G-protein-independent coupling of MC4R to Kir7.1 in hypothalamic neurons.

The regulated release of anorexigenic α-melanocyte stimulating hormone (α-MSH) and orexigenic Agouti-related protein (AgRP) from discrete hypothalamic arcuate neurons onto common target sites in the central nervous system has a fundamental role in the regulation of energy homeostasis. Both peptides bind with high affinity to the melanocortin-4 receptor (MC4R); existing data show that α-MSH is an agonist that couples the receptor to the Gαs signalling pathway, while AgRP binds competitively to block α-MSH binding and blocks the constitutive activity mediated by the ligand-mimetic amino-terminal domain of the receptor. Here we show that, in mice, regulation of firing activity of neurons from the paraventricular nucleus of the hypothalamus (PVN) by α-MSH and AgRP can be mediated independently of Gαs signalling by ligand-induced coupling of MC4R to closure of inwardly rectifying potassium channel, Kir7.1. Furthermore, AgRP is a biased agonist that hyperpolarizes neurons by binding to MC4R and opening Kir7.1, independently of its inhibition of α-MSH binding. Consequently, Kir7.1 signalling appears to be central to melanocortin-mediated regulation of energy homeostasis within the PVN. Coupling of MC4R to Kir7.1 may explain unusual aspects of the control of energy homeostasis by melanocortin signalling, including the gene dosage effect of MC4R and the sustained effects of AgRP on food intake.

The melanocortin-4 receptor is expressed in enteroendocrine L cells and regulates the release of peptide YY and glucagon-like peptide 1 in vivo.

The melanocortin-4 receptor (MC4R) is expressed in the brainstem and vagal afferent nerves and regulates a number of aspects of gastrointestinal function. Here we show that the receptor is also diffusely expressed in cells of the gastrointestinal system, from stomach to descending colon. Furthermore, MC4R is the second most highly enriched GPCR in peptide YY (PYY) and glucagon-like peptide 1 (GLP-1) expressing enteroendocrine L cells. When vectorial ion transport is measured across mouse or human intestinal mucosa, administration of α-MSH induces a MC4R-specific PYY-dependent antisecretory response consistent with a role for the MC4R in paracrine inhibition of electrolyte secretion. Finally, MC4R-dependent acute PYY and GLP-1 release from L cells can be stimulated in vivo by intraperitoneal (i.p.) administration of melanocortin peptides to mice. This suggests physiological significance for MC4R in L cells and indicates a previously unrecognized peripheral role for the MC4R, complementing vagal and central receptor functions.

Further exploration of M1 allosteric agonists: subtle structural changes abolish M1 allosteric agonism and result in pan-mAChR orthosteric antagonism.

This letter describes the further exploration of two series of M(1) allosteric agonists, TBPB and VU0357017, previously reported from our lab. Within the TPBP scaffold, either electronic or steric perturbations to the central piperidine ring led to a loss of selective M(1) allosteric agonism and afforded pan-mAChR antagonism, which was demonstrated to be mediated via the orthosteric site. Additional SAR around a related M(1) allosteric agonist family (VU0357017) identified similar, subtle 'molecular switches' that modulated modes of pharmacology from allosteric agonism to pan-mAChR orthosteric antagonism. Therefore, all of these ligands are best classified as bi-topic ligands that possess high affinity binding at an allosteric site to engender selective M(1) activation, but all bind, at higher concentrations, to the orthosteric ACh site, leading to non-selective orthosteric site binding and mAChR antagonism.

Chemical modification of the M(1) agonist VU0364572 reveals molecular switches in pharmacology and a bitopic binding mode.

We previously reported the discovery of VU0364572 and VU0357017 as M(1)-selective agonists that appear to activate M(1) through actions at an allosteric site. Previous studies have revealed that chemical scaffolds for many allosteric modulators contain molecular switches that allow discovery of allosteric antagonists and allosteric agonists or positive allosteric modulators (PAMs) based on a single chemical scaffold. Based on this, we initiated a series of studies to develop selective M(1) allosteric antagonists based on the VU0364572 scaffold. Interestingly, two lead antagonists identified in this series, VU0409774 and VU0409775, inhibited ACh-induced Ca(2+) responses at rat M(1-5) receptor subtypes, suggesting they are nonselective muscarinic antagonists. VU0409774 and VU0409775 also completely displaced binding of the nonselective radioligand [(3)H]-NMS at M(1) and M(3) mAChRs with affinities similar to their functional IC(50) values. Finally, Schild analysis revealed that these compounds inhibit M(1) responses through a fully competitive interaction at the orthosteric binding site. This surprising finding prompted further studies to determine whether agonist activity of VU0364572 and VU0357017 may also engage in previously unappreciated actions at the orthosteric site on M(1). Surprisingly, both VU0364572 and VU0357017 completely displaced [(3)H]-NMS binding to the orthosteric site of M(1)-M(5) receptors at high concentrations. Furthermore, evaluation of agonist activity in systems with varying levels of receptor reserve and Furchgott analysis using a cell line expressing M(1) under control of an inducible promotor was consistent with an action of these compounds as weak orthosteric partial agonists of M(1). However, consistent with previous studies suggesting actions at a site that is distinct from the orthosteric binding site, VU0364572 or VU0357017 slowed the rate of [(3)H]-NMS dissociation from CHO-rM(1) membranes. Together, these results suggest that VU0364572 and VU0357017 act as bitopic ligands and that novel antagonists in this series act as competitive orthosteric site antagonists.

Novel allosteric agonists of M1 muscarinic acetylcholine receptors induce brain region-specific responses that correspond with behavioral effects in animal models.

M(1) muscarinic acetylcholine receptors (mAChRs) represent a viable target for treatment of multiple disorders of the central nervous system (CNS) including Alzheimer's disease and schizophrenia. The recent discovery of highly selective allosteric agonists of M(1) receptors has provided a major breakthrough in developing a viable approach for the discovery of novel therapeutic agents that target these receptors. Here we describe the characterization of two novel M(1) allosteric agonists, VU0357017 and VU0364572, that display profound differences in their efficacy in activating M(1) coupling to different signaling pathways including Ca(2+) and ß-arrestin responses. Interestingly, the ability of these agents to differentially activate coupling of M(1) to specific signaling pathways leads to selective actions on some but not all M(1)-mediated responses in brain circuits. These novel M(1) allosteric agonists induced robust electrophysiological effects in rat hippocampal slices, but showed lower efficacy in striatum and no measureable effects on M(1)-mediated responses in medial prefrontal cortical pyramidal cells in mice. Consistent with these actions, both M(1) agonists enhanced acquisition of hippocampal-dependent cognitive function but did not reverse amphetamine-induced hyperlocomotion in rats. Together, these data reveal that M(1) allosteric agonists can differentially regulate coupling of M(1) to different signaling pathways, and this can dramatically alter the actions of these compounds on specific brain circuits important for learning and memory and psychosis.

Development of a highly selective, orally bioavailable and CNS penetrant M1 agonist derived from the MLPCN probe ML071.

Herein we report the discovery and SAR of a novel series of M(1) agonists based on the MLPCN probe, ML071. From this, VU0364572 emerged as a potent, orally bioavailable and CNS penetrant M(1) agonist with high selectivity, clean ancillary pharmacology and enantiospecific activity.

Orthosteric- and allosteric-induced ligand-directed trafficking at GPCRs.

Many orthosteric agonists differentially activate downstream effectors of GPCRs. Such defined induction of signaling has strongly supported the hypothesis termed 'ligand-directed trafficking of receptor signaling' (LDTRS). More recently, subtype-selective GPCR activators, such as allosteric agonists and positive allosteric modulators, have also exhibited the capacity to activate specific signaling pathways. Based on this finding, it may be possible to achieve ligand-specific receptor active states that optimize the biological responses specific to GPCRs. This review discusses recent studies in which both orthosteric and allosteric compounds have been demonstrated to induce LDTRS.

Allosteric activators of muscarinic receptors as novel approaches for treatment of CNS disorders.

Muscarinic acetylcholine receptors (mAChRs) represent exciting therapeutic targets for the treatment of multiple CNS disorders. The high degree of conservation of amino acids comprising the orthosteric acetylcholine (ACh) binding site between individual mAChR subtypes has hindered the development of subtype-selective compounds that bind to this site. As a result, many academic and industry researchers are now focusing on developing allosteric activators of mAChRs including both positive allosteric modulators (PAMs) and allosteric agonists. In the past 10 years major advances have been achieved in the discovery of allosteric ligands that possess much greater selectivity for individual mAChR subtypes when compared to previously developed orthosteric agents. These novel allosteric modulators of mAChRs may provide therapeutic potential for treatment of a number of CNS disorders such as Alzheimer's disease and schizophrenia.

The c-terminus of GRK3 indicates rapid dissociation of G protein heterotrimers.

Signals mediated by heterotrimeric G proteins often develop over the course of tens of milliseconds, and could require either conformational rearrangement or complete physical dissociation of Galphabetagamma heterotrimers. Although it is known that some active heterotrimers are dissociated (into Galpha and Gbetagamma) at steady-state, it is not clear that dissociation occurs quickly enough to participate in rapid signaling. Here we show that fusion proteins containing the c-terminus of GPCR kinase 3 (GRK3ct) and either the fluorescent protein cerulean or Renilla luciferase bind to venus-labeled Gbetagamma dimers (Gbetagamma-V), resulting in Förster or bioluminescence resonance energy transfer (FRET or BRET). GRK3ct fusion proteins are freely-diffusible, and do not form preassembled complexes with G proteins. GRK3ct fusion proteins bind to free Gbetagamma-V dimers but not to rearranged heterotrimers, and thus can report G protein dissociation with high temporal resolution. We find that heterotrimer dissociation can occur in living cells in less than 100 ms. Under the conditions of these experiments diffusion and collision of masGRK3ct fusion proteins and Gbetagamma-V were not rate-limiting. These results indicate that G protein heterotrimers can dissociate quickly enough to participate in rapid signaling.

GPR109A is a G-protein-coupled receptor for the bacterial fermentation product butyrate and functions as a tumor suppressor in colon.

Short-chain fatty acids, generated in colon by bacterial fermentation of dietary fiber, protect against colorectal cancer and inflammatory bowel disease. Among these bacterial metabolites, butyrate is biologically most relevant. GPR109A is a G-protein-coupled receptor for nicotinate but recognizes butyrate with low affinity. Millimolar concentrations of butyrate are needed to activate the receptor. Although concentrations of butyrate in colonic lumen are sufficient to activate the receptor maximally, there have been no reports on the expression/function of GPR109A in this tissue. Here we show that GPR109A is expressed in the lumen-facing apical membrane of colonic and intestinal epithelial cells and that the receptor recognizes butyrate as a ligand. The expression of GPR109A is silenced in colon cancer in humans, in a mouse model of intestinal/colon cancer, and in colon cancer cell lines. The tumor-associated silencing of GPR109A involves DNA methylation directly or indirectly. Reexpression of GPR109A in colon cancer cells induces apoptosis, but only in the presence of its ligands butyrate and nicotinate. Butyrate is an inhibitor of histone deacetylases, but apoptosis induced by activation of GPR109A with its ligands in colon cancer cells does not involve inhibition of histone deacetylation. The primary changes in this apoptotic process include down-regulation of Bcl-2, Bcl-xL, and cyclin D1 and up-regulation of death receptor pathway. In addition, GPR109A/butyrate suppresses nuclear factor-kappaB activation in normal and cancer colon cell lines as well as in normal mouse colon. These studies show that GPR109A mediates the tumor-suppressive effects of the bacterial fermentation product butyrate in colon.

A point mutation to Galphai selectively blocks GoLoco motif binding: direct evidence for Galpha.GoLoco complexes in mitotic spindle dynamics.

Heterotrimeric G-protein Galpha subunits and GoLoco motif proteins are key members of a conserved set of regulatory proteins that influence invertebrate asymmetric cell division and vertebrate neuroepithelium and epithelial progenitor differentiation. GoLoco motif proteins bind selectively to the inhibitory subclass (Galphai) of Galpha subunits, and thus it is assumed that a Galphai.GoLoco motif protein complex plays a direct functional role in microtubule dynamics underlying spindle orientation and metaphase chromosomal segregation during cell division. To address this hypothesis directly, we rationally identified a point mutation to Galphai subunits that renders a selective loss-of-function for GoLoco motif binding, namely an asparagine-to-isoleucine substitution in the alphaD-alphaE loop of the Galpha helical domain. This GoLoco-insensitivity ("GLi") mutation prevented Galphai1 association with all human GoLoco motif proteins and abrogated interaction between the Caenorhabditis elegans Galpha subunit GOA-1 and the GPR-1 GoLoco motif. In contrast, the GLi mutation did not perturb any other biochemical or signaling properties of Galphai subunits, including nucleotide binding, intrinsic and RGS protein-accelerated GTP hydrolysis, and interactions with Gbetagamma dimers, adenylyl cyclase, and seven transmembrane-domain receptors. GoLoco insensitivity rendered Galphai subunits unable to recruit GoLoco motif proteins such as GPSM2/LGN and GPSM3 to the plasma membrane, and abrogated the exaggerated mitotic spindle rocking normally seen upon ectopic expression of wild type Galphai subunits in kidney epithelial cells. This GLi mutation should prove valuable in establishing the physiological roles of Galphai.GoLoco motif protein complexes in microtubule dynamics and spindle function during cell division as well as to delineate potential roles for GoLoco motifs in receptor-mediated signal transduction.

Differential dissociation of G protein heterotrimers.

Signalling by heterotrimeric G proteins is often isoform-specific, meaning certain effectors are regulated exclusively by one family of heterotrimers. For example, in excitable cells inwardly rectifying potassium (GIRK) channels are activated by G betagamma dimers derived specifically from G(i/o) heterotrimers. Since all active heterotrimers are thought to dissociate and release free G betagamma dimers, it is unclear why these channels respond primarily to dimers released by G(i/o) heterotrimers. We reconstituted GIRK channel activation in cells where we could quantify heterotrimer expression at the plasma membrane, GIRK channel activation, and heterotrimer dissociation. We find that G(oA) heterotrimers are more effective activators of GIRK channels than G(s) heterotrimers when comparable amounts of each are available. We also find that active G(oA) heterotrimers dissociate more readily than active G(s) heterotrimers. Differential dissociation may thus provide a simple explanation for G alpha-specific activation of GIRK channels and other G betagamma-sensitive effectors.

Some G protein heterotrimers physically dissociate in living cells.

Heterotrimeric G proteins mediate physiological processes ranging from phototransduction to cell migration. In the accepted model of G protein signaling, Galphabetagamma heterotrimers physically dissociate after activation, liberating free Galpha subunits and Gbetagamma dimers. This model is supported by evidence obtained in vitro with purified proteins, but its relevance in vivo has been questioned. Here, we show that at least some heterotrimeric G protein isoforms physically dissociate after activation in living cells. Galpha subunits extended with a transmembrane (TM) domain and cyan fluorescent protein (CFP) were immobilized in the plasma membrane by biotinylation and cross-linking with avidin. Immobile CFP-TM-Galpha greatly decreased the lateral mobility of intracellular Gbeta1gamma2-YFP, indicating the formation of stable heterotrimers. A GTPase-deficient (constitutively active) mutant of CFP-TM-GalphaoA lost the ability to restrict Gbeta1gamma2-YFP mobility, whereas GTPase-deficient mutants of CFP-TM-Galphai3 and CFP-TM-Galphas retained this ability. Activation of cognate G protein-coupled receptors partially relieved the constraint on Gbeta1gamma2-YFP mobility induced by immobile CFP-TM-GalphaoA and CFP-TM-Galphai3 but had no effect on the constraint induced by CFP-TM-Galphas. These results demonstrate the physical dissociation of heterotrimers containing GalphaoA and Galphai3 subunits in living cells, supporting the subunit dissociation model of G protein signaling for these subunits. However, these results are also consistent with the suggestion that G protein heterotrimers (e.g., Galphas) may signal without physically dissociating.