Regulation of Melanocortin-4 Receptor Signaling: Agonist-Mediated Desensitization and Internalization
MELANOCORTIN RECEPTORS BELONG to the superfamily of G protein-coupled seven transmembrane receptors (GPCRs) (1). Each of the five subtypes identified so far couples in a stimulatory fashion to adenylate cyclase. The melanocortin 4 receptor (MC4R) is widely distributed in the brain, particularly in regions of the hypothalamus implicated in appetite and body weight regulation (2, 3). MC4R signaling is modulated by both an endogenous agonist, -melanocyte stimulation hormone (MSH), a peptide cleaved from proopiomelanocortin (POMC) and an endogenous antagonist, agouti-related protein (AGRP) (4). Leptin, an adipocyte-derived hormone, acts on POMC and AGRP neurons in the arcuate nucleus of the hypothalamus, resulting in increased MSH and decreased AGRP formation (5, 6). Several lines of evidence have indicated that activation of MC4R by MSH or synthetic peptide agonists reduce food intake, but suppression of MC4R signaling by AGRP or synthetic antagonists increase food intake and diminish the hypophagic response to leptin (7, 8). Targeted disruption of the MC4R gene in mice causes an obesity-diabetes syndrome characterized by hyperphagia, hyperinsulinemia, and hyperglycemia (9). Noteworthy is that heterozygotes for the null MC4R allele exhibit a phenotype intermediate between that of wild-type and homozygous littermates (9), unlike other monogenic animal models of obesity (10, 11, 12) and obese humans (13, 14, 15, 16). Although heterozygous loss-of-function mutations of MC4R in humans do not always display severe obesity (17, 18), these mutations represent the most common monogenic defect causing human obesity so far reported. Thus, the central melanocortin pathway is extremely important for normal energy homeostasis, and energy homeostasis through this pathway is highly susceptible to quantitative variation in MC4R expression.
Limited information is available to date regarding the changes in MC4R signaling in obesity. It has been reported that diet-induced obesity in rats causes selective changes in the number of MC4R, but not of MC3R, in hypothalamic regions (31). Several mutations within the MC4R of obese patients are associated with decreased binding affinity of agonist to MC4R, resulting in the attenuation of MC4R signals (17, 18, 32, 33). However, it appears that mutations in genes involved in appetite control do not account for obesity in most humans. Taken together, changes in steady-state regulation of MC4R signaling could be involved in the pathogenesis of obesity, as seen with other GPCRs in certain neurological diseases (29) and opiate tolerance and dependence (30). However, little is known about the regulation of MC4R signaling, including termination of the signals, desensitization, and internalization of the receptor, all of which are important for the final status of MC4R signaling. In addition, efforts are underway to develop drugs for the treatment of obesity that act on the MC4R (34). These drugs may have potential problems, however, including rapid disappearance of action by receptor desensitization and internalization as well as tachyphylaxis and down-regulation as a consequence of repeated doses, which are a common and predictable phenomenon for GPCR agonists (35). In this context, we have used mouse hypothalamic GT1–7 cells that express endogenous MC4R to demonstrate for the first time that MC4Rs display agonist-mediated desensitization. Studies using human embryonic kidney (HEK) 293 and COS-1 cells, both of which were transfected with hemagglutinin (HA)-tagged MC4R, have also demonstrated that activation of MC4R by agonist is associated with protein kinase A (PKA) and GRK phosphorylation of serine/threonine residues in the C-terminal tail of MC4R, followed by ß-arrestin and dynamin-dependent internalization of the receptor.
The major findings of the present study are as follows]1) MC4Rs display desensitization in response to agonist, as detected by impaired agonist-mediated cAMP formation after preexposure to the agonist in GT1–7 cells;
2) activation of MC4R by agonist is associated with time-dependent internalization of the receptor in HEK 293 cells;
3) fluorescence microscopy analysis demonstrates the sequestration of GFP-conjugated MC4R from the cell membrane, seen as a distinct punctate pattern, as early as 10 min after MSH exposure;
4) the internalization of MC4R by agonist is partly inhibited by treatment with H89, a specific PKA inhibitor, or overexpression of GRK2-K220R;
5) the internalization of MC4R by agonist is ß-arrestin and dynamin dependent;
6) Thr312 and Ser329/330 in the C-terminal tail of MC4R are potential sites for PKA and GRK phosphorylation and the subsequent recruitment of ß-arrestin to the activated receptor; and
7) AGRP, an endogenous antagonist of MC4R, increases cell surface MC4R.
RT-PCR analysis showed that hypothalamic GT1–7 cells predominantly express mRNA for MC4R, compared with MC3R, whereas medial hypothalamic tissues express both MC3R and MC4R mRNA, suggesting that GT1–7 cells are a suitable model for the evaluation of MC4R signaling. The desensitization of MC4R progressed rapidly during the first 30 min, and cAMP formation was reduced to less than 10% of control following 3-h preexposure of 100 nM MSH (Fig. 1D). Because forskolin-mediated cAMP formation was not influenced by the pretreatment with MSH (Fig. 2B), the desensitization by MSH in GT1–7 cells is probably due to the decrease in cell surface MC4R, uncoupling of G-protein from MC4R, or both. In our data, cAMP formation in response to 100 nM MSH was approximately 16 times less in GT1–7 cells than HEK 293 cells (Fig. 1A vs. Fig. 3A), whereas the magnitude of the desensitization caused by 100 nM MSH was greater in the former than the latter (Fig. 1D vs. Fig. 3B). The reason for the difference remains unclear at present, but it might be possible that, because MC4R in HEK 293 cells was overexpressed by transient transfection of MC4R cDNA, the expression level of MC4R was much greater in HEK 293 cells than GT1–7 cells; therefore, the endogenously expressed G protein that binds to the receptor was not sufficient to desensitize the receptor efficiently in HEK 293 cells. Of particular interest is that desensitization was observed by MSH at concentrations that produce minimal increase in cAMP formation (Figs. 1E and 2A). This may indicate that MC4R is readily desensitized by a small increase of cAMP associated with low receptor occupancy, as seen in ß2-adrenoceptor, which is triggered at very low receptor occupancy, and only subtle increases in cAMP are needed to fully activate PKA (35).
It might be argued that the agonist-mediated decrease in cell-surface MC4R is caused not only by the internalization of the agonist-activated MC4R but also by a decrease of synthesis or translocation to the membrane of de novo synthesized receptors. It is not clear how many de novo receptors are newly synthesized and translocated to the cell membrane during a 60-min test period that was performed 48 h after the transfection of HA-hMC4R cDNA. It appears evident that MSH causes the rapid sequestration of GFP-conjugated MC4R from the cell membrane, as shown in Fig. 4. Furthermore, there was no obvious difference in the amounts of cell surface HA-epitope in HEK 293 cells between 48 h and 49 h after cDNA transfection (data not shown). Thus, although receptors might be synthesized de novo during a 60-min test period, it is unlikely that the 40–50% decrease in cell surface HA-MC4R would result from changes in this rate. To explore whether the translocation of MC4R to cell membrane is influenced by agonist, further studies would be required.
A large body of evidence has suggested that phosphorylation of serine/threonine residues in the third internal loop or C-terminal tail of GPCRs by serine/threonine kinases is involved in the desensitization and internalization of many GPCRs (21, 22, 48, 49). In our data, overexpression of GRK2-K220R lacking kinase activity and that of wild-type GRK2 had little or minimal effect on the agonist-promoted internalization of MC4R in HEK 293 cells. However, in COS-1 cells, overexpression of GRK2-K220R inhibited the agonist-mediated internalization of MC4R, whereas coexpression of wild-type GRK2 had little effect on the internalization. These findings suggest that GRKs play a crucial role in the agonist-mediated MC4R internalization, presumably through the phosphorylation of agonist-occupied receptor. The inability of GRK2-K220R to inhibit MC4R internalization in HEK 293 cells might result from a high affinity of endogenous GRKs for activated MC4R, resulting in insufficient competition of GRK2-K220R for receptor binding because HEK 293 cells are reported to express the highest level of endogenous GRKs and ß-arrestins, compared with other cell lines such as COS-7 and Chinese hamster ovary cells (44), as confirmed by Western blot analysis (Fig. 7A). Many in vitro studies have shown that most GPCRs can be phosphorylated by one or more different GRKs (22), although the extent of contribution or isoform specificity of GRK for the phosphorylation of the receptor remains unclear in most cases. The RT-PCR analysis indicates that mRNA for GRK2–6 is expressed both in hypothalamus and GT1–7 cells (Fig. 6). Future studies should shed light on which subtypes of GRKs are involved in the phosphorylation of MC4R expressed in the relevant hypothalamic neurons.
In HEK 293 cells expressing HA-hMC4R, although the exposure of the cells to forskolin at a concentration that increases cAMP 1.5-fold, compared with 100 nM MSH, this had little effect on the internalization of MC4R. In contrast, agonist-mediated internalization was suppressed by cotreatment with the specific PKA inhibitor H89, consistent with the fact that Thr312 (LRKTF) has the phosphorylation consensus motifs for PKA (XRRXS/T where X means any amino acid,* means hydrophobic amino acid). The activation of MC4R by agonist causes PKA activation, as demonstrated by phosphorylation of CREB in GT1–7 cells. These results suggest that the agonist-mediated activation of G protein/adenylate cyclase and subsequent cAMP formation, a common stimulus that provokes activation of PKA, contribute to the desensitization and internalization of MC4R. Similar findings have been observed for the desensitization of MC2R]
Several reports have established that GRK-mediated phosphorylation followed by ß-arrestin binding may represent a common mechanism required for the sequestration of many GPCRs (24, 50). ß-Arrestins can serve as adaptor proteins between the phosphorylated receptor and component of the endocytic machinery, such as adaptor protein 2 and clathrin (51, 52). In our data, overexpression of dominant-negative mutants of ß-arrestin1 and dynamin I inhibited the agonist-mediated internalization of MC4R by 54% and 90%, respectively, suggesting that ß-arrestin- and dynamin-dependent processes are involved in the internalization of MC4R. Because ß-arrestin1-V53D binds better to clathrin than ß-arrestin but is significantly impaired in its interaction with phosphorylated GPCRs (46), it competes with the wild-type ß-arrestin for clathrin binding. The incomplete blocking of DN-ß-arrestin1 on the agonist-mediated internalization might be due to the insufficient expression of DN-ß-arrestin1 to block the interaction of MC4R-bound ß-arrestin to clathrin. Alternatively, processes independent of ß-arrestin may also contribute to the internalization of the agonist-activated receptors. However, the latter is more likely because the MC4R-T312A/S329A/S330A mutant that lacks potential binding sites for ß-arrestin still undergoes internalization more than half of wild-type MC4R. RT-PCR analysis showed that GT1–7 cells expressed mRNAs of both ß-arrestins (Fig. 6), consistent with the finding that both ß-arrestin 1 and 2 are predominantly localized in neuronal tissues (53). ß-Arrestins share 78% homology and bind many receptors with comparable affinity (53). Dynamin I, a neuron-specific isoform, was equally expressed in GT1–7 cells and medial hypothalamic tissues (Fig. 6). Therefore, it is reasonable to assume that both ß-arrestin and dynamin are involved in the desensitization and internalization of MC4R in neurons, although it is still unclear which subtypes of arrestins contribute to the processes in the hypothalamic neuronal cells.
Replacement by alanine(s) of Thr312 and Ser329/330 in the C-terminal tail resulted in an impaired sequestration of mutated receptors to agonist, whereas mutations of Thr232 or Ser306 did not. This indicates that phosphorylation of these residues by kinases is critical for the internalization of MC4R. Although certain GPCRs are known to be internalized in a phosphorylation-independent manner (54) or in the absence of detectable agonist-induced phosphorylation (55), these results are consistent with the notion that, in many GPCRs, phosphorylation by GRK of serine/threonine residues in the C-terminal tail of GPCRs is a critical step for the desensitization and internalization (25, 56, 57). Because the Ser329/330 is consistent with the phosphorylation consensus motifs for GRK2 and GRK3 (the presence of acidic residue localized on the N-terminal side of target serines/threonines) (58), these serines may be phosphorylated by GRK, although the motif is not an essential determinant in directing phosphorylation. Gurevich et al. have demonstrated that only 2 mol of phosphate/mol are required for the high-affinity arrestin binding to rhodopsin (59), m2 chorinergic (60), and ß2-adrenergic receptors (61) using various truncated and chimeric arrestins. In contrast to other GPCRs that have several serine/threonine residues to be phosphorylated, the only two serine/threonine residues located in the C terminus are critical for the agonist-promoted internalization of MC4R. Taken together, it seems likely that ß-arrestin is recruited with high affinity to the C terminus of activated MC4R to phosphorylated Thr312 and Ser329/330. This hypothesis arises from the finding that the magnitude of internalization of MC4R-T312A/S329A/S330A mutant by agonist was almost identical with that of MC4R-T312A mutant, not synergically potentiated, compared with that of T312A or S329A/S330A mutant.
The MC4R-T312A/S329A/S330A mutant that lacks potential ß-arrestin binding sites still underwent agonist-mediated internalization by approximately 60% of wild-type MC4R, indicating that, in HEK 293 cells, less than half of agonist-mediated internalization is serine/threonine and arrestin dependent, and the rest is dynamin dependent but serine/threonine and arrestin independent. It might be therefore reasonable that the inhibition of the internalization by H89 (Fig. 5), GRK2 K220R (Fig. 7), and ß-arrestin1-V53D (Fig. 8) was incomplete (50% or less). The internalization of some GPCRs is less sensitive to the effects of ß-arrestin mutant. For example, the internalization of AT1-angiotensin (62) and m2 muscarinic (63) receptors is not blocked by DN-ß-arrestins. Caveolae have also been reported to mediate the internalization of several GPCRs (20). Caveolae are morphologically distinct from clathrin-coated vesicles, but their formation is reported to be dynamin dependent. Most GPCRs have a conserved NPXXY motif in the seventh transmembrane helices, and this motif is known as an internalization signal in ß2-adrenergic receptors (64); initial asparagine is substituted to aspartic acid in all five subtypes of melanocortin receptors. Thus, alternative endocytic processes, in addition to the GRK/ß-arrestin-dependent process, may participate in the internalization of MC4R.
AGRP has been identified as competitively antagonizing the both MC4R and MC3R in the brain. Recently it has been suggested that AGRP acts as an inverse agonist for the MC4R in addition to a competitive antagonist (65, 66). In our data, preexposure of AGRP (83–132) to GT1–7 cells for 24 h potentiated the cAMP formation in response to MSH, whereas 30-min exposure did not. Furthermore, exposure of AGRP (83–132) alone for 6 h increased cell surface MC4R in HEK 293 cells expressing HA-hMC4R. These findings suggest that MC4R expressed in GT1–7 and HEK 293 cells internalizes spontaneously, even in the absence of agonist, and that AGRP (83–132) inhibits the internalization of the receptor from or recruits the receptor to the membrane, thereby leading to the increased cell surface MC4R number as well as cAMP formation in response to MSH. Thus, AGRP has a potential to up-regulate MC4R by stabilizing the inactive conformation of MC4R because inverse agonists have the ability to stabilize the inactive conformation of a receptor (67). The mechanism underlying this up-regulation is unclear at present and requires further investigation.
Although it has become clear from our data that MC4R signaling is regulated by desensitization and internalization of the receptor, there is no evidence that altered desensitization and internalization of MC4R are involved in the pathogenesis of obesity. Recently Pierroz et al. (68) have shown that treatment of mice with diet-induced obesity with MTII, an MSH analog, suppresses feeding during the first few days, with food intake returned to the level of nontreated mice thereafter. This may indicate that, even if leptin resistance is present, MC4R and downstream pathway is responsive to agonist given exogenously and that MC4R signaling may be readily desensitized by repeated doses. Several reports have shown that high-fat diet- or overfeeding-induced obesity in mice or rats is associated with increases in plasma leptin level and hypothalamic POMC expression (36, 69, 70), presumably leading to the enhanced MC4R signaling. Sustained rises in MC4R signaling may reduce the cell surface MC4R because of the enhanced desensitization and internalization of the receptor, thereby resulting in a sustained decrease in MC4R signaling, as reported by Harrold et al. (31), who showed that diet-induced obesity was associated with decreased hypothalamic MC4R expression in rats. In contrast, MTII is effective in reducing food intake and body weight in Zucker fatty rats, and the effects are more marked in obese rats than lean control rats (71). Zucker fatty rats are reported to show increased hypothalamic MC4R expression (31), probably because of decreased hypothalamic POMC expression associated with impaired leptin signaling.
Our data also suggest that AGRP has a potential to increase cell surface MC4R expression and cAMP formation in response to agonist. Taken together, the elevated melanocortin tone by overfeeding or a high-fat diet may facilitate the desensitization and internalization of MC4R, thereby leading to the down-regulation of MC4R. In addition, treatment with synthetic MC4R agonist may be effective in reducing food intake and body weight, but MC4R signaling may readily cause tachyphylaxis, especially in conditions of increased melanocortin tone. By contrast, it is tempting to speculate that the decreased melanocortin tone by food restriction or impaired leptin signaling may result in up-regulation of MC4R.
In summary, this study provides evidence that MC4R undergoes desensitization and internalization in response to agonist. MC4R desensitization and internalization involve PKA-, GRK-, ß-arrestin-, and dynamin-dependent pathways, indicating that these proteins play important roles in controlling the amplitude and duration of the MC4R signaling. The present study provides fresh insight into the regulation of MC4R from the viewpoint of both rapid (desensitization and internalization) and chronic (down-regulation) regulation of MC4R signaling. Impaired regulation of desensitization and internalization is a potential etiology of certain human diseases. Elevated GRK expression has been reported to accompany chronic heart failure (72) and hypertension (73). Recently Barak et al. (74) have found a naturally occurring loss-of-function mutation in the vasopressin receptor that is associated with familial nephrogenic diabetes insipidus by inducing constitutive arrestin-mediated desensitization. Thus, excessive GRK- and ß-arrestin-mediated desensitization and internalization of MC4R would be responsible for certain cases of human obesity. The regulation of central MC4R signaling is critical for the normal energy homeostasis. The present study provides a more complete understanding of the regulation of MC4R signaling and suggests new directions for research into MC4R signaling in the regulation of energy homeostasis.
Effects of leptin and melanocortin signaling interactions on pubertal development and reproduction.
2012
Abstract
Leptin and melanocortin signaling control ingestive behavior, energy balance, and substrate utilization, but only leptin signaling defects cause hypothalamic hypogonadism and infertility. Although GnRH neurons do not express leptin receptors, leptin influences GnRH neuron activity via regulation of immediate downstream mediators including the neuropeptides neuropeptide Y and the melanocortin agonist and antagonist, ?-MSH, agouti-related peptide, respectively. Here we show that modulation of melanocortin signaling in female db/db mice through ablation of agouti-related peptide, or heterozygosity of melanocortin 4 receptor, restores the timing of pubertal onset, fertility, and lactation. Additionally, melanocortin 4 receptor activation increases action potential firing and induces c-Fos expression in GnRH neurons, providing further evidence that melanocortin signaling influences GnRH neuron activity. These studies thus establish melanocortin signaling as an important component in the leptin-mediated regulation of GnRH neuron activity, initiation of puberty and fertility.
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