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.

Endocytosis and intracellular trafficking of receptors are pivotal to maintain physiological functions and drug action; however, robust quantitative approaches are lacking to study such processes in live cells. Here we present new bioluminescence resonance energy transfer (BRET) sensors to quantitatively monitor G protein-coupled receptors (GPCRs) and β-arrestin trafficking. These sensors are based on bystander BRET and use the naturally interacting chromophores luciferase (RLuc) and green fluorescent protein (rGFP) from Renilla. The versatility and robustness of this approach are exemplified by anchoring rGFP at the plasma membrane or in endosomes to generate high dynamic spectrometric BRET signals on ligand-promoted recruitment or sequestration of RLuc-tagged proteins to, or from, specific cell compartments, as well as sensitive subcellular BRET imaging for protein translocation visualization. These sensors are scalable to high-throughput formats and allow quantitative pharmacological studies of GPCR trafficking in real time, in live cells, revealing ligand-dependent biased trafficking of receptor/β-arrestin complexes.

G protein–coupled receptors (GPCRs) are important therapeutic targets that exhibit functional selectivity (biased signaling), in which different ligands or receptor variants elicit distinct downstream signaling. Understanding all the signaling events and biases that contribute to both the beneficial and adverse effects of GPCR stimulation by given ligands is important for drug discovery. Here, we report the design, validation, and use of pathway-selective bioluminescence resonance energy transfer (BRET) biosensors that monitor the engagement and activation of signaling effectors downstream of G proteins, including protein kinase C (PKC), phospholipase C (PLC), p63RhoGEF, and Rho. Combined with G protein and β-arrestin BRET biosensors, our sensors enabled real-time monitoring of GPCR signaling at different levels in downstream pathways in both native and engineered cells. Profiling of the responses to 14 angiotensin II (AngII) type 1 receptor (AT1R) ligands enabled the clustering of compounds into different subfamilies of biased ligands and showed that, in addition to the previously reported functional selectivity between Gαq and β-arrestin, there are also biases among G protein subtypes. We also demonstrated that biases observed at the receptor and G protein levels propagated to downstream signaling pathways and that these biases could occur through the engagement of different G proteins to activate a common effector. We also used these tools to determine how naturally occurring AT1R variants affected signaling bias. This suite of BRET biosensors provides a useful resource for fingerprinting biased ligands and mutant receptors and for dissecting functional selectivity at various levels of GPCR signaling.

Previously, D 2 dopamine receptors (D 2 DARs) have been shown to undergo G-protein-coupled receptor kinase phosphorylation in an agonist-specific fashion. We have now investigated the ability of the second messenger-activated protein kinases, protein kinase A (PKA) and protein kinase C (PKC), to mediate phosphorylation and desensitization of the D 2 DAR. HEK293T cells were transiently transfected with the D 2 DAR and then treated with intracellular activators and inhibitors of PKA or PKC. Treatment with agents that increase cAMP, and activate PKA, had no effect on the phosphorylation state of the D 2 DAR, suggesting that PKA does not phosphorylate the D 2 DAR in HEK293T cells. In contrast, cellular treatment with phorbol 12-myristate 13-acetate (PMA), a PKC activator, resulted in an ϳ3-fold increase in D 2 DAR phosphorylation. The phosphorylation was specific for PKC as the PMA effect was mimicked by phorbol 12,13-dibutyrate, but not by 4␣-phorbol 12,13-didecanoate, active and inactive, phorbol diesters, respectively. The PMA-mediated D 2 DAR phosphorylation was completely blocked by co-treatment with the PKC inhibitor, bisindolylmaleimide II, and augmented by co-transfection with PKC␤I. In contrast, PKC inhibition had no effect on agonist-promoted phosphorylation, suggesting that PKC is not involved in this response. PKC phosphorylation of the D 2 DAR was found to promote receptor desensitization as reflected by a decrease in agonist potency for inhibiting cAMP accumulation. Most interestingly, PKC phosphorylation also promoted internalization of the D 2 DAR through a ␤-arrestin-and dynamin-dependent pathway, a response not usually associated with PKC phosphorylation of G-protein-coupled receptors. Site-directed mutagenesis experiments resulted in the identification of two domains of PKC phosphorylation sites within the third intracellular loop of the receptor. Both of these domains are involved in regulating sequestration of the D 2 DAR, whereas only one domain is involved in receptor desensitization. These results indicate that PKC can mediate phosphorylation of the D 2 DAR, resulting in both functional desensitization and receptor internalization.

Abstract-We directly examined the role of the Ca v 1.3 (␣ 1D ) Ca 2ϩ channel in the sinoatrial (SA) node by using Ca v 1.3 Ca 2ϩ channel-deficient mice. A previous report has shown that the null mutant (Ca v 1.3 Ϫ/Ϫ ) mice have sinus bradycardia with a prolonged PR interval. In the present study, we show that spontaneous action potentials recorded from the SA nodes show a significant decrease in the beating frequency and rate of diastolic depolarization in Ca v 1.3

Multiple Ca2+ channels confer diverse functions to hair cells of the auditory and vestibular organs in the mammalian inner ear. We used gene-targeting technology to generate a 1D Ca 2+ channel-deficient mice to determine the physiological role of these Ca 2+ channels in hearing and balance. Analyses of auditory-evoked brainstem recordings confirmed that a 1D )/) mice were deaf and revealed that heterozygous (a 1D +/) ) mice have increased hearing thresholds. However, hearing deficits in a 1D +/) mice were manifested mainly by the increase in threshold of low-frequency sounds. In contrast to impaired hearing, a 1D )/) mice have balance performances equivalent to their wild-type littermates. Light and electron microscope analyses of the inner ear revealed outer hair cell loss at the apical cochlea, but no apparent abnormality at the basal cochlea and the vestibule. We determined the mechanisms underlying the auditory function defects and the normal vestibular functions by examining the Ba 2+ currents in cochlear inner and outer hair cells versus utricular hair cells in a 1D +/) mice. Whereas the whole-cell Ba 2+ currents in inner hair cells consist mainly of the nimodipine-sensitive current (85%), the utricular hair cells express only 50% of this channel subtype. Thus, differential expression of a 1D channels in the cochlear and utricular hair cells confers the phenotype of the a 1D null mutant mice. Because vestibular and cochlear hair cells share common features and null deletion of several genes have yielded both deafness and imbalance in mice, a 1D null mutant mice may serve as a model to disentangle vestibular from auditory-specific functions.

In addition to G protein-coupled receptor (GPCR) desensitization and endocytosis, β-arrestin recruitment to ligand-stimulated GPCRs promotes non-canonical signalling cascades. Distinguishing the respective contributions of β-arrestin recruitment to the receptor and β-arrestin-promoted endocytosis in propagating receptor signalling has been limited by the lack of selective analytical tools. Here, using a combination of virtual screening and cell-based assays, we have identified a small molecule that selectively inhibits the interaction between β-arrestin and the β2-adaptin subunit of the clathrin adaptor protein AP2 without interfering with the formation of receptor/β-arrestin complexes. This selective β-arrestin/β2-adaptin inhibitor (Barbadin) blocks agonist-promoted endocytosis of the prototypical β2-adrenergic (β2AR), V2-vasopressin (V2R) and angiotensin-II type-1 (AT1R) receptors, but does not affect β-arrestin-independent (transferrin) or AP2-independent (endothelin-A) receptor internalization. Interestingly, Barbadin fully blocks V2R-stimulated ERK1/2 activation and blunts cAMP accumulation promoted by both V2R and β2AR, supporting the concept of β-arrestin/AP2-dependent signalling for both G protein-dependent and -independent pathways.

We investigated the role of G protein-coupled receptor kinase (GRK)-mediated phosphorylation in agonist-induced desensitization, arrestin association, endocytosis, and intracellular trafficking of the D 2 dopamine receptor (DAR). Agonist activation of D 2 DARs results in rapid and sustained receptor phosphorylation that is solely mediated by GRKs. A survey of GRKs revealed that only GRK2 or GRK3 promotes D 2 DAR phosphorylation. Mutational analyses resulted in the identification of eight serine/threonine residues within the third cytoplasmic loop of the receptor that are phosphorylated by GRK2/3. Simultaneous mutation of these eight residues results in a receptor construct, GRK(؊), that is completely devoid of agonist-promoted GRK-mediated receptor phosphorylation. We found that both wild-type (WT) and GRK(؊) receptors underwent a similar degree of agonist-induced desensitization as assessed using [ 35 S]GTP␥S binding assays. Similarly, both receptor constructs internalized to the same extent in response to agonist treatment. Furthermore, using bioluminescence resonance energy transfer assays to directly assess receptor association with arrestin3, we found no differences between the WT and GRK(؊) receptors. Thus, phosphorylation is not required for arrestin-receptor association or agonist-induced desensitization or internalization. In contrast, when we examined recycling of the D 2 DARs to the cell surface, subsequent to agonist-induced endocytosis, the GRK(؊) construct exhibited less recycling in comparison with the WT receptor. This impairment appears to be due to a greater propensity of the GRK(؊) receptors to down-regulate once internalized. In contrast, if the receptor is highly phosphorylated, then receptor recycling is promoted. These results reveal a novel role for GRK-mediated phosphorylation in regulating the post-endocytic trafficking of a G protein-coupled receptor. Dopamine receptors (DARs)3 are members of the GPCR superfamily and consist of five structurally distinct subtypes (1, 2). These can be divided into two subfamilies on the basis of their structure and pharmacological and transductional properties (3). The "D 1 -like" subfamily includes the D 1 and D 5 receptors, which couple to the heterotrimeric G proteins G S or G OLF to stimulate adenylyl cyclase activity and raise intracellular levels of cAMP. The D 2 -like subfamily includes the D 2 , D 3 , and D 4 receptors, which couple to inhibitory G i/o proteins to reduce adenylyl cyclase activity as well as modulate voltagegated K ϩ or Ca 2ϩ channels. Within the central nervous system, these receptors modulate movement, learning and memory, reward and addiction, cognition, and certain neurendocrine functions. As with other GPCRs, the DARs are subject to a wide variety of regulatory mechanisms, which can either positively or negatively modulate their expression and functional activity (4).Upon agonist activation, most GPCRs undergo desensitization, a homeostatic process that results in a waning of receptor response despite continued agonist stimu...