B). Immunofluorescence staining, confocal imaging, and quantification of the data were done as described in SI Methods (C) The fractions of total cell receptor at the cell surface and endosomes from the experiments shown in A and B were measured as described in SI Methods [WT HA-MC4R-GFP cotransfected with EV, number of cells (n) = 22; WT ER–α-MSH, n = 91; ER–α-MSH–Trp, n = 60; ER–α-MSH–delta, n = 53. I316S; HA-MC4R-GFP cotransfected with EV, n = 38; ER–α-MSH-WT, n = 127; ER–α-MSH–Trp, n = 136; ER–α-MSH–delta, n = 95). Quantification was done from three independent experiments. Each experiment was done three times. (D) N2A cells were cotransfected as in B. cAMP was measured in the absence (light bars) or in the presence (dark bars) of exogenous 100 nM α-MSH. The graph shows a representative experiment in which samples were run in replicates of eight. Each experiment was done three times. Data are expressed as mean ± SD. **P < 0.001, ***P < 0.0001; one-way ANOVA; ns, nonsignificant.

Next, we determined that the HA-MC4R-GFP I316S coexpressed with ER–α-MSH signals constantly, as does the WT receptor coexpressed with ER–α-MSH (Fig. 4D, lanes 1 and 3). However, unlike the signal generated by WT HA-MC4R-GFP, the signal generated by HA-MC4R-GFP I316S in a complex with ER–α-MSH was more elevated than that generated by the receptor variant acutely exposed to extracellular α-MSH (Fig. 4D, lanes 2 and 3), most likely because ER–α-MSH concomitantly increases the pool of receptors exiting the biosynthetic pathway to reach the plasma membrane/endosomes. In conclusion, the data in Fig. 4 indicate that interaction with ER–α-MSH and ER–α-MSH–Trp induces changes in conformation of an obesity-linked variant of MC4R in the ER. Such changes appear to underlie the ability of the receptor variant to exit the biosynthetic pathway and to signal constantly.

Expression of Intracellular α-MSH Induces cAMP Signaling at Maximal Amplitude in Immortalized GT1-7 Hypothalamic Cells.

Expression of intracellular α-MSH in neuronal cells with exogenous MC4R induces constant cAMP signaling at the amplitude induced by acute exposure to extracellular α-MSH. This constant activity over a prolonged period is unexpected, because it has been reported that prolonged exposure to extracellular α-MSH induces desensitization of MC4R, which is detectable by the disappearance of a population of MC4R from the cell surface and, functionally, by decreased amplitude of cAMP signaling upon a rechallenge with the hormone (22, 24, 25). As is consistent with these reports, pretreatment of N2A cells transiently expressing HA-MC4R-GFP with 1 μM α-MSH for 4 h blunted the response to acute challenge with α-MSH by ∼70% (Fig. 5A, lanes 2 and 4). Conversely, the level of cAMP constantly generated by HA-MC4R-GFP coexpressed with ER–α-MSH was similar to the level induced by the acute, 15-min exposure to extracellular α-MSH in N2A cells that were not pretreated with the hormone (Fig. 5A, lanes 2 and 5), as is consistent with data in Fig. 2, rather than to the level observed in response to the rechallenge with α-MSH after the prolonged pretreatment with the hormone (Fig. 5B, lanes 4 and 5). These data indicate that MC4R in a complex with α-MSH initiated in the ER signals constantly at maximal amplitude, unlike MC4R in a complex with extracellular α-MSH, which appears to become desensitized over time. However, it is possible that ability of ER–α-MSH to induce maximal signaling by HA-MC4R-GFP is an artifact caused by the overexpression of MC4R. To test this possibility, ER–α-MSH was expressed in N2A cells and GT1-7 immortalized hypothalamic neurons (Fig. 5 B and C), both of which express endogenous MC4R (25, 51–53). The GT1-7 cells were transduced with lentiviral particles to achieve ER–α-MSH expression in most of the cells (Fig. S4). In both N2A and GT1-7 cells, acute stimulation with extracellular α-MSH was induced by 60-min incubation with the hormone, which led to a 50% increase in intracellular cAMP (Fig. 5 B and C, lanes 1 and 2). The magnitude of this signal is similar to that found previously in the GT1-7 cells (52). Conversely, when N2A and GT1-7 cells were pretreated with extracellular α-MSH for 4 h and then were rechallenged with the hormone for 1 h, the amount of cAMP did not increase (Fig. 5 B and C, lanes 1 and 3). This result indicates that cells expressing endogenous MC4R undergo profound desensitization upon prolonged incubation with the agonist. However, when N2A and GT1-7 cells expressed ER–α-MSH, the abundance of cAMP was increased by ∼50%, to the same extent as in cells acutely exposed to extracellular α-MSH (Fig. 5 B and C, lanes 2 and 4). These experiments indicate that when endogenous MC4R binds to α-MSH in the ER, the receptor signals constantly and at maximal amplitude, as observed with overexpressed HA-MC4R-GFP, and suggest that the receptor does not become desensitized.

Fig. 5.

Expression of intracellular α-MSH induces constant cAMP and AMPK signaling in neuronal N2A cells and immortalized GT1-7 hypothalamic cells expressing endogenous MC4R. (A) N2A cells were cotransfected with HA-MC4R-GFP and either EV or ER–α-MSH. Cells were incubated in the absence or presence of 1 µM α-MSH for 4 h (pretreatment). Cells were washed and stimulated by incubating the cells with 100 nM α-MSH for 15 min in the presence of 3-isobutyl-1-methylxanthine (IBMX). The upper panel shows a schematic diagram of the experiment shown in the lower panel. (B) N2A cells were transfected with empty vector (EV) or ER–α-MSH. Cells were incubated in the absence or presence of 1 µM α-MSH for 4 h (pretreatment). Cells were stimulated by incubating the cells with 100 nM α-MSH for 60 min in the presence of IBMX. The upper panel is as in A. (C) GT1-7 cells were either mock-transduced or transduced with ER–α-MSH–plenti6 lentiviral particles at a final concentration of 10⁶ IU/mL in the complete culture medium, and cells were incubated at 37 °C for 72 h. The experiment was done as in B. (D) N2A cells were transfected with either empty vector (EV) or ER–α-MSH and were incubated at 37 °C in the presence or absence of 1 μM α-MSH for 4 h (pretreatment). Cells were washed and treated with or without 100 nM α-MSH for 15 min (stimulation). Expression of phospho-AMPK and total AMPK were measured by Western blot as described in SI Methods. (Left) A schematic diagram of the experiment. (Center) A representative Western blot from three independent experiments. (Right) The quantification of three independent experiments. Data are expressed as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001; one-way ANOVA; ns, nonsignificant.

In vivo experiments indicate that MC4R signaling leads to inhibition of AMP kinase (AMPK) activity in the hypothalamus, which is necessary to reduce food intake (54). Consistent with these data are reports that exposure of immortalized hypothalamic neurons to α-MSH induces dephosphorylation of AMPK at Thr172, resulting in a loss of AMPK activity (55). When N2A cells were exposed for 15 min to α-MSH added to the medium, phosphorylation of AMPKα at Thr¹⁷² was reduced by ∼40% compared with cells that were not treated with the agonist (Fig. 5D, lanes 1 and 2). This result indicates that the α-MSH signaling to decrease AMPK activity is reproduced in the N2A cells. However, when N2A cells were treated with α-MSH for 4 h, the level of phosphorylated AMPK did not change, whether or not cells were washed to be acutely reexposed to extracellular α-MSH (Fig. 5D, lanes 3 and 4). This result again indicates profound desensitization of MC4R. Conversely, when ER–α-MSH was expressed in N2A cells, the level of AMPK phosphorylation at Thr¹⁷² was reduced to that seen in cells acutely exposed to the hormone (Fig. 5D, lanes 2 and 5). These data indicate that endogenous MC4R in a complex with of ER–α-MSH signals through the AMPK pathway at maximal amplitude, suggesting that MC4R does not become desensitized.

MC4R Exposed to Intracellular α-MSH Does Not Become Desensitized.

Formation of the MC4R/intracellular α-MSH complex in the ER appears to decrease the propensity of the receptor to become desensitized. However, constant signaling by MC4R/ER–α-MSH instead might result from the continuous delivery of newly synthesized complexes to the cell surface/endosomes and/or from signaling along the biosynthetic pathway. To test these possibilities, cells were treated for 2 h with cycloheximide to block protein synthesis and with 1 μM α-MSH to compare the effects of prolonged exposure to extracellular α-MSH on HA-MC4R-GFP in a complex with ER–α-MSH and on unoccupied HA-MC4R-GFP. After 2-h incubation with extracellular α-MSH, more than 80% of the ER–α-MSH immunoreactivity disappeared from the plasma membrane of N2A cells cotransfected with ER–α-MSH and HA-MC4R-GFP, indicating that exposure to the extracellular α-MSH displaced most of the intracellular agonist from the MC4R binding site (Fig. 6 B and C). Upon prolonged exposure to extracellular α-MSH, cells without coexpressed ER–α-MSH had ∼40% less HA-MC4R-GFP at the plasma membrane, and cells with coexpressed ER–α-MSH had ∼20% less HA-MC4R-GFP (Fig. 6D, lanes 1 and 2 and lanes 3 and 4). This result indicates that the expression of intracellular α-MSH blunts the loss of receptor abundance at the plasma membrane caused by prolonged exposure to extracellular α-MSH. Functionally, when rechallenged with the hormone, cAMP levels were reduced by ∼40% in cells incubated with extracellular α-MSH without coexpressed ER–α-MSH and were unchanged in cells incubated with extracellular α-MSH with coexpressed ER–α-MSH (Fig. 6 E and F). The data indicate that when the MC4R/intracellular α-MSH complex initially forms in the ER, the propensity of the receptor to become desensitized is decreased. In addition, blocking protein synthesis by cycloheximide seems not to disrupt the ability of HA-MC4R-GFP in a complex with intracellular α-MSH to generate cAMP at the amplitude generated by the receptor when acutely exposed to extracellular α-MSH (Fig. 6F, lanes 1 and 3). This result suggests that continuous delivery of newly synthesized MC4R/ER–α-MSH complexes does not contribute to the constant signaling of the receptor. Moreover it appears that the MC4R/intracellular α-MSH complex signals after exiting the biosynthetic pathway. These observations are consistent with the finding that MC4R in a complex with ER–α-MSH signals at same level as MC4R acutely exposed to extracellular α-MSH.

Fig. 6.

HA-MC4R-GFP in a complex with intracellular α-MSH does not become desensitized. (A) Schematic diagram of the experiments shown in B–D. N2A cells were cotransfected with WT HA-MC4R-GFP and either EV or ER–α-MSH. CHX, cycloheximide. (B) Staining of ER–α-MSH at the cell surface (anti-Myc, Cy3, red fluorescence) and HA-MC4R-GFP at the cell surface (anti-HA, Cy5, blue fluorescence) was done as described in SI Methods. (Scale bar, 5 μm.) The figure shows a representative sample from two independent experiments. (C) The graph shows the quantification of the experiments in B [without α-MSH, number of cells (n) = 30; with 1 μMα-MSH, n = 35]. (D) Cells were incubated in the absence or presence of 1 µM α-MSH for 2 h. Cell surface HA-MC4R-GFP was measured as described in SI Methods. The graph shows a representative experiment in which samples were run in replicates of four. Each experiment was done three times. (E) Schematic diagram of the experiments shown in F. (F) Cells were incubated in the absence or presence of 1 µM α-MSH for 2 h. Intracellular cAMP was measured in the presence of 100 nM α-MSH. The graph shows a representative experiment in which samples were run in replicates of eight. Each experiment was done three times. Data are expressed as mean ± SD. Statistical significance was assessed with a Student t test (C) or one-way ANOVA (D and F), ***P < 0.001; ns, nonsignificant.

MC4R Reaching the Cell Surface in a Complex with Intracellular α-MSH Is Stabilized in an Active Conformation Different from That of the Receptor in a Complex with Extracellular α-MSH.

The data shown in this paper indicate that, when MC4R binds to intracellular α-MSH, the receptor is stabilized into an active conformation that signals constantly and does not become desensitized. These observations open the possibility that MC4R can have different functional properties at the cell surface, depending on whether the receptor initially binds α-MSH in the ER or at the plasma membrane. Because MC4R cycles constantly between the plasma membrane and the endosomes, dissecting possible differences in the function of the receptor residing at the cell surface requires an assay with temporal resolution. In this respect, we have found that expression of ER–α-MSH does not change the fraction of recycling HA-MC4R-GFP localized at the cell surface (Fig. S5B). Expression of ER–α-MSH also does not change the rate of internalization of HA-MC4R-GFP (t_1/2 ∼10 min) compared with that of the unoccupied receptor (Fig. S5A). Thus, the distribution and dynamics of HA-MC4R-GFP associated with intracellular α-MSH along the recycling compartments are similar to those of the unoccupied receptor. We measured cAMP generation by using a real-time FRET-based approach with the genetically encoded sensor m-Turquoise-^TEpac^VV-YFP (^TEpac^VV), which is composed of the cAMP-responsive protein Epac sandwiched between two fluorescent tags, mTurquoise (donor) and YFP (acceptor) (56). When N2A_MC4R cells were transfected with ^TEpac^VV and incubated with forskolin, which directly activates adenylate cyclase, the fluorescence of mTurquoise increased and that of YFP decreased (Fig. 7A), thus increasing the ratio of mTurquoise fluorescence to YFP fluorescence (the FRET ratio) (Fig. 7B), as is consistent with a previous report (56). These data indicate that ^TEpac^VV can detect changes in intracellular cAMP concentration over time in N2A_MC4R cells. When N2A_MC4R cells expressing ^TEpac^VV were exposed to extracellular α-MSH, the FRET ratio increased, indicating that binding of the agonist to HA-MC4R-GFP induces an increase of intracellular cAMP. This increase occurred within ∼2.5 min, and the ratio remained elevated over the next 15 min. The addition of α-MSH at 100 nM appeared to be sufficient to bind to the entire population of cell receptors, because subsequent additions of the hormone at higher concentrations did not increase further the amount of intracellular cAMP being generated (Fig. S6). When AgRP was added to the medium of N2A_MC4R cells previously exposed to extracellular α-MSH, the FRET ratio dropped (Fig. 7E). This decrease indicates that AgRP, by displacing extracellular α-MSH bound to HA-MC4R-GFP, leads to a decrease in the intracellular concentration of cAMP, consistent with the role of AgRP as an antagonist (12–16). The AgRP-dependent decrease in cAMP level also occurred within ∼2.5 min, an interval during which the population of HA-MC4R-GFP receptors at the cell surface changes by less than 15%. [This value is derived from modeling, as described in SI Methods, the internalization data of HA-MC4R-GFP, which we previously found to be unchanged by binding to extracellular α-MSH (24).] Conversely, in the N2A_MC4R cells cotransfected with ER–α-MSH, the ^TEpac^VV FRET ratio already was elevated to the level seen in N2A_MC4R cells acutely stimulated with α-MSH and, as is consistent with data in Fig. 2, did not change with the addition of extracellular α-MSH to the medium (Fig. 7D). Importantly, the addition of AgRP did not decrease the cAMP level in cells expressing ER–α-MSH (Fig. 7F). Thus, it appears that AgRP does not displace α-MSH from MC4R at the cell surface when the complex initially was formed in the ER but does so when the complex initially was formed at the cell surface. However, the possibility still existed that AgRP is unable to displace the intracellular α-MSH from HA-MC4R-GFP because of changes in the α-MSH protein itself, such as the Myc epitope at the C terminus. To exclude this possibility, ER–α-MSH at the cell surface was replaced by extracellular α-MSH by pretreating cells with 1 μM α-MSH, as in Fig. 6B, and then exposing them to 100 nM α-MSH for the duration of the assay (Fig. 7G). Importantly, even when ER–α-MSH bound to HA-MC4R-GFP was replaced by extracellular α-MSH, the addition of AgRP did not decrease the cAMP level in cells expressing ER–α-MSH (Fig. 7H). The data suggest that MC4R reaching the cell surface in a complex with intracellular α-MSH is stabilized in an active conformation that is different from that of the receptor in a complex with extracellular α-MSH. These data also indicate that, when MC4R folds in the presence of α-MSH in the ER, there is a persistent change in conformation of the receptor, and this change makes it resistant to antagonism by AgRP, whether or not the intracellular α-MSH has been replaced by the extracellular hormone.

Fig. 7.

At the cell surface, the sensitivity of MC4R in a complex with intracellular α-MSH to antagonism by AgRP is different from that of MC4R in a complex with extracellular α-MSH. (A and B) N2A_MC4R were transfected with mTurquoise-_TEPAC_VV-YFP. Cells were pretreated with 100 mM IBMX for 10 min and subsequently were treated with 1 mM forskolin at the indicated time. The fluorescence of mTurquoise was excited at 458 nm, and the intensities of fluorescence emitted at 480–495 nm (mTurquoise) and at 535–565 nm (YFP) were recorded over time (A), along with the ratio of the intensity of mTurquoise fluorescence to the intensity of fluorescence YFP (B). (C–F) N2A_MC4R cells were transfected with mTurquoise-_TEPAC_VV-YFP and with either empty vector (EV) (C and E) or ER–α-MSH (D and F). Cells were treated with 100 nM α-MSH and with or without 600 nM AgRP as indicated. Fluorescent ratios were calculated as in B. Graphs shown are of representative cells for each condition. (G) Schematic diagram of the experiments shown in H. (H) N2A_MC4R cells transfected with mTurquoise-_TEPAC_VV-YFP and ER–α-MSH were incubated in the presence of 1 µM α-MSH for 2 h. Cells were washed once in DMEM and then were incubated in DMEM with 100 nM α-MSH throughout the experiment. Cells were treated with 600 nM AgRP as indicated in the figure. Fluorescent intensities were recorded, and fluorescent ratios were calculated as described above. For each condition, at least 15 cells were quantified in at least three separate experiments.

MC4R in a Complex with Intracellular α-MSH, Unlike MC4R in a Complex with Extracellular α-MSH, Recycles Efficiently to the Plasma Membrane.

MC4R desensitization occurs because a population of constantly recycling receptors, when bound to extracellular α-MSH, are retained along the endosomal pathway and do not reappear at the plasma membrane by being routed to lysosomes (22–25, 57). We have shown here that upon coexpression of ER–α-MSH the fraction of recycling HA-MC4R-GFP residing at the cell surface is the same as that of the ligand-free receptor (Fig. S5B). Therefore it is possible that when α-MSH bound to MC4R in the ER, the receptor, although stabilized in an active conformation, is not retained in the endosomal/lysosomal compartment and therefore recycles to the plasma membrane as efficiently as if it were unoccupied. To visualize the dynamics of HA-MC4R-GFP at the cell surface, we used total internal reflectance fluorescence (TIRF) microscopy. By this approach, HA-MC4R-GFP appeared as diffuse fluorescence and as brighter puncta (Fig. 8A), as is consistent with data obtained previously (24). Time-lapse experiments carried out over an interval of 3 min indicated that, although most of the HA-MC4R-GFP puncta remained visible at the cell surface and changed their position minimally, some puncta appeared, and others disappeared (Fig. 8B). We labeled these appearing/disappearing puncta as “mobile GFP puncta.” In the N2A_MC4R cells kept in the basal state, the percentage of mobile puncta either appearing or leaving the field of vision was the same, 50% (Fig. 8C, lanes 1 and 2). This observation is consistent with our previous finding that the receptor recycles constitutively so that, at the steady state in the absence of agonist, the same number of receptors arrives at and leaves the plasma membrane. However, when α-MSH was added to the medium, the percentages of mobile GFP puncta appearing and disappearing from the field of vision were significantly different, with fewer puncta appearing at the plasma membrane (Fig. 8C, lanes 3 and 4). This result is consistent with our finding that binding to the extracellular agonist inhibits recycling of MC4R back to the cell surface (24). In the N2A_MC4R cells coexpressing ER–α-MSH, the percentage of mobile GFP puncta appearing and disappearing from the plasma membrane was the same regardless of the addition of α-MSH to the medium (Fig. 8C, lanes 5–8). These observations indicate that MC4R in a complex with ER–α-MSH does not become desensitized because the receptor recycles efficiently to the plasma membrane. The result is consistent with the concept that a difference in the conformation of MC4R in a complex with intracellular and extracellular α-MSH, respectively, underlies specific properties, such the ability to cycle efficiently to the cell surface, as shown in Fig. 8, and resistance to AgRP antagonism at the cell surface, as shown in Fig. 7.

Fig. 8.

At the cell surface, MC4R in a complex with intracellular α-MSH has dynamics similar to those of the unoccupied receptor. (A) A frame from a TIRF time-lapse experiment (0–3 min) showing puncta of HA-MC4R-GFP at the cell surface of live N2A_MC4R cells. (Scale bar, 1.5 μm.) (B) Puncta corresponding to HA-MC4R-GFP and visualized during a 3-min interval are divided in three categories: White arrows indicate puncta remaining stably at the cell surface; red arrows indicate puncta disappearing from the cell surface; and green arrows indicate puncta appearing at the cell surface. A detailed description of the TIRF experiments and quantification can be found in SI Methods. (Scale bar, 1.5 μm.) (C) N2A_MC4R cells were either mock-transduced or transduced with ER–α-MSH–plenti6 lentiviral particles. Cells were imaged in the presence or absence of 100 nM α-MSH. Quantification was done from three independent experiments as described in SI Methods. [mock, number of cells (n) = 13; mock + 100 nM α-MSH, n = 16; ER–α-MSH, n = 15; ER–α-MSH + 100 nM α-MSH = 6]. Data are expressed as mean ± SD. ***P < 0.001; one-way ANOVA; ns, nonsignificant.

MC4R in a Complex with Intracellular α-MSH, Unlike MC4R in a Complex with Extracellular α-MSH, Does Not Route to Lysosomes.

As described above, we have found that when MC4R initially binds to α-MSH in the ER, the receptor does not become desensitized under prolonged exposure to extracellular α-MSH (Fig. 6) and cycles to the plasma membrane as efficiently as the unoccupied receptor (Fig. 8). These observations suggest that the complex MC4R/intracellular α-MSH, unlike MC4R/extracellular α-MSH, does not route to lysosomes when internalized. To evaluate this possibility, we transiently transfected hypothalamic GT1-7 and N2A cells with HA-MC4R and the lysosomal marker lysosomal-associated membrane protein 1 LAMP1-GFP. Cells were treated with extracellular α-MSH or were left untreated in the presence of anti-HA antibody for 1 h at 37 °C to label the entire pool of receptors constitutively trafficking between the plasma membrane and the endosomal compartment (24). The localization of this pool of recycling receptors was visualized by staining fixed and permeabilized cells with secondary antibodies conjugated to Cy3 (Fig. 9A and Fig. S7A, red fluorescence). In GT1-7 and N2A cells not treated with α-MSH, colocalization of HA-MC4R with LAMP1-GFP was ∼10% (Fig. 9B and Fig. S7B). Conversely, when GT1-7 and N2A cells were incubated with α-MSH, colocalization of HA-MC4R with the lysosomal marker increased by approximately sixfold. Importantly, when HA-MC4R was coexpressed with ER–α-MSH, the amount of receptor colocalizing with LAMP1-GFP did not increase significantly compared with cells that were not treated with α-MSH. These results indicate that, although the binding of MC4R to ER–α-MSH along the biosynthetic pathway stabilizes an active conformation of the receptor, it does not induce routing to lysosomes. These data are consistent with the efficient cycling of the receptor in a complex with the intracellular α-MSH to the cell surface observed in Fig. 8.

Fig. 9.

MC4R in a complex with intracellular α-MSH does not route to lysosomes. (A) GT1-7 cells were cotransfected with LAMP1-GFP, WT HA-MC4R, and either empty vector (EV) or ER–α-MSH. Cells were incubated with anti-HA antibodies for 1 h at 37 °C. Immunofluorescence staining, confocal imaging, and quantification of the data were done as described in SI Methods. LAMP1-GFP (GFP, green fluorescence) and HA-MC4R at the cell surface and endosomes (CS + end; anti-HA, Cy3, red fluorescence) are shown. White indicates colocalization pixels. Arrows indicate the portion of the images enlarged in the Insets. (B) The graph shows the quantification of the experiments in A [EV, number of cells (n) = 16; EV + 100 nM α-MSH, n = 24; ER–α-MSH, n = 20]. Quantification was done from two independent experiments. Data are expressed as mean ± SD. ***P < 0.001; one-way ANOVA; ns, nonsignificant.

It has been reported the N-glycosylation site at position 187 in the β-2 adrenergic receptor is necessary for desensitization by routing the receptor to the lysosomes after exposure to the agonist (58). This observation suggested that the change in N-glycosylation of MC4R caused by the binding to ER–α-MSH results in an inability of MC4R to become desensitized. However, all the HA-MC4R-GFP receptors mutated at N-glycosylation sites, including N17Q, disappeared from the plasma membrane in response to extracellular α-MSH to the same extent as the WT receptor (Fig. S7B). Thus, the change in N-glycosylation of MC4R resulting from coexpression with ER–α-MSH does not seem, by itself, to be sufficient to confer receptor resistance to desensitization.

Conclusions

The most important result of this paper is that MC4R can exist in a fully active conformation that does not become desensitized and that such a conformation is achieved when MC4R forms a complex with α-MSH in the ER. Under these conditions, the receptor is poised to signal constantly at the same amplitude as MC4R acutely exposed to extracellular α-MSH. Constant signaling by MC4R in a complex with the intracellular agonist likely occurs outside the biosynthetic pathway because inhibition of protein synthesis does not affect this function, which instead appears to be dependent on an acquired resistance to desensitization. In this respect, we find that MC4R in a complex with intracellular α-MSH cycles efficiently to the cell surface and does not route to lysosomes, similar to unoccupied MC4R and unlike MC4R in a complex with the extracellular α-MSH (22–25, 57). Our data suggest that the resistance of MC4R to desensitization in a complex with intracellular α-MSH most likely is caused by the receptor’s being stabilized in an active conformation different from that of the receptor bound to extracellular α-MSH. This conclusion is based on data gathered using our temporally resolved FRET-based assays to measure real-time changes in intracellular cAMP levels. Using this assay, we show that AgRP antagonizes signaling by MC4R in a complex with extracellular α-MSH, as is consistent with previous reports (12–16). However, when the receptor/agonist complex is formed along the secretory pathway, AgRP does not attenuate MC4R signaling at the cell surface, thus suggesting a change in the conformation of the receptor. Such change in conformation appears to be an intrinsic property of the receptor folded in the presence of α-MSH because it remains insensitive to antagonism by AgRP, even when intracellular α-MSH is replaced with extracellular α-MSH. We find that intracellular α-MSH interacts with WT-MC4R in the ER because N-glycosylation at Asn¹⁷ of the receptor is abolished. Consistent with that finding, expression of the intracellular α-MSH promotes folding of an obesity-linked variant of MC4R that otherwise is retained in the ER, allowing it to exit the biosynthetic pathway and signal constantly. For other GPCRs, it has been found that different classes of drugs can alter the active conformation of the receptors so that they become desensitized to different extents (36–39, 59). Our results suggest that, at least for MC4R, the cellular localization where agonist binding initially takes place affects the conformation and the desensitization properties of the receptor. In this respect, protein folding in the ER is dependent on the surrounding environment (60), which here is modified by coexpression of the MC4R ligand. Several cell-permeable inverse agonist/antagonists have been shown to increase the exit of obesity-linked variants of MC4R from the biosynthetic pathway (32–34); here we offer evidence that targeting an agonist to the ER can have a similar effect. Our data suggest that a membrane-permeable small-molecule agonist would be a better approach than an antagonist to rescue MC4R function of variants because it would lead to constant/maximal signaling by the rescued receptors, as shown here, and it would not have to be displaced by α-MSH to stimulate receptor activity (29).

It has been reported that peripheral administration of potent MC4R agonists to mice that are obese because of prolonged treatment with high-fat diet leads to tachyphylaxis after few days of treatment, an effect that may occur by compensatory up-regulation of anorexigenic hormones such as AgRP and/or by desensitization of the receptor by prolonged exposure to the agonist (26, 27). The data in this paper indicate that intracellular delivery of α-MSH blunts both desensitization of MC4R and antagonism by AgRP. This work offers evidence that small-molecule or peptide-based agonists that are able to reach the ER may constitute a therapeutic approach to promote constant signaling by MC4R, and perhaps by other GPCRs, by stabilizing them in an active conformation that is resistant to desensitization.

Methods

Reagents, antibodies, and constructs, fluorescence microscopy, determination of HA-MC4R-GFP at the cell surface by enzyme-linked immunoassay, immunoprecipitation of HA-MC4R-GFP, and phosphorylation of AMPK are described in SI Methods.

Cell Culture, Transfection, and Transduction.

Neuroblastoma N2A (American Type Culture Collection) and immortalized hypothalamic GT1-7 cells were cultured in DMEM with 10% (vol/vol) FBS and penicillin/streptomycin. (For details, see SI Methods.)

Assay to Determine cAMP.

Intracellular cAMP was measured by using the direct cAMP enzyme immunoassay kit from Enzo Life Sciences following the manufacturer’s instructions. (For details, see SI Methods.)

Deglycosylation of MC4R.

PNGase F was purchased from New England Biolabs. (For details, see SI Methods.)

Statistical Analysis.

Data are expressed as mean ± SD. The experiments were performed in triplicate and done at least three times. Data were analyzed by using GraphPad Prism version 5.0 Software and were compared using the Student t test and one-way ANOVA as indicated.