The E92K Melanocortin 1 Receptor Mutant Induces cAMP Production and Arrestin Recruitment but Not ERK Activity Indicating Biased Constitutive Signaling

BACKGROUND: The melanocortin 1 receptor (MC1R) constitutes a key regulator of melanism. Consequently, many naturally-occurring MC1R mutations are associated with a change in color. An example is the Glu-to-Lys substitution found at position II:20/2.60 in the top of transmembrane helix II which has been identified in melanic mice and several other species. This mutation induces a pronounced increase in MC1R constitutive activity suggesting a link between constitutive activity and melanism which is corroborated by the attenuation of α-melanocyte stimulating hormone (αMSH) induced activation. However, the mechanism by which the mutation induces constitutive activity is currently not known. METHODOLOGY/PRINCIPAL FINDINGS: Here we characterize the constitutive activity, cell surface expression and internalization of the mouse mutant, Mc1r E92K. As previously reported, only positively charged residues at position II:20/2.60 induced an increase in constitutive activity as measured by cAMP accumulation and CREB activation. Furthermore, the mutation induced a constitutive recruitment of β-arrestin. This phenomenon is only observed in MC1R, however, as the equivalent mutations in MC2-5R had no effect on receptor signaling. Interestingly, the mutation did not induce constitutive ERK1/2 phosphorylation or increase the internalization rate indicating the constitutive activity to be biased. Finally, to identify regions of importance for the increased constitutive activity of Mc1r E92K, we employed a chimeric approach and identified G102 and L110 in the extracellular loop 1 to be selectively important for the constitutive activity as this, but not αMSH-mediated activation, was abolished upon Ala substitution. CONCLUSIONS/SIGNIFICANCE: It is concluded that the E92K mutation induces an active conformation distinct from that induced by αMSH and that the extracellular loop 1 is involved in maintaining this conformational state. In turn, the results suggest that in MC1R, which lacks an extracellular loop 2, the first extracellular loop may play a more prominent role during receptor activation than in general

Biased Gs versus Gq proteins and ?-arrestin signaling in the NK1 receptor determined by interactions in the water hydrogen bond network

X-ray structures, molecular dynamics simulations, and mutational analysis have previously indicated that an extended water hydrogen bond network between trans-membranes I-III, VI, and VII constitutes an allosteric interface essential for stabilizing different active and inactive helical constellations during the seven-trans-membrane receptor activation. The neurokinin-1 receptor signals efficiently through Gq, Gs, and ?-arrestin when stimulated by substance P, but it lacks any sign of constitutive activity. In the water hydrogen bond network the neurokinin-1 has a unique Glu residue instead of the highly conserved AspII:10 (2.50). Here, we find that this GluII:10 occupies the space of a putative allosteric modulating Na+ ion and makes direct inter-helical interactions in particular with SerIII:15 (3.39) and AsnVII:16 (7.49) of the NPXXY motif. Mutational changes in the interface between GluII:10 and AsnVII:16 created receptors that selectively signaled through the following: 1) Gqonly; 2) ?-arrestin only; and 3) Gqand ?-arrestinbutnot through Gs. Interestingly, increased constitutive Gs but not Gq signaling was observed by Ala substitution of four out of the six core polar residues of the network, in particular SerIII:15. Three residues were essential for all three signaling pathways, i.e. the water-gating micro-switch residues TrpVI:13 (6.48) of the CWXP motif and TyrVII:20 (7.53) of the NPXXY motif plus the totally conserved AsnI:18 (1.50) stabilizing the kink in trans-membrane VII. It is concluded that the interface between position II:10 (2.50), III:15 (3.39), and VII:16 (7.49) in the center of the water hydrogen bond network constitutes a focal point for fine-tuning seven transmembrane receptor conformations activating different signal transduction pathways

The orphan G protein-coupled receptor GPR139 is activated by the peptides:Adrenocorticotropic hormone (ACTH), α-, and β-melanocyte stimulating hormone (α-MSH, and β-MSH), and the conserved core motif HFRW

GPR139 is an orphan G protein-coupled receptor that is expressed primarily in the brain. Not much is known regarding the function of GPR139. Recently we have shown that GPR139 is activated by the amino acids l-tryptophan and l-phenylalanine (EC(50) values of 220 μM and 320 μM, respectively), as well as di-peptides comprised of aromatic amino acids. This led us to hypothesize that GPR139 may be activated by peptides. Sequence alignment of the binding cavities of all class A GPCRs, revealed that the binding pocket of the melanocortin 4 receptor is similar to that of GPR139. Based on the chemogenomics principle “similar targets bind similar ligands”, we tested three known endogenous melanocortin 4 receptor agonists; adrenocorticotropic hormone (ACTH) and α- and β-melanocyte stimulating hormone (α-MSH and β-MSH) on CHO-k1 cells stably expressing the human GPR139 in a Fluo-4 Ca(2+)-assay. All three peptides, as well as their conserved core motif HFRW, were found to activate GPR139 in the low micromolar range. Moreover, we found that peptides consisting of nine or ten N-terminal residues of α-MSH activate GPR139 in the submicromolar range. α-MSH(1-9) was found to correspond to the product of a predicted cleavage site in the pre-pro-protein pro-opiomelanocortin (POMC). Our results demonstrate that GPR139 is a peptide receptor, activated by ACTH, α-MSH, β-MSH, the conserved core motif HFRW as well as a potential endogenous peptide α-MSH(1-9). Further studies are needed to determine the functional relevance of GPR139 mediated signaling by these peptides

The aromatic amino acid sensor GPR142 controls metabolism through balanced regulation of pancreatic and gut hormones.

OBJECTIVES: GPR142, which is highly expressed in pancreatic islets, has recently been deorphanized as a receptor for aromatic amino acids; however, its physiological role and pharmacological potential is unclear. METHODS AND RESULTS: We find that GPR142 is expressed not only in β- but also in α-cells of the islets as well as in enteroendocrine cells, and we confirm that GPR142 is a highly selective sensor of essential aromatic amino acids, in particular Trp and oligopeptides with N-terminal Trp. GPR142 knock-out mice displayed a very limited metabolic phenotype but demonstrated that L-Trp induced secretion of pancreatic and gut hormones is mediated through GPR142 but that the receptor is not required for protein-induced hormone secretion. A synthetic GPR142 agonist stimulated insulin and glucagon as well as GIP, CCK, and GLP-1 secretion. In particular, GIP secretion was sensitive to oral administration of the GPR142 agonist an effect which in contrast to the other hormones was blocked by protein load. Oral administration of the GPR142 agonist increased [3H]-2-deoxyglucose uptake in muscle and fat depots mediated through insulin action while it lowered liver glycogen conceivably mediated through glucagon, and, consequently, it did not lower total blood glucose. Nevertheless, acute administration of the GPR142 agonist strongly improved oral glucose tolerance in both lean and obese mice as well as Zucker fatty rat. Six weeks in-feed chronic treatment with the GPR142 agonist did not affect body weight in DIO mice, but increased energy expenditure and carbohydrate utilization, lowered basal glucose, and improved insulin sensitivity. CONCLUSIONS: GPR142 functions as a sensor of aromatic amino acids, controlling GIP but also CCK and GLP-1 as well as insulin and glucagon in the pancreas. GPR142 agonists could have novel interesting potential in modifying metabolism through a balanced action of gut hormones as well as both insulin and glucagon

Allosteric and orthosteric sites in CC chemokine receptor (CCR5), a chimeric receptor approach

Chemokine receptors play a major role in immune system regulation and have consequently been targets for drug development leading to the discovery of several small molecule antagonists. Given the large size and predominantly extracellular receptor interaction of endogenous chemokines, small molecules often act more deeply in an allosteric mode. However, opposed to the well described molecular interaction of allosteric modulators in class C 7-transmembrane helix (7TM) receptors, the interaction in class A, to which the chemokine receptors belong, is more sparsely described. Using the CCR5 chemokine receptor as a model system, we studied the molecular interaction and conformational interchange required for proper action of various orthosteric chemokines and allosteric small molecules, including the well known CCR5 antagonists TAK-779, SCH-C, and aplaviroc, and four novel CCR5 ago-allosteric molecules. A chimera was successfully constructed between CCR5 and the closely related CCR2 by transferring all extracellular regions of CCR2 to CCR5, i.e. a Trojan horse that resembles CCR2 extracellularly but signals through a CCR5 transmembrane unit. The chimera bound CCR2 (CCL2 and CCL7), but not CCR5 chemokines (CCL3 and CCL5), with CCR2-like high affinities and potencies throughout the CCR5 signaling unit. Concomitantly, high affinity binding of small molecule CCR5 agonists and antagonists was retained in the transmembrane region. Importantly, whereas the agonistic and antagonistic properties were preserved, the allosteric enhancement of chemokine binding was disrupted. In summary, the Trojan horse chimera revealed that orthosteric and allosteric sites could be structurally separated and still act together with transmission of agonism and antagonism across the different receptor units

Modulation of constitutive activity and signaling bias of the ghrelin receptor by conformational constraint in the second extracellular loop

Based on a rare, natural Glu for Ala-204(C+6) variant located six residues after the conserved Cys residue in extracellular loop 2b (ECL2b) associated with selective elimination of the high constitutive signaling of the ghrelin receptor, this loop was subjected to a detailed structure functional analysis. Introduction of Glu in different positions demonstrated that although the constitutive signaling was partly reduced when introduced in position 205(C+7) it was only totally eliminated in position 204(C+6). No charge-charge interaction partner could be identified for the Glu(C+6) variant despite mutational analysis of a number of potential partners in the extracellular loops and outer parts of the transmembrane segments. Systematic probing of position 204(C+6) with amino acid residues of different physicochemical properties indicated that a positively charged Lys surprisingly provided phenotypes similar to those of the negatively charged Glu residue. Computational chemistry analysis indicated that the propensity for the C-terminal segment of extracellular loop 2b to form an extended α-helix was increased from 15% in the wild type to 89 and 82% by introduction in position 204(C+6) of a Glu or a Lys residue, respectively. Moreover, the constitutive activity of the receptor was inhibited by Zn(2+) binding in an engineered metal ion site, stabilizing an α-helical conformation of this loop segment. It is concluded that the high constitutive activity of the ghrelin receptor is dependent upon flexibility in the C-terminal segment of extracellular loop 2 and that mutations or ligand binding that constrains this segment and thereby conceivably the movements of transmembrane domain V relative to transmembrane domain III inhibits the high constitutive signaling

Overlapping binding site for the endogenous agonist, small-molecule agonists, and ago-allosteric modulators on the ghrelin receptor

A library of robust ghrelin receptor mutants with single substitu-tions at 22 positions in the main ligand-binding pocket was em-ployed tomap binding sites for six different agonists: two peptides (the 28-amino-acid octanoylated endogenous ligand ghrelin and the hexapeptide growth hormone secretagogue GHRP-6) plus four nonpeptide agonists—the original benzolactam L-692,429 [3-amino-3-methyl-N-(2,3,4,5-tetrahydro-2-oxo-1-([2-(1H-tetrazol

Overlapping Binding Site for the Endogenous Agonist, Small-Molecule Agonists, and Ago-allosteric Modulators on the Ghrelin Receptor

ABSTRACT A library of robust ghrelin receptor mutants with single substitutions at 22 positions in the main ligand-binding pocket was employed to map binding sites for six different agonists: two peptides (the 28-amino-acid octanoylated endogenous ligand ghrelin and the hexapeptide growth hormone secretagogue GHRP-6) plus four nonpeptide agonists-the original benzolactam L-692,429 [3-amino-3-methyl-N-(2,3,4,5- The strongest mutational effect with respect to decrease in potency for stimulation of inositol phosphate turnover was for all six agonists the GluIII:09-to-Gln substitution in the extracellular segment of TM-III. Likewise, all six agonists were affected by substitutions of PheVI:16, ArgVI:20, and PheVI:23 on the opposing face of transmembrane domain (TM) VI. Each of the agonists was also affected selectively by specific mutations. The mutational map of the ability of L-692,429 and GHRP-6 to act as allosteric modulators by increasing ghrelin's maximal efficacy overlapped with the common mutational map for agonism but it was not identical with the map for the agonist property of these small-molecule ligands. In molecular models, built over the inactive conformation of rhodopsin, low energy conformations of the nonpeptide agonists could be docked to satisfy many of their mutational hits. It is concluded that although each of the ligands in addition exploits other parts of the receptor, a large, common binding site for both small-molecule agonists-including ago-allosteric modulators-and the endogenous agonist is found on the opposing faces of TM-III and -VI of the ghrelin receptor. The gastrointestinal peptide hormone ghrelin is an important regulator of appetite, energy expenditure, and acute growth hormone secretion through interaction with the ghrelin receptors located mainly in the central nervous system The initial agonists synthesized for the ghrelin receptor were peptides, sharing common structural features, including a central hydrophobic motif and a positive charge in the amidated C-terminal end. Despite relatively poor bioavailability and low therapeutic index/window, these peptides efficiently induced GH secretion in vitro as well as in vivo both in animal and human model