Despite the discovery of heterotrimeric αβγ G proteins ∼25 years ago, their selective perturbation by cell-permeable inhibitors remains a fundamental challenge. Here we report that the plant-derived depsipeptide FR900359 (FR) is ideally suited to this task. Using a multifaceted approach we systematically characterize FR as a selective inhibitor of Gq/11/14 over all other mammalian Gα isoforms and elaborate its molecular mechanism of action. We also use FR to investigate whether inhibition of Gq proteins is an effective post-receptor strategy to target oncogenic signalling, using melanoma as a model system. FR suppresses many of the hallmark features that are central to the malignancy of melanoma cells, thereby providing new opportunities for therapeutic intervention. Just as pertussis toxin is used extensively to probe and inhibit the signalling of Gi/o proteins, we anticipate that FR will at least be its equivalent for investigating the biological relevance of Gq.

G protein-independent, arrestin-dependent signaling is a paradigm that broadens the signaling scope of G protein-coupled receptors (GPCRs) beyond G proteins for numerous biological processes. However, arrestin signaling in the collective absence of functional G proteins has never been demonstrated. Here we achieve a state of “zero functional G” at the cellular level using HEK293 cells depleted by CRISPR/Cas9 technology of the Gs/q/12 families of Gα proteins, along with pertussis toxin-mediated inactivation of Gi/o. Together with HEK293 cells lacking β-arrestins (“zero arrestin”), we systematically dissect G protein- from arrestin-driven signaling outcomes for a broad set of GPCRs. We use biochemical, biophysical, label-free whole-cell biosensing and ERK phosphorylation to identify four salient features for all receptors at “zero functional G”: arrestin recruitment and internalization, but—unexpectedly—complete failure to activate ERK and whole-cell responses. These findings change our understanding of how GPCRs function and in particular of how they activate ERK1/2.

The glycine transporter subtype 2 (GlyT2) is localized in the axon terminals of glycinergic neurons. Mice deficient in GlyT2 are normal at birth but during the second postnatal week develop a lethal neuromotor deficiency that resembles severe forms of human hyperekplexia (hereditary startle disease) and is characterized by spasticity, tremor, and an inability to right. Histological and immunological analyses failed to reveal anatomical or biochemical abnormalities, but the amplitudes of glycinergic miniature inhibitory currents (mIPSCs) were strikingly reduced in hypoglossal motoneurons and dissociated spinal neurons from GlyT2-deficient mice. Thus, postnatal GlyT2 function is crucial for efficient transmitter loading of synaptic vesicles in glycinergic nerve terminals, and the GlyT2 gene constitutes a candidate disease gene in human hyperekplexia patients.

(4) disclose previously undetected features of GPCR behavior. Significant impact of DMR is therefore anticipated in the emerging areas of systems biology and systems pharmacology but also for the discovery of mechanistically novel drugs.3

Members of the muscarinic acetylcholine receptor family (M1-M5) are known to be involved in a great number of important central and peripheral physiological and pathophysiological processes. Because of the overlapping expression patterns of the M1-M5 muscarinic receptor subtypes and the lack of ligands endowed with sufficient subtype selectivity, the precise physiological functions of the individual receptor subtypes remain to be elucidated. To explore the physiological roles of the M2 muscarinic receptor, we have generated mice lacking functional M2 receptors by using targeted mutagenesis in mouse embryonic stem cells. The resulting mutant mice were analyzed in several behavioral and pharmacologic tests. These studies showed that the M2 muscarinic receptor subtype, besides its well documented involvement in the regulation of heart rate, plays a key role in mediating muscarinic receptor-dependent movement and temperature control as well as antinociceptive responses, three of the most prominent central muscarinic effects. These results offer a rational basis for the development of novel muscarinic drugs.Muscarinic acetylcholine receptors are known to regulate numerous fundamental physiological processes, including the muscarinic actions of acetylcholine on peripheral effector tissues and a multitude of central sensory, vegetative, and motor functions (1-4). In addition, disturbances in central muscarinic neurotransmission have been implicated in a variety of pathophysiological conditions, including Alzheimer's and Parkinson's diseases (1-4).Molecular cloning studies have revealed the existence of five molecularly distinct muscarinic receptor subtypes referred to as M1-M5 (5-7). The M1-M5 receptors are prototypical members of the superfamily of G protein-coupled receptors. Although the odd-numbered muscarinic receptor subtypes (M1, M3, and M5) are selectively linked to G q͞11 proteins, the even-numbered receptors (M2 and M4) are preferentially coupled to G proteins of the G i͞o family (5-7).The M1-M4 receptors are widely expressed throughout the central nervous system and the body periphery (6,(8)(9)(10). Studies with subtype-selective antibodies and in situ mRNA hybridization experiments have shown that most brain regions express several different muscarinic receptor subtypes (8-10). Based on this observation, it has been extremely difficult to assign specific central functions to individual muscarinic receptor subtypes.In addition, the lack of muscarinic agonists and antagonists with pronounced subtype selectivity also has represented a major limitation in studying the physiological roles of the M1-M5 receptors (5-7). This problem is accentuated further in the case of in vivo studies in which the actual concentrations of drugs at their sites of action are difficult to determine because of pharmacokinetic factors.In the body periphery, muscarinic receptors mediate the well known functions of acetylcholine at parasympathetically innervated effector organs, including contraction of smooth muscle, stimulatio...