The melanocortin-4 receptor (MC4R) is a member of the G-protein-coupled receptor (GPCR) superfamily, which has been extensively studied in obesity pathogenesis due to its critical role in regulating energy homeostasis. Both the Gs-cAMP and ERK1/2 cascades are known as important intracellular signaling pathways initiated by the MC4R. The DRYxxI motif at the end of transmembrane domain 3 and the intracellular loop 2 (ICL2) are thought to be crucial for receptor function in several GPCRs. To study the functions of this domain in MC4R, we performed alanine-scanning mutagenesis on seventeen residues. We showed that one residue was critical for receptor cell surface expression. Eight residues were important for ligand binding. Mutations of three residues impaired Gs-cAMP signaling without changing the binding properties. Investigation on constitutive activities of all the mutants in the cAMP pathway revealed that six residues were involved in constraining the receptor in inactive states and five residues were important for receptor activation in the absence of an agonist. In addition, mutations of four residues impaired the ligand-stimulated ERK1/2 signaling pathway without affecting the binding properties. We also showed that some mutants were biased to the Gs-cAMP or ERK1/2 signaling pathway. In summary, we demonstrated that the DRYxxI motif and ICL2 were important for MC4R function.
Obesity, associated with several adverse health conditions, such as type 2 diabetes mellitus, hypertension, cardiovascular disease, and certain types of cancer [1], has become a critical health issue worldwide. Obesity is commonly caused by imbalanced energy intake and expenditure. Many genetic factors are involved in the development of obesity [2]. The melanocortin-4 receptor (MC4R), highly expressed in the central nervous system (CNS), is one of the major factors in obesity pathogenesis. MC4R is crucial in the regulation of both food intake and energy expenditure [3]. Targeted deletion of Mc4r in mice causes maturity-onset obesity associated with hyperphagia, hyperinsulinemia, and hyperglycemia [3]. Human genetic studies also revealed that 5.8% of subjects with severe obesity commencing at childhood have mutations in MC4R, demonstrating dysfunction of MC4R to be the most common cause of monogenic obesity [4].
As a member of family-A G-protein-coupled receptors (GPCRs), MC4R has seven hydrophobic transmembrane domains (TMDs) connected by several intracellular and extracellular loops (ICLs and ECLs) [5]. Activation of MC4R results in GDP/GTP exchange in the α-subunit of stimulatory G (Gs) protein. The α-subunit disassociates from βγ heterodimer and activates adenylyl cyclase (AC) to increase the intracellular cyclic AMP (cAMP) level and subsequently enhance protein kinase A (PKA) activity. This conventional Gs-cAMP signaling pathway is crucial in inducing anorexigenic effect to result in a negative energy balance. In addition, MC4R also activates ERK1/2 [6,7,8,9], one of three mitogen-activated protein kinases (MAPK) pathways. The ERK1/2 activation through MC4R has been shown to regulate energy homeostasis by inhibiting food intake [10,11]. Defects in ERK1/2 signaling may also contribute to obesity pathogenesis in MC4R mutation carriers [12]. Therefore, both the Gs-cAMP and ERK1/2 signaling pathways are related to the MC4R function of energy homeostasis.
Constitutive activation of GPCRs is characterized by signaling in the absence of agonist stimulation. The constitutive activity of MC4R is essential for maintaining normal energy homeostasis in humans. It has been suggested that defects in constitutive activity of MC4R in the cAMP pathway attenuate the tonic satiety signal resulting in dysfunctional energy balance and obesity [13,14]. A recent study suggested that constitutive and agonist-induced MC4R activations differentially modulate signal to impact on distinct subtypes of voltage-gated calcium channels [15]. The constitutive activity of MC4R can affect the specific channels controlling transcriptional activity coupled to depolarization and neurotransmitter release [15]. Moreover, MC4R can also be constitutively active in the ERK1/2 pathway [9], suggesting that the constitutive activation of the ERK1/2 pathway may be involved in maintaining normal energy homeostasis.
The interactions involving highly conserved residues at the cytoplasmic surface are crucial for signaling properties in GPCRs. Crystal structural studies in several family-A GPCRs reveal that the DRYxxI motif in the end of TMD3 is of importance for receptor function. Conversed amino acids in the DRYxxI motif and TMD6 form polar interactions (commonly termed as ‘ionic lock’), bridging two transmembrane domains to stabilize the receptor in an inactive conformation [16,17]. Once the receptor binds to ligands, this ‘ionic lock’ is broken and the new interaction forms between DRYxxI motif and TMD5, triggering the receptor into an active conformation [17,18]. Therefore, the DRYxxI motif is critical in constraining the receptor in inactive conformation. The ICL2, linking TMD3 and TMD4, serves as a platform for hydrogen-bonding interaction between a conserved tyrosine on the ICL2 and DRYxxI motifs [17]. ICL2 participates in G-protein coupling and β-arrestin binding, indicating that this loop is significant in receptor activation and desensitization [19].
One of our previous studies based on alanine-scanning mutagenesis has shown that the DRYxxI motif and ICL2 are critical for MC3R function [20]. However, systematic study of this domain in MC4R is still lacking. In order to enhance the understanding of the structure–function relationship of MC4R, we generated seventeen mutants in total using alanine-scanning mutagenesis to investigate the functional roles of residues in this domain. Cell surface expression, ligand binding and signaling properties of the Gs-cAMP and ERK1/2 pathways of these seventeen mutants were investigated in the present study.
To investigate the function of each residue of DRYxxI motif and ICL2 of human MC4R, alanine-scanning mutagenesis was performed to mutate each residue to alanine or alanine to glycine. A total of seventeen mutant MC4Rs were generated. NDP-MSH, a superpotent analog of endogenous agonist α-MSH [21], was used in the present study.
It is known that GPCR mutations may impact receptors’ cell surface expression with defective protein synthesis or failure to pass through the quality control, especially in endoplasmic reticulum [22]. To quantitate the cell surface expression of mutant MC4Rs, flow cytometry technique was used in the present study. As shown, T150A had significantly lower cell surface expression (57.98 ± 10.03% of WT). Four mutants (A154G, Q156A, Y157A, and M161A) had slightly increased cell surface expression with statistical significance. All the other mutants were expressed at similar levels at the cell surface as WT MC4R.
Ligand-binding properties of the mutant MC4Rs were determined using unlabeled NDP-MSH to displace radiolabeled NDP-MSH. shows representative results and data from three independent experiments are summarized in Table 1. Of the mutant MC4Rs, D146A, Y148A, M160A, and M161A, had significantly decreased IC 50 s compared with the WT MC4R, indicating that these four mutants had increased affinities for the ligand. However, T150A had significantly increased IC 50 (decreased affinity) compared to the WT MC4R. In addition, seven mutants (D146A, Y148A, Y153A, Q156A, Y157A, M161A, and T162A) had significantly decreased maximal binding compared to the WT MC4R whereas two mutants (F149A and N159A) exhibited increased maximal binding with statistical significance (Table 1 and Figure 3).
To investigate both constitutive and ligand-induced signaling properties of MC4R mutants in the cAMP pathway, HEK293T cells expressing WT or mutant MC4Rs were stimulated without or with different concentrations of NDP-MSH. Five mutants (R147A, T150A, I151A, L155A, and Y157A) had significantly decreased basal cAMP levels compared to WT MC4R (Table 1 and Figure 4A). The basal cAMP production of T150A, which had reduced cell surface expression, was only 19.34 ± 4.05% of WT. However, six mutants (D146A, Y148A, F149A, F152A, Y153A, and H158A) displayed increased basal cAMP levels compared with WT (Table 1 and Figure 4A).
To further confirm that these mutants were indeed constitutively active, three inverse agonists, including the endogenous ligand AgRP (83-132) and two small molecule ligands, Ipsen 5i and ML00253764, were used to treat the cells expressing these six mutants. As shown in Figure 4B, AgRP (83-132) (10−8 M) could decrease the basal cAMP levels of WT and all six mutants with the inhibition ranging from 36 to 63%. The basal cAMP levels of WT and five mutants (Y148A, F149A, F152A, Y153A, and H158A) could be reduced by treatment of Ipsen 5i (10−6 M) or ML00253764 (10−5 M), with the inhibition ranging from 60 to 86%. In contrast, D146A showed activation rather than inhibition after treatment of these two small molecules.
NDP-MSH-stimulated cAMP levels were also measured in WT MC4R and mutants. As shown in Table 1 and Figure 5, we found that NDP-MSH dose-dependently increased intracellular cAMP accumulation in cells transfected with WT MC4R with an EC 50 of 0.38 ± 0.07 nM. Seven mutants (D146A, R147A, T150A, I151A, L155A, Q156A, and Y157A) were less capable of producing cAMP reflected by their significantly increased EC 50 s. Two mutants (F152A and H158A) showed decreased EC 50 s, suggesting that they could respond to NDP-MSH stimulation more potently. When maximal responses were analyzed, only one mutant, T150A, was demonstrated to have remarkably decreased maximal response (17.62 ± 3.60% of WT) despite normal maximal binding. Two mutants, M161A and T162A, which had very low maximal binding, showed significantly, although very slightly, increased maximal response.
To investigate the signaling properties of mutant MC4Rs on the ERK1/2 pathway, pERK1/2 levels were measured by western blot. Our data showed that all alanine mutants did not exhibit statistically significant alterations on basal pERK1/2 levels relative to WT basal pERK1/2 level (Figure 6A,C). WT MC4R could respond to NDP-MSH stimulation in the ERK1/2 pathway with more than two-fold increased pERK1/2 level. The mutants also had significantly increase
