Ligand rescue of constitutively active mutant receptors

Constitutively active mutants (CAMs) of G protein-coupled receptors are found naturally in disease states and they can be generated by point mutation. As these mutants are able to activate G proteins in the absence of a ligand, they are useful tools in the study of conformational changes leading to receptor activation and in the drug discovery process. Early studies on CAMs noted that they are often expressed at lower levels than their wild-type forms. In this review we discuss the mechanisms responsible for this reduced expression and also provide an explanation for the observation that challenging cells with receptor ligands can increase the CAM expression level. The application of these observations to the development of a high-throughput reporter assay suitable for ligand identification is also discussed.     

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.     

Antibodies against the melanocortin-4 receptor act as inverse agonists in vitro and in vivo

Functionally active antibodies (Abs) against central G-protein-coupled receptors have not yet been reported. We selected the hypothalamic melanocortin-4 receptor (MC4-R) as a target because of its crucial role in the regulation of energy homeostasis. A 15 amino acid sequence of the N-terminal (NT) domain was used as an antigen. This peptide showed functional activity in surface plasmon resonance experiments and in studies on HEK-293 cells overexpressing the human MC4-R (hMC4-R). Rats immunized against the NT peptide produced specific antibodies, which were purified and characterized in vitro. In HEK-293 cells, rat anti-NT Abs showed specific immunofluorescence labeling of hMC4-R. They reduced the production of cAMP under basal conditions and after stimulation with a synthetic MC4-R agonist. Rats immunized against the NT peptide developed a phenotype consistent with MC4-R blockade, that is, increased food intake and body weight, increased liver and fat pad weight, and elevated plasma triglycerides. In a separate experiment in rats, an increase in food intake could be produced after injection of purified Abs into the third ventricle. Similar results were obtained in rats injected with anti-NT Abs raised in rabbits. Our data show for the first time that active immunization of rats against the NT sequence of the MC4-R results in specific Abs, which appear to stimulate food intake by acting as inverse agonists in the hypothalamus.     

Pyrrolidines as potent functional agonists of the human melanocortin-4 receptor

A series of pyrrolidine derivatives were synthesized and characterized as potent agonists of the human melanocortin-4 receptor. For example, 28c had a K(i) of 13 nM in binding affinity and EC(50) of 6.9 nM in agonist potency with an intrinsic activity of 100% of the endogenous ligand alpha-MSH.     

Activation of protein kinase C by naturally occurring ether-linked diglycerides

Recent studies have demonstrated that ether-linked diglycerides are endogenous constituents of biologic tissues and accumulate during agonist stimulation (Daniel, L. W., Waite, M., and Wykle, R. L. (1986) J. Biol. Chem. 261, 9128-9132) and myocardial ischemia (Ford, D. A., and Gross, R. W. (1989) Circ. Res. 64, 173-177). Although protein kinase C previously had been thought to specifically require 1,2-diacyl-sn-glycerol (DAG) molecular species for activation, the present study demonstrates that purified rat brain protein kinase C is activated by naturally occurring ether-linked diglycerides (e.g. 1-O-hexadec-1'-enyl-2-octa-dec-9'-enoyl-sn-glycerol and 1-O-hexadecyl-2-octa-dec-9'-enoyl-sn-glycerol) with a similar dose response curve to that for DAG molecular species. Although in vitro assays demonstrated that DAG could partially activate protein kinase C in the absence of free calcium, activation by ether-linked diglycerides required free calcium concentrations found only in stimulated cells (greater than 1 microM [Ca2+]free). To substantiate these findings the alpha and beta isoforms of protein kinase C from rat brain cortical grey matter were resolved by hydroxylapatite chromatography. Although the beta isoform of protein kinase C was substantially activated by DAG in the absence of free calcium, activation by ether-linked diglycerides had an absolute requirement for physiologic increments in free calcium ion found in stimulated cells. Since ether lipids are localized in specific subcellular membrane compartments, accumulate during several pathophysiologic perturbations and are effective activators of protein kinase C with separate and distinct calcium requirements in comparison to DAG, these results suggest that ether-linked diglycerides are important and potentially specific biologic activators of one or more isoforms of protein kinase C.     

Constitutive internalization of constitutively active agiotensin II AT(1A) receptor mutants is blocked by inverse agonists.

As constitutively active mutants (CAMs) mimic an active conformation, they can be used to characterize the process of G protein-coupled receptor activation. Here, we used CAMs to study the link between activation and internalization of the angiotensin II AT(1A) receptor. The cellular localization of fluorescently tagged N111A, I245T, and L305Q mutants was determined by confocal microscopy. In the absence of ligand, CAMs were mostly located in intracellular vesicles, whereas the wild-type AT(1A) was found at the cell surface. After 2 h incubation with inverse agonist, losartan, CAMs were translocated to the plasma membrane. Similar observations were made in H295, a human adrenocortical cell line which expresses physiologically the AT(1) receptor. This phenomenon, which was not dependent on protein synthesis and the pharmacology and kinetics of which were similar to the recycling of the wild-type receptor, was called "externalization". After externalization and losartan removal, the L305Q CAM underwent rapid ligand-independent endocytosis, with the same kinetics and temperature sensitivity as the angiotensin II-induced internalization of the wild-type AT(1A). Moreover, the addition of a second mutation known to block internalization (Delta 329 truncation) prevented intracellular localization of the CAM. These data show that AT(1A) CAMs are constitutively and permanently internalized and recycled. This mechanism is different from the down-regulation observed for CAMs of other G protein-coupled receptors and thus defines a new paradigm for the cellular regulation of CAMs.     

Identification of agonists and antagonists of the human melanocortin-4 receptor from piperazinebenzylamines

SAR studies of a series of piperazinebenzylamines resulted in identification of potent agonists and antagonists of the human melanocortin-4 receptor. Thus, the 1,2,3,4-tetrahydroisoquinolin-1-ylacetyl compound 12e and the quinolin-3-ylcarbonyl analogue 12l possessed K(i) values of 6.3 and 4.5 nM, respectively. Interestingly, 12e was a full agonist with an EC(50) value of 31 nM, and 12l was a weak partial agonist (IA=17%) and functioned as an antagonist (IC(50)=300 nM).     

Stabilization of human haemoglobin by naturally occurring osmolytes

The influence of the naturally occurring osmolytes xylitol, glycine and betaine on the thermal stability of human haemoglobin was investigated. Experiments were made in the temperature range of 55–70 °C, adding up to 30% w/w of osmolytes to the protein solution. All the additives stabilized haemoglobin, with xylitol and glycine appearing more effective. A kinetic analysis based on the Lumry–Eyring inactivation scheme showed that the denaturation process can be described by a second-order rate expression, with an apparent activation energy ranging from 45 to 82 kcal/mol.     

Activation of purified cardiac ryanodine receptors by dihydropyridine agonists

Prior observations have raised the possibility that dihydropyridine (DHP) agonists directly affect the sarcoplasmic reticulum (SR) cardiac Ca(2+) release channel [i.e., ryanodine receptor (RyR)]. In single-channel recordings of purified canine cardiac RyR, both DHP agonists (-)-BAY K 8644 and (+)-SDZ202-791 increased the open probability of the RyR when added to the cytoplasmic face of the channel. Importantly, the DHP antagonists nifedipine and (-)-SDZ202-791 had no competitive blocking effects either alone or after channel activation with agonist. Thus there is a stereospecific effect of SDZ202-791, such that the agonist activates the channel, whereas the antagonist has little effect on channel activity. Further experiments showed that DHP agonists changed RyR activation by suppressing Ca(2+)-induced inactivation of the channel. We concluded that DHP agonists can also influence RyR single-channel activity directly at a unique allosteric site located on the cytoplasmic face of the channel. Similar results were obtained in human purified cardiac RyR. An implication of these data is that RyR activation by DHP agonists is likely to cause a loss of Ca(2+) from the SR and to contribute to the negative inotropic effects of these agents reported by other investigators. Our results support this notion that the negative inotropic effects of DHP agonists result in part from direct alteration in the activity of RyRs.     

The natural inverse agonist agouti-related protein induces arrestin-mediated endocytosis of melanocortin-3 and -4 receptors

Agouti-related protein (Agrp), one of the two naturally occurring inverse agonists known to inhibit G protein-coupled receptor activity, regulates energy expenditure by decreasing basal and blocking agonist-promoted melanocortin receptor (MCR) signaling. Here we report that, in addition to its inverse agonistic activities, Agrp exhibits agonistic properties on the endocytosis pathway of melanocortin receptors. Sustained exposure of human embryonic kidney 293 cells to Agrp induced endocytosis of the MC3R or the MC4R. The extent and kinetics of Agrp-promoted MCR endocytosis were similar to the endocytosis induced by melanocortins. Using the bioluminescence resonance energy transfer technique, we further showed that after binding of Agrp both MCRs interacted with beta-arrestins. In line with this observation, in COS-7 cells co-expression of beta-arrestins enhanced Agrp-induced MCR endocytosis, whereas in human embryonic kidney 293 cells co-transfection of beta-arrestin-specific small interference RNAs diminished Agrp-promoted endocytosis. This new regulatory mechanism was likewise detectable in a cell line derived from murine hypothalamic neurons endogenously expressing MC4R, pointing to the physiological relevance of Agrp-promoted receptor endocytosis. In conclusion, we demonstrated that Agrp does not solely act by directly blocking MCR signaling but also by reducing the amount of MCR molecules accessible to melanocortins at the cell surface. This beta-arrestin-dependent mechanism reveals a new aspect of MCR signaling in particular and refines the concept of G protein-coupled receptor antagonism in general.     

Activation of the NaCl- and drought-induced RD29A and RD29B promoters by constitutively active Arabidopsis MAPKK or MAPK proteins

Mitogen-activated protein (MAP) kinases mediate cellular responses to a wide variety of stimuli. Activation of a MAP kinase (MAPK) occurs after phosphorylation by an upstream MAP kinase kinase (MAPKK). The Arabidopsis thaliana genome encodes 10 MKKs, but few of these have been shown directly to activate any of the 20 Arabidopsis MAPKs (AtMPKs) and NaCl-, drought- or abscisic acid (ABA)-induced genes RD29A or RD29B. We have constructed the constitutively activated form for nine of the 10 AtMKK proteins, and tested their ability to activate the RD29A and RD29B promoters and also checked the ability of the nine activated AtMKK proteins to phosphorylate 11 of the AtMPK proteins in transient assays. The results show that three proteins, AtMKK1, AtMKK2 and AtMKK3, could activate the RD29A promoter, while these three and two additional AtMKK6/8 proteins could activate the RD29B promoter. Four other proteins, AtMKK7/AtMKK9 and AtMKK4/AtMKK5, can cause hypersensitive response (HR) in tobacco leaves using transient analysis. The activation of the RD29A promoter correlated with four uniquely activated AtMPK proteins. A novel method of activating AtMPK proteins by fusion to a cis-acting mutant of a human MAPK kinase MEK1 was used to confirm that specific members of the AtMPK gene family can activate the RD29A stress pathway.     

Structure-activity relationships of piperazinebenzylamines as potent and selective agonists of the human melanocortin-4 receptor

SAR studies on a series of piperazinebenzenes directed toward the human melanocortin-4 receptor resulted in potent MC4R agonists. Replacement of the triazole moiety of an initial lead 4 by a basic nitrogen baring a lipophilic side-chain increased the binding affinities of these compounds. Analogs bearing an additional hetero-atom in the side-chain possessed good agonist potency. Thus, 11h had a Ki of 11 nM, and 13g exhibited an EC50 of 3.8 nM and a Ki of 6.4 nM.     

Synthesis and characterization of pyrrolidine derivatives as potent agonists of the human melanocortin-4 receptor

A series of trans-4-phenylpyrrolidine-3-carboxamides were synthesized and characterized as potent ligands of the human melanocortin-4 receptor. Interestingly, a pair of diastereoisomers 20f-1 and 20f-2 displayed potent functional agonist and antagonist activity, respectively. Thus, the 3S,4R-compound 20f-1 possessed a K(i) of 11nM and an EC(50) of 24nM, while its 3R,4S-isomer 20f-2 exhibited a K(i) of 8.6 and an IC(50) of 65nM. Both compounds were highly selective over other melanocortin receptor subtypes. The MC4R agonist 20f-1 also demonstrated efficacy in diet-induced obese rats.     

Biased signaling in naturally occurring mutations of G protein-coupled receptors associated with diverse human diseases

G protein-coupled receptors (GPCRs) play critical roles in transmitting a variety of extracellular signals into the cells and regulate diverse physiological functions. Naturally occurring mutations that result in dysfunctions of GPCRs have been known as the causes of numerous diseases. Significant progresses have been made in elucidating the pathophysiology of diseases caused by mutations. The multiple intracellular signaling pathways, such as G protein-dependent and β-arrestin-dependent signaling, in conjunction with recent advances on biased agonism, have broadened the view on the molecular mechanism of disease pathogenesis. This review aims to briefly discuss biased agonism of GPCRs (biased ligands and biased receptors), summarize the naturally occurring GPCR mutations that cause biased signaling, and propose the potential pathophysiological relevance of biased mutant GPCRs associated with various endocrine diseases.     

Regulation of neuropeptide Y release from hypothalamic slices by melanocortin-4 agonists and leptin

We have examined the regulation of the orexigenic neurotransmitter, NPY, in hypothalamic slices of rat brain to discover whether the leptin or melanocortin receptor-4 (MCR-4) agonists, which act as satiety signals, can influence the release of this neurotransmitter. Basal and potassium-stimulated NPY release from hypothalamic slices was not significantly altered by the addition of recombinant murine leptin. However, the melanocortin-4 agonists, alpha-MSH and MT-II, significantly inhibited potassium-stimulated NPY release (p < 0.01) without significantly altering basal NPY release. However, the MCR-4 antagonist, agouti-related protein, did not significantly alter either basal or stimulated NPY release. In conclusion, hypothalamic NPY release can be attenuated by MCR-4 agonists, but not by leptin, suggesting that the activation of MCR-4 receptors leading to satiety can also further inhibit food intake through an inhibition of orexigenic NPYergic activity.     

Substituted NDP-MSH peptides paired with mutant melanocortin-4 receptors demonstrate the role of transmembrane 6 in receptor activation

The melanocortin-4 receptor (MC4R) is involved in regulating energy homeostasis and is a potential therapeutic target for obesity and cachexia. Molecular interactions between peptide ligands and MC4R have been studied in detail. Less is known regarding the role of these interactions in the mechanism of MC4R activation. The aim of this study was to investigate the molecular mechanism of human MC4R activation by [Nle4, d-Phe7]alpha-melanocyte-stimulating hormone (NDP-MSH), by first defining the role of the His6-d-Phe7-Arg8-Trp9 residues in receptor activation (Emax for stimulation of cAMP accumulation) using modified peptides, then understanding how their interaction with the receptor modulates activation using site-directed mutagenesis and a molecular model of NDP-MSH bound to the active state of the receptor. Alanine substitution indicated that the d-Phe7, Arg8, and Trp9 side chains contribute binding energy but are not essential for the receptor activation event. Conversely, His6 to Ala6 substitution reduced receptor activation but did not affect affinity. Chlorine substitutions on the d-Phe7 side chain also inhibited receptor activation. F261(6.51)A and F284(7.35)A receptor mutations acted as gain-of-function mutations, restoring efficacy to the His6 and d-Phe7 substituted peptides that had lost efficacy at the wild-type receptor. Based on a model of NDP-MSH and MC4R interaction, the antagonist behavior of these peptides is consistent with the prevention of transmembrane 6 (TM6) rotation. This data supports the hypothesis that increasing the size of d-Phe7 directly interferes with TM6 rotation, preventing receptor activation. We further propose that removing the interaction with the His6 side chain reorients the peptide within the binding pocket, indirectly impeding TM6 rotation by strengthening peptide interaction with F261(6.51) and F284(7.35). These findings refine the molecular basis for the mechanism of ligand-stimulated hMC4R activation and will be useful for the development of hMC4R agonists and antagonists.     

A cell surface inactive mutant of the human lutropin receptor (hLHR) attenuates signaling of wild-type or constitutively active receptors via heterodimerization

The D405N and Y546F mutations of the human lutropin receptor (hLHR) have previously been shown to partially attenuate hCG-stimulated cAMP synthesis despite normal cell surface expression and hCG binding affinity (Min, L. and Ascoli, M. Mol. Endocrinol. 14:1797–1810, 2000). We now show that these mutations each stabilize a resting state of the hLHR. A combined mutant D405N,Y546F is similarly expressed at the cell surface and exhibits normal ligand-binding, but is profoundly signaling impaired. Introduction of hLHR(wt) into cells stably expressing the signaling inactive D405N,Y546F resulted in the attenuation of hCG-stimulated cAMP production by hLHR(wt) even if excess Gs is co-expressed. Similarly, co-expression of D405N,Y546F with hLHR constitutively active mutants (CAMs) attenuated their constitutive activity. Quantitative bioluminescence resonance energy transfer (BRET) analyses demonstrated that D405N,Y546F formed heterodimers with both wt and CAM hLHR. In contrast hLHR(D405N,Y546F) did not heterodimerize with the melanocortin 3 receptor (MC3R) and agonist-stimulated cAMP production through the MC3R was not attenuated when these two receptors were co-expressed. Taken altogether, our data demonstrate that a signaling inactive hLHR mutant (that is trafficked normally to the plasma membrane) attenuates the signaling of the cell surface localized wt or the constitutively active hLHR due to receptor heterodimerization. Our studies, therefore, suggest a novel ramification of GPCR signaling resulting from receptor dimerization.     

Activation of extracellular-regulated kinases by normal and mutant EGF receptors

Glioblastoma cells express a mutant EGF receptor (EGFRvIII) that has constitutive tyrosine kinase activity and enhances their tumorigenicity. Here we show that EGFRvIII promotes constitutive phosphorylation of extracellular regulated kinases (ERKs) in glioblastoma cells in the absence of EGF. EGFRvIII also promoted constitutive activation of phosphoinositide 3-kinase in these cells, as assessed by phosphorylation of protein kinase B/akt. As expected, phosphorylation of protein kinase B/akt was blocked by the phosphoinositide 3-kinase inhibitors wortmannin and LY294002. Less expectedly, we found that this treatment also blocked EGFRvIII-induced phosphorylation of ERKs. In contrast, ERK phosphorylation induced by EGF-activated normal EGF receptor in the same cells was largely unaffected by treatment with phosphoinositide 3-kinase inhibitors. This difference in behavior between the normal receptor and EGFRvIII was not due to differences in the levels of activated EGFRvIII and wild-type EGF receptor, as the two types of receptor were tyrosine phosphorylated to a similar extent under the experimental conditions used. EGFRvIII activation of ERKs was also sensitive to the phospholipase C inhibitor U73122, whereas ERK activation by normal EGF receptor was not. These results show that EGFRvIII and wild-type EGF receptor preferentially use different signaling pathways to induce ERK phosphorylation. The different mechanisms of ERK activation used by normal and mutant EGF receptors may be important in understanding the potent tumorigenic activity of EGFRvIII.     

Activation of central melanocortin-4 receptor suppresses lipopolysaccharide-induced fever in rats

Activation of central melanocortin receptors (MCR) inhibits fever, but the identity of the MCR subtype(s) mediating this antipyretic effect is unknown. To determine whether selective central melanocortin receptor-4 (MC4R) activation produces antipyretic effects, the MC4R selective agonist MRLOB-0001 (CO-His-d-Phe-Arg-Trp-Dab-NH(2)) was administered intracerebroventricularly to rats treated with Escherichia coli lipopolysaccharide (LPS, 30 microg/kg ip). Treatment with MRLOB-0001 (150 ng icv) did not lower core body temperature (T(c)) in afebrile rats but did suppress LPS-induced increases in T(c) and associated decreases in tail skin temperature (T(sk)), an indicator of vasomotor thermoeffector function. In contrast, systemic treatment with MRLOB-0001 (150 ng iv) did not produce similar antipyretic effects. Coadministration of the selective MC4R antagonist HS014 (1 microg icv) blocked the antipyretic effects of MRLOB-0001. HS014 alone (1 microg icv) had no significant effect on LPS-induced increases in T(c) or decreases in T(sk) and in afebrile rats had no significant effects on T(c) or T(sk). We conclude that pharmacological activation of central MC4R suppresses febrile increases in T(c) and that inhibition of heat conservation pathways may contribute to this effect. These findings suggest that the central MC4R may mediate the long-recognized antipyretic effects of centrally administered melanocortins.