Genetic studies in mice and humans have established the essential role of the central leptin-melanocortin axis in the maintenance of long-term energy homeostasis (35
) and identified MC4R as a prime target for therapeutic intervention in obesity. Our study demonstrates that the N-terminus of MC4R is required for the constitutive activity of the receptor and that loss of this constitutive activity is associated with obesity in humans (Figure ). Deletion and trans
-rescue experiments demonstrate that the N-terminal domain of MC4R functions as a tethered intramolecular ligand that maintains the constitutive activity of this receptor. The constitutive activation by the N-terminal domain is independent of the full activation provided by α-MSH, since the N-terminal domain is not required for the α-MSH–mediated effects.
Figure 6 Model depicting reduced constitutive activity (CA) of N-terminal domain MC4R mutants and its effect on energy balance. The constitutive activity of WT MC4R is responsible for the tonic inhibition of food intake and determines the set point for normal (more ...)
The role of the N-terminal domain in receptor activation has been clearly established for several GPCRs. For instance, N-termini of the PAR receptor family act as tethered full agonists after protease cleavage by thrombin or other serine/threonine proteases (36
). To our knowledge, however, MC4R is the first example of a receptor whose N-terminal domain is required to maintain constitutive signaling and may have therapeutic implications for the pharmacological treatment of obesity (Figure ). Our studies support the notion that a ligand that elicits a low, sustained level of MC4R activation (mimicking its constitutive activity) would be more effective than a potent ligand that induces receptor internalization and desensitization.
The only MC4R mutational hot spot found to date in severely obese patients (Figure ) is located at position R18 with three different missense substitutions in the N-terminal domain that decrease the constitutive activity of the receptor. Two different obesity-associated mutations have been identified at position T11 (13
). None of these N-terminal domain mutations have been found in nonobese controls (15
). Taken together, these findings argue in favor of a physiological role for constitutive activity in the maintenance of the anorexigenic catabolic state required for normal body weight in humans (Figure ). Point mutations in the N-terminal domain from obese patients reduce MC4R constitutive activity 50% or greater. The physiological significance of the decrease in constitutive activity observed in vitro is strengthened by the demonstration that variations of the same order in the level of constitutive activation of the closely related MC1R lead to drastic changes in coat color in mammals (34
). Indeed, the preservation of MC1R signaling in the absence of ligand, as judged by eumelanin synthesis in Pomc–/–
mice, strongly suggests a physiological role for its constitutive activity in rodents (37
). In humans, numerous studies have shown the pathological consequences of abnormally high basal signaling, most notably in the thyroid-stimulating hormone receptor (TSHR) and the lutenizing hormone receptor (LHR) leading to adenomas associated with hyperthyroidism and male precocious puberty, respectively (38
). By showing that mutations causing decreased basal signaling in MC4R result in a pathological state, our data provide the first evidence for the essential role of constitutive GPCR signaling in normal human physiology.
Since the constitutive activity of MC4R is inhibited by AGRP in vitro, our findings support a model in which the in vivo effects of AGRP are also mediated through its inverse agonist effects on MC4R. Precise comparisons of the obesity phenotypes of Pomc–/–
mice in the same background, as well as the study of double-null Pomc–/–Agrp–/–
mice, expected from our results to be less obese than the Pomc–/–
mice, should provide valuable information to support this model. Interestingly, a recent report indicates that Pomc–/–
mice did not demonstrate elevated neuropeptide Y (NPY) levels in the dorsomedial hypothalamus as seen in Mc4r–/–
), suggesting that constitutive activity of MC4R might be sufficient to maintain NPY signaling in the dorsomedial hypothalamus.
It should also be noted that while studies in rodents have been key for the discovery and study of the leptin-melanocortin system and genetic studies in humans have been largely confirmatory, subtle differences such as the degree of in vivo constitutive activity of MC4R and its importance for the maintenance of the long-term energy balance might reflect species-specific differences in this regulatory system. The study of the effects of naturally occurring obesity-causing mutations offers a unique approach to unraveling the molecular mechanisms underlying this regulation in humans in whom therapies will ultimately be used.