A Novel Melanocortin-4 Receptor Mutation MC4R-P272L Associated with Severe Obesity Has Increased Propensity To Be Ubiquitinated in the ER in the Face of Correct Folding

Heterozygous mutations in the melanocortin-4 receptor (MC4R) gene represent the most frequent cause of monogenic obesity in humans. MC4R mutation analysis in a cohort of 77 children with morbid obesity identified previously unreported heterozygous mutations (P272L, N74I) in two patients inherited from their obese mothers. A rare polymorphism (I251L, allelic frequency: 1/100) reported to protect against obesity was found in another obese patient. When expressed in neuronal cells, the cell surface abundance of wild-type MC4R and of the N74I and I251L variants and the cAMP generated by these receptors in response to exposure to the agonist, α-MSH, were not different. Conversely, MC4R P272L was retained in the endoplasmic reticulum and had reduced cell surface expression and signaling (by ≈3-fold). The chemical chaperone PBA, which promotes protein folding of wild-type MC4R, had minimal effects on the distribution and signaling of the P272L variant. In contrast, incubation with UBE-41, a specific inhibitor of ubiquitin activating enzyme E1, inhibited ubiquitination of MC4R P272L and increased its cell surface expression and signaling to similar levels as wild-type MC4R. UBE41 had much less profound effects on MC4R I316S, another obesity-linked MC4R variant trapped in the ER. These data suggest that P272L is retained in the ER by a propensity to be ubiquitinated in the face of correct folding, which is only minimally shared by MC4R I316S. Thus, studies that combine clinical screening of obese patients and investigation of the functional defects of the obesity-linked MC4R variants can identify specific ways to correct these defects and are the first steps towards personalized medicine.     

Functional characterization of mutations in melanocortin-4 receptor associated with human obesity

Melanocortin-4 receptor (MC4R) is a G protein-coupled receptor implicated in the regulation of body weight. Genetic studies in humans have identified two frameshift mutations of MC4R associated with a dominantly inherited form of obesity. We have generated and expressed the corresponding MC4R mutants in 293T cells and found that cells transfected with the truncation mutants failed to exhibit agonist binding or responsiveness despite retention of structural motifs potentially sufficient for binding and signaling. Immunofluorescence studies showed that the mutant proteins were expressed and localized in the intracellular compartment but absent from the plasma membrane, suggesting that these mutations disrupted the proper cellular transport of MC4R. Further studies identified a sequence in the cytoplasmic tail of MC4R necessary for the cell surface targeting. We further investigated a possible dominant-negative activity of the mutants on wild-type receptor function. Co-transfection studies showed that the mutants affected neither signaling nor cell surface expression of wild-type MC4R. We also characterized three human sequence variants of MC4R, but these exhibited identical affinities for peptide ligands and identical agonist responsiveness. Thus, unlike the obesity-associated MC4R truncation mutants, the polymorphisms of MC4R are unlikely to be contributors to human obesity.     

Functional characterization of nine novel naturally occurring human melanocortin-3 receptor mutations

The melanocortin-3 receptor (MC3R) is a member of family A rhodopsin-like G protein-coupled receptors. Mouse genetic studies suggested that MC3R and the related MC4R are non-redundant regulators of energy homeostasis. Lack of Mc3r leads to higher feed efficiency and fat mass. However, until now only a few MC3R mutations have been identified in humans and the role of MC3R in the pathogenesis of obesity was unclear. In the present study, we performed detailed functional studies on nine naturally occurring MC3R mutations recently reported. We found that all nine mutants had decreased cell surface expression. A260V, M275T, and L297V had decreased total expression whereas the other six mutants had normal total expression. Mutants S69C and T280S exhibited significant defects in ligand binding and signaling. The dramatic defects of T280S might be partially caused by decreased cell surface expression. In addition, we found mutants M134I and M275T had decreased maximal binding but displayed similar signaling properties as wild-type MC3R. All the other mutants had normal binding and signaling activities. Co-expression studies showed that all mutants except L297V did not affect wild-type MC3R signaling. Multiple mutations at T280 demonstrated the necessity of Thr for cell surface expression, ligand binding, and signaling. In summary, we provided detailed data of these novel human MC3R mutations leading to a better understanding of structure-function relationship of MC3R and the role of MC3R mutation in obesity.     

Functional characterization of novel melanocortin-3 receptor mutations identified from obese subjects

It is controversial whether mutation in the melancortin-3 receptor (MC3R) gene is a cause for monogenic obesity in humans. Three novel mutations in the MC3R, A293T, I335S, and X361S, were identified from morbidly obese subjects. We investigated whether these mutations caused loss-of-function and the molecular defects if any. Ligand binding, signaling, and cell surface expression of the mutant MC3Rs were studied. I335S resulted in a complete loss of ligand binding and signaling due to intracellular retention. A293T and X361S MC3Rs had normal ligand binding and signaling as wild type MC3R. Co-expression studies showed that the mutants did not affect wild type MC3R signaling. Hence the I335S variant previously identified from obese patients is not expressed at the cell surface when expressed in vitro, suggesting that it might contribute to obesity in carriers of this variant. Whether A293T and X361S cause obesity remains to be investigated. Additional mutations at I335 showed that I335, part of the highly conserved N/DPxxY motif, was critical for multiple aspects of the MC3R function, including cell surface expression, ligand binding, and signaling.     

The melanocortin-1 receptor carboxyl terminal pentapeptide is essential for MC1R function and expression on the cell surface

The pigmentary actions of the melanocortins are mediated by the melanocortin-1 receptor (MC1R), a seven transmembrane domains receptor positively coupled to Gs and the cAMP cascade. In order to define the structure-function relationships of potentially relevant domains in MC1R, particularly its C-terminal cytosolic tail, we generated and analyzed several variants with C-terminal deletions, as well as point mutants in selected residues of the human MC1R. We show that the MC1R C-terminal pentapeptide is essential for proper receptor expression on the plasma membrane, and that residues Thr314, Cys315 and Trp317 are at least partially responsible for this effect.     

Signaling from the human melanocortin 1 receptor to ERK1 and ERK2 mitogen-activated protein kinases involves transactivation of cKIT

Melanocortin 1 receptor (MC1R), a Gs protein-coupled receptor expressed in melanocytes, is a major determinant of skin pigmentation, phototype and cancer risk. Upon stimulation by αMSH, MC1R triggers the cAMP and ERK1/ERK2 MAPK pathways. In mouse melanocytes, ERK activation by αMSH binding to Mc1r depends on cAMP, and melanocytes are considered a paradigm for cAMP-dependent ERK activation. However, human MC1R variants associated with red hair, fair skin [red hair color (RHC) phenotype], and increased skin cancer risk display reduced cAMP signaling but activate ERKs as efficiently as wild type in heterologous cells, suggesting independent signaling to ERKs and cAMP in human melanocytes. We show that MC1R signaling activated the ERK pathway in normal human melanocytes and melanoma cells expressing physiological levels of endogenous RHC variants. ERK activation was comparable for wild-type and mutant MC1R and was independent on cAMP because it was neither triggered by stimulation of cAMP synthesis with forskolin nor blocked by the adenylyl cyclase inhibitor 2',5'-dideoxyadenosine. Stimulation of MC1R with αMSH did not lead to protein kinase C activation and ERK activation was unaffected by protein kinase C inhibitors. Conversely, pharmacological interference, small interfering RNA studies, expression profiles, and functional reconstitution experiments showed that αMSH-induced ERK activation resulted from Src tyrosine kinase-mediated transactivation of the stem cell factor receptor, a receptor tyrosine kinase essential for proliferation, differentiation, and survival of melanocyte precursors, thus demonstrating a functional link between the stem cell factor receptor and MC1R. Moreover, this transactivation phenomenon is unique because it is unaffected by natural mutations impairing canonical MC1R signaling through the cAMP pathway.     

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

Although mutations in the melanocortin-4 receptor (MC4R) gene cause severe early-onset obesity, we still do not have effective approaches to correct the defects of these mutations. Several antagonists have been identified as pharmacoperones of the MC4R whereas no agonist of the MC4R has been reported. In the present study, we investigated the effect of a small molecule agonist of the MC4R, THIQ, on the cell surface expression and signaling of ten intracellularly retained MC4R mutants using different cell lines. We showed that THIQ increased the cell surface expression of three mutants (N62S, C84R, and C271Y) and two of them (N62S and C84R) had increased signaling in HEK293 cells. Interestingly, THIQ increased the signaling of two other mutants (P78L and P260Q) without increasing their cell surface expression in HEK293 cells. In neuronal cells, THIQ exhibited a more potent effect, correcting the cell surface expression and signaling of seven mutants (N62S, I69R, P78L, C84R, W174C, P260Q, and C271Y). Other mutants were not rescued by THIQ. We also showed that THIQ did not rescue MC4R mutants defective in ligand binding or signaling or one intracellularly retained mutant of the melanocortin-3 receptor. In summary, we demonstrated that a small molecule agonist acted as a pharmacoperone of the MC4R rescuing the cell surface expression and signaling of some intracellularly retained MC4R mutants.     

Mechanisms of melanocortin-2 receptor (MC2R) internalization and recycling in human embryonic kidney (hek) cells: identification of Key Ser/Thr (S/T) amino acids

ACTH is the most important stimulus of the adrenal cortex. The precise molecular mechanisms underlying the ACTH response are not yet clarified. The functional ACTH receptor includes melanocortin-2 receptor (MC2R) and MC2R accessory proteins (MRAP). In human embryonic kidney 293/Flp recombinase target cells expressing MC2R, MRAP1 isoforms, and MRAP2, we found that ACTH induced a concentration-dependent and arrestin-, clathrin-, and dynamin-dependent MC2R/MRAP1 internalization, followed by intracellular colocalization with Rab (Ras-like small guanosine triphosphate enzyme)4-, Rab5-, and Rab11-positive recycling endosomes. Preincubation of cells with monensin and brefeldin A revealed that 28% of the internalized receptors were recycled back to the plasma membrane and participated in total accumulation of cAMP. Moreover, certain intracellular Ser and Thr (S/T) residues of MC2R were found to play important roles not only in plasma membrane targeting and function but also in promoting receptor internalization. The S/T residues T131, S140, T204, and S280 were involved in MRAP1-independent cell-surface MC2R expression. Other mutants (S140A, S208A, and S202D) had lower cell-surface expressions in absence of MRAPβ. In addition, T143A and T147D drastically impaired cell-surface expression and function, whereas T131A, T131D, and S280D abrogated MC2R internalization. Thus, the modification of MC2R intracellular S/T residues may positively or negatively regulate its plasma membrane expression and the capacity of ACTH to induce cAMP accumulation. Mutations of T131, T143, T147, and S280 into either A or D had major repercussions on cell-surface expression, cAMP accumulation, and/or internalization parameters, pointing mostly to the second intracellular loop as being crucial for MC2R expression and functional regulation.     

Impaired desensitization of a mutant adrenocorticotropin receptor associated with apparent constitutive activity

A naturally occurring ACTH receptor [melanocortin 2 receptor (MC2R)] mutation (F278C) has been identified in a subject with ACTH-independent Cushing's syndrome. Functional characterization of this mutant receptor reveals that it is associated with elevated basal cAMP accumulation when compared with wild-type receptor-expressing cell lines. Dose responsiveness is similar between wild-type and mutant receptors in cell lines expressing similar numbers of binding sites. In view of the location of this mutation in the C-terminal tail of the MC2R, desensitization and internalization were investigated and found to be impaired. Inhibition of protein kinase A by H89 blocks wild-type MC2R desensitization and also results in increased basal activity, as does alanine substitution of Ser 280 in the C-terminal tail. Alanine substitution of Ser 208, the consensus protein kinase A phosphorylation target in the third cytoplasmic loop also results in a reduction in desensitization without significant change in basal activity or internalization. These findings suggest a novel mechanism is involved in the apparently constitutive activation of the MC2R in which failure of desensitization appears to be associated with enhanced basal receptor activity.     

Pharmacological chaperones increase the cell-surface expression of intracellularly retained mutants of the melanocortin 4 receptor with unique rescuing efficacy profiles

Mutated versions of membrane proteins often fail to express at the plasma membrane, but instead are trapped in the secretory pathway, resulting in disease. The retention of these mutant proteins is thought to result from local misfolding, which prevents export from the ER (endoplasmic reticulum), targeting the receptor for degradation via the ER-associated quality control system. The rhodopsin-like G-protein-coupled MC4R (melanocortin 4 receptor) is an example of such a membrane protein. Over 100 natural MC4R mutations are linked with an obese phenotype and to date represent the most common monogenic cause of severe early-onset obesity. More than 80% of these mutations result in a substantial proportion of MC4R being retained intracellularly. If these receptors were expressed at the plasma membrane, many could be functional, as mutations often occur in regions distinct from those associated with ligand or G-protein binding. Our aim is to show proof of concept that selective compounds can rescue the function of MC4R mutants by increasing their cell-surface expression, and further to this, examine whether the rescue profile differs between mutants. Whole-cell ELISA and 96-well fluorescence-based assays with N-terminally HA (haemagglutinin)-tagged and C-terminally mCherry-tagged mutant MC4Rs were used to screen a number of novel MC4R-selective compounds. A total of four related compounds increased the cell-surface expression of wild-type and three intracellularly retained mutant MC4Rs, thus acting as pharmacological chaperones. There appears to be a unique rescue efficacy profile for each compound that does not correlate with potency, suggesting distinct receptor conformations induced by the different mutations. A degree of functionality of V50M and S58C was also rescued following relocation to the cell surface.     

Activation of type I cyclic AMP-dependent protein kinases with defective cyclic AMP-binding sites

Two S49 mouse lymphoma cell variants hemizygous for expression of mutant regulatory (R) subunits of type I cyclic AMP-dependent protein kinase were used to investigate functional consequences of lesions in the putative cAMP-binding sites of R subunit. Kinase activation properties of wild-type and mutant enzymes were compared using cAMP and six site-selective analogs of cAMP. Kinases from both mutant sublines were relatively resistant to cyclic nucleotide-dependent activation, but they were fully activable by at least some effectors. Relative resistances of the mutant kinases varied from about 5-fold for analogs selective for their nonmutated sites to as much as 700-fold for analogs selective for their mutated sites; resistance to cAMP was intermediate. Apparent affinities of wild-type and mutant R subunits for [3H]cAMP were not appreciably different, but competition experiments with site-selective analogs of cAMP suggested that binding of cAMP to mutant R subunits was primarily to their nonmutated sites. Analyses of cooperativity in cyclic nucleotide-dependent activation of mutant kinases, synergism between site I- and site II-selective analogs in activating the mutant enzymes, and dissociation of bound cAMP from mutant R subunits provided additional evidence that the mutations in these strains selectively inactivated single classes of cAMP-binding sites: phenomena attributable in wild-type enzyme to intrachain interactions between sites I and II were always absent or severely diminished in experiments with the mutant enzymes. These results confirm that R subunit sequences implicated in cAMP binding by homology with other cyclic nucleotide-binding proteins actually correspond to functional cAMP-binding sites. Furthermore, occupation of either cAMP-binding site I or II is apparently sufficient for activation of cAMP-dependent protein kinase. The presence of four functional cAMP-binding sites in wild-type kinase enhances the cooperativity and sensitivity of cAMP-mediated activation.     

Mutations that prevent cyclic nucleotide binding to binding sites A or B of type I cyclic AMP-dependent protein kinase

Cyclic nucleotide binding and activation properties of cAMP-dependent protein kinases from five independent mutants of S49 mouse lymphoma cells were studied. These mutants were all hemizygous for expression of mutant regulatory (R) subunits of the type I kinase with lesions that altered the electrostatic charge of R subunit: lesions in three of the mutants mapped to cAMP-binding site A, and those in two of the mutants mapped to cAMP-binding site B. A nucleotide mismatch assay using 32P-labeled cRNA and ribonuclease A confirmed and refined localization of the mutations to single amino acid residues implicated in cAMP binding. R subunits from all mutants retained the ability to bind cAMP, but binding behaved as if it were entirely to nonmutated sites: 1) relative affinities of 11 adenine-modified derivatives of cAMP for mutant enzymes were identical to their relative affinities for the site of wild-type kinase that corresponded to the nonmutated site of the mutant; 2) the potencies of these analogs as activators of mutant kinases were strictly correlated with their binding affinities (for wild-type enzyme activation potencies were correlated with mean affinities of the analogs for cAMP-binding sites A and B); 3) combinations of analogs with strong preferences for opposite cAMP-binding sites in wild-type kinase showed no synergism in activating mutant kinases; 4) dissociation of cAMP from mutant kinases was monophasic; and 5) high salt accelerated dissociation of cAMP from kinases with site B lesions but retarded dissociation from those with site A lesions.     

Poor cell surface expression of human melanocortin-4 receptor mutations associated with obesity

The melanocortin-4 receptor (MC4R) plays an important role in the regulation of body weight in rodents. Mutations in the coding region of the MC4R are found more frequently in obese individuals, supporting the hypothesis that also in humans deficient melanocortin signaling may lead to obesity. Family studies that were carried out to demonstrate the relevance of single mutations for obesity were mostly inconclusive, most likely due to small sample size and complexity of the trait. In addition, the existing pharmacological data of the mutant receptors are limited in that for most mutations the effect on receptor expression level and Agouti-related protein (AgRP) pharmacology have not been studied. The aim of the present study was to gain further insight into the impact of the MC4R mutations on receptor function. Eleven missense mutations were tested for cell surface expression, affinity for alpha-melanocyte-stimulating hormone (alpha-MSH) and AgRP-(83-132), and the biological response to alpha-MSH. All mutants were poorly expressed at the cell surface, as measured by 125I-[Nle4-D-Phe7]alpha-MSH binding, and only a few mutants showed altered pharmacology for alpha-MSH and AgRP. Hemagglutinin-tagged mutant receptors were retained in the intracellular environment. These pharmacological data provide a basis to estimate the quantitative effect of MC4R mutations for the development of obesity.     

Regulation of human melanocortin 1 receptor signaling and trafficking by Thr-308 and Ser-316 and its alteration in variant alleles associated with red hair and skin cancer

The melanocortin 1 receptor (MC1R), a G protein-coupled receptor (GPCR) positively coupled to adenylyl cyclase, is a key regulator of melanocyte proliferation and differentiation and a determinant of pigmentation, skin phototype, and skin cancer risk. MC1R activation stimulates melanogenesis and increases the ratio of black, strongly photoprotective eumelanins to yellowish and poorly photoprotective pheomelanin pigments. Desensitization and internalization are key regulatory mechanisms of GPCR signaling. Agonist-induced desensitization usually depends on phosphorylation by a GPCR kinase (GRK) followed by receptor internalization in endocytic vesicles. We have shown that MC1R desensitization is mediated by two GRKs expressed in melanocytes and melanoma cells, GRK2 and GRK6. Here we show that in contrast with this dual specificity for desensitization, GRK6 but not GRK2 mediated MC1R internalization. Mutagenesis studies suggested that the targets of GRK6 are two residues located in the MC1R cytosolic C terminus, Thr-308 and Ser-316. A T308D/S316D mutant mimicking their phosphorylated state was constitutively desensitized and associated with endosomes, whereas a T308A/S316A mutant was resistant to desensitization and internalization. We studied the desensitization and internalization of three variant MC1R forms associated with red hair and increased skin cancer risk: R151C, R160W, and D294H. These variants showed a less efficient desensitization. Moreover, D294H was resistant to internalization, thus accounting for its abnormally high surface expression. Co-expression of variant and wild type MC1R modified its desensitization and internalization behavior. These data suggest that MC1R might be regulated by novel mechanisms including differential effects of GRKs and altered desensitization rates of certain allelic combinations.     

The cytosolic chaperone Hsc70 promotes traffic to the cell surface of intracellular retained melanocortin-4 receptor mutants

Inherited modifications in protein structure frequently cause a loss-of-function by interfering with protein synthesis, transport, or stability. For the obesity-linked melanocortin-4 receptor (MC4R) and other G protein-coupled receptors, many mutants are intracellular retained. The biogenesis and trafficking of G protein-coupled receptors are regulated by multiple factors, including molecular chaperone networks. Here, we have investigated the ability of the cytosolic cognate 70-kDa heat-shock protein (Hsc70) chaperone system to modulate cell surface expression of MC4R. Clinically occurring MC4R mutants S58C, P78L, and D90N were demonstrated to have reduced trafficking to the plasma membrane and to be retained at the endoplasmic reticulum (ER). Analyses by fluorescence recovery after photobleaching revealed that the mobility of MC4R mutant protein at the ER was reduced, implying protein misfolding. In cells expressing MC4R, overexpression of Hsc70 resulted in increased levels of wild-type and mutant receptors at the cell surface. MC4R and Hsc70 coimmunoprecipitated, and fluorescence recovery after photobleaching analyses showed that increasing cellular levels of Hsc70 promoted the mobility of ER retained MC4R. Moreover, expression of HSJ1b, a cochaperone that enhances degradation of Hsc70 clients, reduced cellular levels of MC4R. Hsp70 and Hsp90 chaperone systems collaborate in the cellular processing of clients. For MC4R, inhibition of endogenous Hsp90 by geldanamycin reduced receptor levels. By contrast, expression of the Hsp90 cochaperone Aha1 (activator of Hsp90 ATPase) increased cellular levels of MC4R. Finally, we demonstrate that signaling of intracellular retained MC4R mutants is increased in cells overexpressing Hsc70. These data indicate that cytosolic chaperone systems can facilitate rescue of intracellular retained MC4R by improving folding. They also support proteostasis networks as a potential target for MC4R-linked obesity.     

Study of second-site suppression in the pheP gene for the phenylalanine transporter of Escherichia coli

Site-directed mutagenesis was used to investigate a region of the PheP protein corresponding to the postulated consensus amphipathic region (CAR) in the GabP protein. Whereas some critical residues are conserved in both proteins, there are major differences between the two proteins which may reflect different functions for this region. Replacement of R317, Y313, or P341 by a number of other amino acids destroyed the PheP function. An R317E-E234R double mutant exhibited low levels of PheP transport activity, indicating that there is a possible interaction between these two residues in the wild-type protein. E234 is highly conserved in members of the superfamily of amino acid-polyamine-organocation transporters and also is critical for PheP function in the wild-type protein. Second-site suppressors were isolated for mutants with mutations in E234, Y313, R317, and P341. Most suppressor mutations were found to cluster towards the extracellular face of spans III, IX, and X. Some mutations, such as changes at M116, were able to suppress each of the primary changes at positions E234, Y313, R317, and P341 but were unable to restore function to a number of other primary mutants. The possible implications of these results for the tertiary structure of the protein are discussed.     

Functional status and relationships of melanocortin 1 receptor signaling to the cAMP and extracellular signal-regulated protein kinases 1 and 2 pathways in human melanoma cells

Melanocortin 1 receptor (MC1R), a major determinant of skin phototype frequently mutated in melanoma, is a Gs protein-coupled receptor that regulates pigment production in melanocytes. MC1R stimulation activates cAMP synthesis and the extracellular signal-regulated (ERK) ERK1 and ERK2. In human melanocytes, ERK activation by MC1R relies on cAMP-independent transactivation of the c-KIT receptor. Thus MC1R functional coupling to the cAMP and ERK pathways may involve different structural requirements giving raise to biased effects of skin cancer-associated mutations. We evaluated the impact of MC1R mutations on ERK activation, cAMP production and agonist binding. We found that MC1R mutations impair cAMP production much more often than ERK activation, suggesting less stringent requirements for functional coupling to the ERK pathway. We examined the crosstalk of the cAMP and ERK pathways in HBL human melanoma cells (wild-type for MC1R, NRAS and BRAF). ERK activation by constitutively active upstream effectors or pharmacological inhibition had little effect on MC1R-stimulated cAMP synthesis. High cAMP levels were compatible with normal ERK activation but, surprisingly, the adenylyl cyclase activator forskolin abolished ERK activation by MC1R, most likely by a cAMP-independent mechanism. These results indicate little crosstalk of the cAMP and ERK pathways in HBL melanoma cells. Finally, we studied cAMP accumulation in a panel of 22 human melanoma cell lines stimulated with MC1R agonists or forskolin. cAMP synthesis was often inhibited, even in cells wild-type for MC1R and NRAS. Therefore, the cAMP pathway is more frequently impaired in melanoma than could be predicted by the MC1R or NRAS genotype.     

A membrane-proximal basic domain and cysteine cluster in the C-terminal tail of CCR5 constitute a bipartite motif critical for cell surface expression

We examined the structural requirements for cell surface expression, signaling, and human immunodeficiency virus co-receptor activity for the chemokine receptor, CCR5. Serial C-terminal truncation of CCR5 resulted in progressive loss of cell surface expression; mutants truncated at the 317th position and shorter were not detected at the cell surface. Alanine substitution of basic residues in the membrane-proximal domain (residues 314-322) in the context of a full-length C-tail resulted in severe reduction in surface expression. C-terminal truncation that excised the three cysteines in this domain reduced surface expression, but further truncation of upstream basic residue(s) abolished surface expression. Substituting the carboxyl-terminal domain of CXCR4 for that of CCR5 failed to rectify the trafficking defect of the tailless CCR5. In contrast, tailless CXCR4 or a CXCR4 chimera that exchanged the native cytoplasmic domain for that of wild type CCR5 was expressed at the cell surface. Deletion mutants that expressed at the cell surface responded to chemokine stimulation and mediated human immunodeficiency virus entry. Substitution of all serine and threonine residues in the C-terminal tail of CCR5 abolished chemokine-mediated receptor phosphorylation but preserved downstream signaling (Ca(2+) flux), while substitutions of tyrosine residues in the C-tail affected neither phenotype. CCR5 mutants that failed to traffic to the plasma membrane did not exhibit obvious changes in metabolic turnover and were retained in the Golgi or pre-Golgi compartments(s). Thus, the basic domain (-KHIAKRF-) and the cysteine cluster (-CKCC-) in the C-terminal tail of CCR5 function cooperatively for optimal surface expression.     

Mutationally induced disulfide bond formation within the third extracellular loop causes melanocortin 4 receptor inactivation in patients with obesity

By screening patients with severe early onset obesity for mutations within the melanocortin 4 receptor (MC4R) gene, we have identified a missense mutation (C271R) that occurs homozygous in two siblings with obesity. In-depth functional characterization of C271R revealed a right-shifted concentration response curve due to lower affinity to natural and synthetic MC4R agonists and a reduced cell surface expression. Cys-271 is located in the third extracellular loop. Here, we provide evidence that Cys-271 forms an intra-loop disulfide bond with Cys-277. Unexpectedly, we found that loss of receptor function is not only caused by the disruption of this disulfide bridge. Our data strongly support a new mechanism in which the receptor malfunction in the C271R mutant is induced by formation of a functionally disastrous disulfide bridge between Cys-277 and a third Cys residue at position 279. Mutational and chemical disruption of this improper disulfide bond was able to restore normal receptor potency. By demonstrating that a loss of a disulfide bond-participating Cys residue can favor a functionally disastrous disulfide bond, we now add a new mechanism of how Cys residues can be involved in G-protein-coupled receptor malfunction.     

Role of third intracellular loop of the melanocortin 4 receptor in the regulation of constitutive activity

The melanocortin 4 receptor (MC4R) has been reported to display constitutive activity, which is probably relevant to the maintenance of a normal energy balance. Among the clinically reported mutants of MC4R in human obesity patients, we investigated the functional characteristics of seven mutants characterized by mutations in the third intracellular (i3) loop of MC4R. Via a CRE (cAMP responsive element)-mediated luciferase reporter gene assay, we show that most of these mutants displayed significantly reduced basal activity with reduced reporter gene activity, whereas the P230L mutant manifested significantly increased basal activity. When the dominant negative G_s mutant was co-expressed, the majority of the mutants, including the P230L mutant, showed reduced basal activity. These results suggest that the i3 loop of MC4R is essential not only for the functional activity but also for the regulation and maintenance of an optimal constitutive activity of MC4R in association with G protein coupling, in the control of energy homeostasis.