GPCR-G Protein-ß-Arrestin Super-Complex Mediates Sustained G Protein Signaling.

Classically, G protein-coupled receptor (GPCR) stimulation promotes G protein signaling at the plasma membrane, followed by rapid ß-arrestin-mediated desensitization and receptor internalization into endosomes. However, it has been demonstrated that some GPCRs activate G proteins from within internalized cellular compartments, resulting in sustained signaling. We have used a variety of biochemical, biophysical, and cell-based methods to demonstrate the existence, functionality, and architecture of internalized receptor complexes composed of a single GPCR, ß-arrestin, and G protein. These super-complexes or "megaplexes" more readily form at receptors that interact strongly with ß-arrestins via a C-terminal tail containing clusters of serine/threonine phosphorylation sites. Single-particle electron microscopy analysis of negative-stained purified megaplexes reveals that a single receptor simultaneously binds through its core region with G protein and through its phosphorylated C-terminal tail with ß-arrestin. The formation of such megaplexes provides a potential physical basis for the newly appreciated sustained G protein signaling from internalized GPCRs.

Mechanism of intracellular allosteric ß2AR antagonist revealed by X-ray crystal structure.

G-protein-coupled receptors (GPCRs) pose challenges for drug discovery efforts because of the high degree of structural homology in the orthosteric pocket, particularly for GPCRs within a single subfamily, such as the nine adrenergic receptors. Allosteric ligands may bind to less-conserved regions of these receptors and therefore are more likely to be selective. Unlike orthosteric ligands, which tonically activate or inhibit signalling, allosteric ligands modulate physiologic responses to hormones and neurotransmitters, and may therefore have fewer adverse effects. The majority of GPCR crystal structures published to date were obtained with receptors bound to orthosteric antagonists, and only a few structures bound to allosteric ligands have been reported. Compound 15 (Cmpd-15) is an allosteric modulator of the ß2 adrenergic receptor (ß2AR) that was recently isolated from a DNA-encoded small-molecule library. Orthosteric ß-adrenergic receptor antagonists, known as beta-blockers, are amongst the most prescribed drugs in the world and Cmpd-15 is the first allosteric beta-blocker. Cmpd-15 exhibits negative cooperativity with agonists and positive cooperativity with inverse agonists. Here we present the structure of the ß2AR bound to a polyethylene glycol-carboxylic acid derivative (Cmpd-15PA) of this modulator. Cmpd-15PA binds to a pocket formed primarily by the cytoplasmic ends of transmembrane segments 1, 2, 6 and 7 as well as intracellular loop 1 and helix 8. A comparison of this structure with inactive- and active-state structures of the ß2AR reveals the mechanism by which Cmpd-15 modulates agonist binding affinity and signalling.

Allosteric nanobodies reveal the dynamic range and diverse mechanisms of G-protein-coupled receptor activation.

G-protein-coupled receptors (GPCRs) modulate many physiological processes by transducing a variety of extracellular cues into intracellular responses. Ligand binding to an extracellular orthosteric pocket propagates conformational change to the receptor cytosolic region to promote binding and activation of downstream signalling effectors such as G proteins and ß-arrestins. It is well known that different agonists can share the same binding pocket but evoke unique receptor conformations leading to a wide range of downstream responses ('efficacy'). Furthermore, increasing biophysical evidence, primarily using the ß2-adrenergic receptor (ß2AR) as a model system, supports the existence of multiple active and inactive conformational states. However, how agonists with varying efficacy modulate these receptor states to initiate cellular responses is not well understood. Here we report stabilization of two distinct ß2AR conformations using single domain camelid antibodies (nanobodies)­a previously described positive allosteric nanobody (Nb80) and a newly identified negative allosteric nanobody (Nb60). We show that Nb60 stabilizes a previously unappreciated low-affinity receptor state which corresponds to one of two inactive receptor conformations as delineated by X-ray crystallography and NMR spectroscopy. We find that the agonist isoprenaline has a 15,000-fold higher affinity for ß2AR in the presence of Nb80 compared to the affinity of isoprenaline for ß2AR in the presence of Nb60, highlighting the full allosteric range of a GPCR. Assessing the binding of 17 ligands of varying efficacy to the ß2AR in the absence and presence of Nb60 or Nb80 reveals large ligand-specific effects that can only be explained using an allosteric model which assumes equilibrium amongst at least three receptor states. Agonists generally exert efficacy by stabilizing the active Nb80-stabilized receptor state (R80). In contrast, for a number of partial agonists, both stabilization of R80 and destabilization of the inactive, Nb60-bound state (R60) contribute to their ability to modulate receptor activation. These data demonstrate that ligands can initiate a wide range of cellular responses by differentially stabilizing multiple receptor states.

ß-Arrestin-Biased Allosteric Modulator Potentiates Carvedilol-Stimulated ß Adrenergic Receptor Cardioprotection.

ß 1 adrenergic receptors (ß 1ARs) are central regulators of cardiac function and a drug target for cardiac disease. As a member of the G protein-coupled receptor family, ß 1ARs activate cellular signaling by primarily coupling to Gs proteins to activate adenylyl cyclase, cAMP-dependent pathways, and the multifunctional adaptor-transducer protein ß-arrestin. Carvedilol, a traditional ß-blocker widely used in treating high blood pressure and heart failure by blocking ß adrenergic receptor-mediated G protein activation, can selectively stimulate Gs-independent ß-arrestin signaling of ß adrenergic receptors, a process known as ß-arrestin-biased agonism. Recently, a DNA-encoded small-molecule library screen against agonist-occupied ß 2 adrenergic receptors (ß 2ARs) identified Compound-6 (Cmpd-6) to be a positive allosteric modulator for agonists on ß 2ARs. Intriguingly, it was further discovered that Cmpd-6 is positively cooperative with the ß-arrestin-biased ligand carvedilol at ß 2ARs. Here we describe the surprising finding that at ß 1ARs unlike ß 2ARs, Cmpd-6 is cooperative only with carvedilol and not agonists. Cmpd-6 increases the binding affinity of carvedilol for ß 1ARs and potentiates carvedilol-stimulated, ß-arrestin-dependent ß 1AR signaling, such as epidermal growth factor receptor transactivation and extracellular signal-regulated kinase activation, whereas it does not have an effect on Gs-mediated cAMP generation. In vivo, Cmpd-6 enhances the antiapoptotic, cardioprotective effect of carvedilol in response to myocardial ischemia/reperfusion injury. This antiapoptotic role of carvedilol is dependent on ß-arrestins since it is lost in mice with myocyte-specific deletion of ß-arrestins. Our findings demonstrate that Cmpd-6 is a selective ß-arrestin-biased allosteric modulator of ß 1ARs and highlight its potential clinical utility in enhancing carvedilol-mediated cardioprotection against ischemic injury. SIGNIFICANCE STATEMENT: This study demonstrates the positive cooperativity of Cmpd-6 on ß1ARs as a ß-arrestin-biased positive allosteric modulator. Cmpd-6 selectively enhances the affinity and cellular signaling of carvedilol, a known ß-arrestin-biased ß-blocker for ß1ARs, whereas it has minimal effect on other ligands tested. Importantly, Cmpd-6 enhances the ß-arrestin-dependent in vivo cardioprotective effect of carvedilol during ischemia/reperfusion injury-induced apoptosis. The data support the potential therapeutic application of Cmpd-6 to enhance the clinical benefits of carvedilol in the treatment of cardiac disease.

Unique Positive Cooperativity Between the ß-Arrestin-Biased ß-Blocker Carvedilol and a Small Molecule Positive Allosteric Modulator of the ß2-Adrenergic Receptor.

Among ß-blockers that are clinically prescribed for heart failure, carvedilol is a first-choice agent with unique pharmacological properties. Carvedilol is distinct from other ß-blockers in its ability to elicit ß-arrestin-biased agonism, which has been suggested to underlie its cardioprotective effects. Augmenting the pharmacologic properties of carvedilol thus holds the promise of developing more efficacious and/or biased ß-blockers. We recently identified compound-6 (cmpd-6), the first small molecule positive allosteric modulator of the ß2-adrenergic receptor (ß2AR). Cmpd-6 is positively cooperative with orthosteric agonists at the ß2AR and enhances agonist-mediated transducer (G-protein and ß-arrestin) signaling in an unbiased manner. Here, we report that cmpd-6, quite unexpectedly, displays strong positive cooperativity only with carvedilol among a panel of structurally diverse ß-blockers. Cmpd-6 enhances the binding affinity of carvedilol for the ß2AR and augments its ability to competitively antagonize agonist-induced cAMP generation. Cmpd-6 potentiates ß-arrestin1- but not Gs-protein-mediated high-affinity binding of carvedilol at the ß2AR and ß-arrestin-mediated cellular functions in response to carvedilol including extracellular signal-regulated kinase phosphorylation, receptor endocytosis, and trafficking into lysosomes. Importantly, an analog of cmpd-6 that selectively retains positive cooperativity with carvedilol acts as a negative modulator of agonist-stimulated ß2AR signaling. These unprecedented cooperative properties of carvedilol and cmpd-6 have implications for fundamental understanding of G-protein-coupled receptor (GPCR) allosteric modulation, as well as for the development of more effective biased beta blockers and other GPCR therapeutics. SIGNIFICANCE STATEMENT: This study reports on the small molecule-mediated allosteric modulation of the ß-arrestin-biased ß-blocker, carvedilol. The small molecule, compound-6 (cmpd-6), displays an exclusive positive cooperativity with carvedilol among other ß-blockers and enhances the binding affinity of carvedilol for the ß2-adrenergic receptor. Cooperative effects of cmpd-6 augment the ß-blockade property of carvedilol while potentiating its ß-arrestin-mediated signaling functions. These findings have potential implications in advancing G-protein-coupled receptor allostery, developing biased therapeutics and remedying cardiovascular ailments.

Allosteric activation of proto-oncogene kinase Src by GPCR-beta-arrestin complexes.

G protein-coupled receptors (GPCRs) initiate signaling cascades via G-proteins and beta-arrestins (ßarr). ßarr-dependent actions begin with recruitment of ßarr to the phosphorylated receptor tail and are followed by engagement with the receptor core. ßarrs are known to act as adaptor proteins binding receptors and various effectors, but it is unclear whether in addition to the scaffolding role ßarrs can allosterically activate their downstream targets. Here we demonstrate the direct allosteric activation of proto-oncogene kinase Src by GPCR-ßarr complexes in vitro and establish the conformational basis of the activation. Whereas free ßarr1 had no effect on Src activity, ßarr1 in complex with M2 muscarinic or ß2-adrenergic receptors reconstituted in lipid nanodiscs activate Src by reducing the lag phase in Src autophosphorylation. Interestingly, receptor-ßarr1 complexes formed with a ßarr1 mutant, in which the finger-loop, required to interact with the receptor core, has been deleted, fully retain the ability to activate Src. Similarly, ßarr1 in complex with only a phosphorylated C-terminal tail of the vasopressin 2 receptor activates Src as efficiently as GPCR-ßarr complexes. In contrast, ßarr1 and chimeric M2 receptor with nonphosphorylated C-terminal tail failed to activate Src. Taken together, these data demonstrate that the phosphorylated GPCR tail interaction with ßarr1 is necessary and sufficient to empower it to allosterically activate Src. Our findings may have implications for understanding more broadly the mechanisms of allosteric activation of downstream targets by ßarrs.

Sortase ligation enables homogeneous GPCR phosphorylation to reveal diversity in ß-arrestin coupling.

The ability of G protein-coupled receptors (GPCRs) to initiate complex cascades of cellular signaling is governed by the sequential coupling of three main transducer proteins, G protein, GPCR kinase (GRK), and ß-arrestin. Mounting evidence indicates these transducers all have distinct conformational preferences and binding modes. However, interrogating each transducer's mechanism of interaction with GPCRs has been complicated by the interplay of transducer-mediated signaling events. For example, GRK-mediated receptor phosphorylation recruits and induces conformational changes in ß-arrestin, which facilitates coupling to the GPCR transmembrane core. Here we compare the allosteric interactions of G proteins and ß-arrestins with GPCRs' transmembrane cores by using the enzyme sortase to ligate a synthetic phosphorylated peptide onto the carboxyl terminus of three different receptors. Phosphopeptide ligation onto the ß2-adrenergic receptor (ß2AR) allows stabilization of a high-affinity receptor active state by ß-arrestin1, permitting us to define elements in the ß2AR and ß-arrestin1 that contribute to the receptor transmembrane core interaction. Interestingly, ligation of the identical phosphopeptide onto the ß2AR, the muscarinic acetylcholine receptor 2 and the µ-opioid receptor reveals that the ability of ß-arrestin1 to enhance agonist binding relative to G protein differs substantially among receptors. Furthermore, strong allosteric coupling of ß-arrestin1 correlates with its ability to attenuate, or "desensitize," G protein activation in vitro. Sortase ligation thus provides a versatile method to introduce complex, defined phosphorylation patterns into GPCRs, and analogous strategies could be applied to other classes of posttranslationally modified proteins. These homogeneously phosphorylated GPCRs provide an innovative means to systematically study receptor-transducer interactions.

Allosteric "beta-blocker" isolated from a DNA-encoded small molecule library.

The ß2-adrenergic receptor (ß2AR) has been a model system for understanding regulatory mechanisms of G-protein-coupled receptor (GPCR) actions and plays a significant role in cardiovascular and pulmonary diseases. Because all known ß-adrenergic receptor drugs target the orthosteric binding site of the receptor, we set out to isolate allosteric ligands for this receptor by panning DNA-encoded small-molecule libraries comprising 190 million distinct compounds against purified human ß2AR. Here, we report the discovery of a small-molecule negative allosteric modulator (antagonist), compound 15 [([4-((2S)-3-(((S)-3-(3-bromophenyl)-1-(methylamino)-1-oxopropan-2-yl)amino)-2-(2-cyclohexyl-2-phenylacetamido)-3-oxopropyl)benzamide], exhibiting a unique chemotype and low micromolar affinity for the ß2AR. Binding of 15 to the receptor cooperatively enhances orthosteric inverse agonist binding while negatively modulating binding of orthosteric agonists. Studies with a specific antibody that binds to an intracellular region of the ß2AR suggest that 15 binds in proximity to the G-protein binding site on the cytosolic surface of the ß2AR. In cell-signaling studies, 15 inhibits cAMP production through the ß2AR, but not that mediated by other Gs-coupled receptors. Compound 15 also similarly inhibits ß-arrestin recruitment to the activated ß2AR. This study presents an allosteric small-molecule ligand for the ß2AR and introduces a broadly applicable method for screening DNA-encoded small-molecule libraries against purified GPCR targets. Importantly, such an approach could facilitate the discovery of GPCR drugs with tailored allosteric effects.

Small-Molecule Positive Allosteric Modulators of the ß2-Adrenoceptor Isolated from DNA-Encoded Libraries.

Conventional drug discovery efforts at the ß2-adrenoceptor (ß2AR) have led to the development of ligands that bind almost exclusively to the receptor's hormone-binding orthosteric site. However, targeting the largely unexplored and evolutionarily unique allosteric sites has potential for developing more specific drugs with fewer side effects than orthosteric ligands. Using our recently developed approach for screening G protein-coupled receptors (GPCRs) with DNA-encoded small-molecule libraries, we have discovered and characterized the first ß2AR small-molecule positive allosteric modulators (PAMs)-compound (Cmpd)-6 [(R)-N-(4-amino-1-(4-(tert-butyl)phenyl)-4-oxobutan-2-yl)-5-(N-isopropyl-N-methylsulfamoyl)-2-((4-methoxyphenyl)thio)benzamide] and its analogs. We used purified human ß2ARs, occupied by a high-affinity agonist, for the affinity-based screening of over 500 million distinct library compounds, which yielded Cmpd-6. It exhibits a low micro-molar affinity for the agonist-occupied ß2AR and displays positive cooperativity with orthosteric agonists, thereby enhancing their binding to the receptor and ability to stabilize its active state. Cmpd-6 is cooperative with G protein and ß-arrestin1 (a.k.a. arrestin2) to stabilize high-affinity, agonist-bound active states of the ß2AR and potentiates downstream cAMP production and receptor recruitment of ß-arrestin2 (a.k.a. arrestin3). Cmpd-6 is specific for the ß2AR compared with the closely related ß1AR. Structure-activity studies of select Cmpd-6 analogs defined the chemical groups that are critical for its biologic activity. We thus introduce the first small-molecule PAMs for the ß2AR, which may serve as a lead molecule for the development of novel therapeutics. The approach described in this work establishes a broadly applicable proof-of-concept strategy for affinity-based discovery of small-molecule allosteric compounds targeting unique conformational states of GPCRs.

Conformationally selective RNA aptamers allosterically modulate the ß2-adrenoceptor.

G-protein-coupled receptor (GPCR) ligands function by stabilizing multiple, functionally distinct receptor conformations. This property underlies the ability of 'biased agonists' to activate specific subsets of a given receptor's signaling profile. However, stabilizing distinct active GPCR conformations to enable structural characterization of mechanisms underlying GPCR activation remains difficult. These challenges have accentuated the need for receptor tools that allosterically stabilize and regulate receptor function through unique, previously unappreciated mechanisms. Here, using a highly diverse RNA library combined with advanced selection strategies involving state-of-the-art next-generation sequencing and bioinformatics analyses, we identify RNA aptamers that bind a prototypical GPCR, the ß2-adrenoceptor (ß2AR). Using biochemical, pharmacological, and biophysical approaches, we demonstrate that these aptamers bind with nanomolar affinity at defined surfaces of the receptor, allosterically stabilizing active, inactive, and ligand-specific receptor conformations. The discovery of RNA aptamers as allosteric GPCR modulators significantly expands the diversity of ligands available to study the structural and functional regulation of GPCRs.

Neurological and Motor Disorders: Neuronal Store-Operated Ca2+ Signaling: An Overview and Its Function.

Calcium (Ca2+) is a ubiquitous second messenger that performs significant physiological task such as neurosecretion, exocytosis, neuronal growth/differentiation, and the development and/or maintenance of neural circuits. An important regulatory aspect of neuronal Ca2+ homeostasis is store-operated Ca2+ entry (SOCE) which, in recent years, has gained much attention for influencing a variety of nerve cell responses. Essentially, activation of SOCE ensues following the activation of the plasma membrane (PM) store-operated Ca2+ channels (SOCC) triggered by the depletion of endoplasmic reticulum (ER) Ca2+ stores. In addition to the TRPC (transient receptor potential canonical) and the Orai family of ion channels, STIM (stromal interacting molecule) proteins have been baptized as key molecular regulators of SOCE. Functional significance of the TRPC channels in neurons has been elaborately studied; however, information on Orai and STIM components of SOCE, although seems imminent, is currently limited. Importantly, perturbations in SOCE have been implicated in a spectrum of neuropathological conditions. Hence, understanding the precise involvement of SOCC in neurodegeneration would presumably unveil avenues for plausible therapeutic interventions. We thus review the role of SOCE-regulated neuronal Ca2+ signaling in selecting neurodegenerative conditions.

Impairment of TRPC1-STIM1 channel assembly and AQP5 translocation compromise agonist-stimulated fluid secretion in mice lacking caveolin1.

Neurotransmitter regulation of salivary fluid secretion is mediated by activation of Ca(2+) influx. The Ca(2+)-permeable transient receptor potential canonical 1 (TRPC1) channel is crucial for fluid secretion. However, the mechanism(s) involved in channel assembly and regulation are not completely understood. We report that Caveolin1 (Cav1) is essential for the assembly of functional TRPC1 channels in salivary glands (SG) in vivo and thus regulates fluid secretion. In Cav1(-/-) mouse SG, agonist-stimulated Ca(2+) entry and fluid secretion are significantly reduced. Microdomain localization of TRPC1 and interaction with its regulatory protein, STIM1, are disrupted in Cav1(-/-) SG acinar cells, whereas Orai1-STIM1 interaction is not affected. Furthermore, localization of aquaporin 5 (AQP5), but not that of inositol (1,4,5)-trisphosphate receptor 3 or Ca(2+)-activated K(+) channel (IK) in the apical region of acinar cell was altered in Cav1(-/-) SG. In addition, agonist-stimulated increase in surface expression of AQP5 required Ca(2+) influx via TRPC1 channels and was inhibited in Cav1(-/-) SG. Importantly, adenovirus-mediated expression of Cav1 in Cav1(-/-) SG restored interaction of STIM1 with TRPC1 and channel activation, apical targeting and regulated trafficking of AQP5, and neurotransmitter stimulated fluid-secretion. Together these findings demonstrate that, by directing cellular localization of TRPC1 and AQP5 channels and by selectively regulating the functional assembly TRPC1-STIM1 channels, Cav1 is a crucial determinant of SG fluid secretion.

Beta-arrestin 1 mediated Src activation via Src SH3 domain revealed by cryo-electron microscopy.

Beta-arrestins (ßarrs) are key regulators and transducers of G-protein coupled receptor signaling; however, little is known of how ßarrs communicate with their downstream effectors. Here, we use cryo-electron microscopy to elucidate how ßarr1 recruits and activates non-receptor tyrosine kinase Src. ßarr1 binds Src SH3 domain via two distinct sites: a polyproline site in the N-domain and a non-proline site in the central crest region. At both sites ßarr1 interacts with the aromatic surface of SH3 which is critical for Src autoinhibition, suggesting that ßarr1 activates Src by SH3 domain displacement. Binding of SH3 to the central crest region induces structural rearrangements in the ß-strand V, finger, and middle loops of ßarr1 and interferes with ßarr1 coupling to the receptor core potentially impacting receptor desensitization and downstream signaling.

Structure and dynamics determine G protein coupling specificity at a class A GPCR.

G protein coupled receptors (GPCRs) exhibit varying degrees of selectivity for different G protein isoforms. Despite the abundant structures of GPCR-G protein complexes, little is known about the mechanism of G protein coupling specificity. The ß2-adrenergic receptor is an example of GPCR with high selectivity for Gαs, the stimulatory G protein for adenylyl cyclase, and much weaker for the Gαi family of G proteins inhibiting adenylyl cyclase. By developing a new Gαi-biased agonist (LM189), we provide structural and biophysical evidence supporting that distinct conformations at ICL2 and TM6 are required for coupling of the different G protein subtypes Gαs and Gαi. These results deepen our understanding of G protein specificity and bias and can accelerate the design of ligands that select for preferred signaling pathways.

Activation of TRPC1 by STIM1 in ER-PM microdomains involves release of the channel from its scaffold caveolin-1.

Store-operated Ca(2+) entry (SOCE) is activated by redistribution of STIM1 into puncta in discrete ER-plasma membrane junctional regions where it interacts with and activates store-operated channels (SOCs). The factors involved in precise targeting of the channels and their retention at these specific microdomains are not yet defined. Here we report that caveolin-1 (Cav1) is a critical plasma membrane scaffold that retains TRPC1 within the regions where STIM1 puncta are localized following store depletion. This enables the interaction of TRPC1 with STIM1 that is required for the activation of TRPC1-SOCE. Silencing Cav1 in human submandibular gland (HSG) cells decreased plasma membrane retention of TRPC1, TRPC1-STIM1 clustering, and consequently reduced TRPC1-SOCE, without altering STIM1 puncta. Importantly, activation of TRPC1-SOCE was associated with an increase in TRPC1-STIM1 and a decrease in TRPC1-Cav1 clustering. Consistent with this, overexpression of Cav1 decreased TRPC1-STIM1 clustering and SOCE, both of which were recovered when STIM1 was expressed at higher levels relative to Cav1. Silencing STIM1 or expression of DeltaERM-STIM1 or STIM1((684)EE(685)) mutant prevented dissociation of TRPC1-Cav1 and activation of TRPC1-SOCE. However expression of TRPC1-((639)KK(640)) with STIM1((684)EE(685)) restored function and the dissociation of TRPC1 from Cav1 in response to store depletion. Further, conditions that promoted TRPC1-STIM1 clustering and TRPC1-SOCE elicited corresponding changes in SOCE-dependent NFkB activation and cell proliferation. Together these data demonstrate that Cav1 is a critical plasma membrane scaffold for inactive TRPC1. We suggest that activation of TRPC1-SOC by STIM1 mediates release of the channel from Cav1.

Up-regulation of transient receptor potential canonical 1 (TRPC1) following sarco(endo)plasmic reticulum Ca2+ ATPase 2 gene silencing promotes cell survival: a potential role for TRPC1 in Darier's disease.

The mechanism(s) involved in regulation of store operated calcium entry in Darier's disease (DD) is not known. We investigated the distribution and function of transient receptor potential canonical (TRPC) in epidermal skin cells. DD patients demonstrated up-regulation of TRPC1, but not TRPC3, in the squamous layers. Ca2+ influx was significantly higher in keratinocytes obtained from DD patients and showed enhanced proliferation compared with normal keratinocytes. Similar up-regulation of TRPC1 was also detected in epidermal layers of SERCA2+/- mice. HaCaT cells expressed TRPC1 in the plasma membrane. Expression of sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA)2 small interfering RNA (siRNA) in HaCaT cells increased TRPC1 levels and thapsigargin-stimulated Ca2+ influx, which was blocked by store-operated calcium entry inhibitors. Thapsigargin-stimulated intracellular Ca2+ release was decreased in DD cells. DD keratinocytes exhibited increased cell survival upon thapsigargin treatment. Alternatively, overexpression of TRPC1 or SERCA2-siRNA in HaCaT cells demonstrated resistance to thapsigargin-induced apoptosis. These effects were dependent on external Ca2+ and activation of nuclear factor-kappaB. Isotretinoin reduced Ca2+ entry in HaCaT cells and decreased survival of HaCaT and DD keratinocytes. These findings put forward a novel consequence of compromised SERCA2 function in DD wherein up-regulation of TRPC1 augments cell proliferation and restrict apoptosis. We suggest that the anti-apoptotic effect of TRPC1 could potentially contribute to abnormal keratosis in DD.

Mechanism of ß2AR regulation by an intracellular positive allosteric modulator.

Drugs targeting the orthosteric, primary binding site of G protein-coupled receptors are the most common therapeutics. Allosteric binding sites, elsewhere on the receptors, are less well-defined, and so less exploited clinically. We report the crystal structure of the prototypic ß2-adrenergic receptor in complex with an orthosteric agonist and compound-6FA, a positive allosteric modulator of this receptor. It binds on the receptor's inner surface in a pocket created by intracellular loop 2 and transmembrane segments 3 and 4, stabilizing the loop in an α-helical conformation required to engage the G protein. Structural comparison explains the selectivity of the compound for ß2- over the ß1-adrenergic receptor. Diversity in location, mechanism, and selectivity of allosteric ligands provides potential to expand the range of receptor drugs.

G protein-coupled receptor kinases (GRKs) orchestrate biased agonism at the ß2-adrenergic receptor.

Biased agonists of G protein-coupled receptors (GPCRs), which selectively activate either G protein- or ß-arrestin-mediated signaling pathways, are of major therapeutic interest because they have the potential to show improved efficacy and specificity as drugs. Efforts to understand the mechanistic basis of this phenomenon have focused on the hypothesis that G proteins and ß-arrestins preferentially couple to distinct GPCR conformations. However, because GPCR kinase (GRK)-dependent receptor phosphorylation is a critical prerequisite for the recruitment of ß-arrestins to most GPCRs, GRKs themselves may play an important role in establishing biased signaling. We showed that an alanine mutant of the highly conserved residue tyrosine 219 (Y219A) in transmembrane domain five of the ß2-adrenergic receptor (ß2AR) was incapable of ß-arrestin recruitment, receptor internalization, and ß-arrestin-mediated activation of extracellular signal-regulated kinase (ERK), whereas it retained the ability to signal through G protein. We found that the impaired ß-arrestin recruitment in cells was due to reduced GRK-mediated phosphorylation of the ß2AR Y219A C terminus, which was recapitulated in vitro with purified components. Furthermore, in vitro ligation of a synthetically phosphorylated peptide onto the C terminus of ß2AR Y219A rescued both the initial recruitment of ß-arrestin and its engagement with the intracellular core of the receptor. These data suggest that the Y219A mutation generates a G protein-biased state primarily by conformational selection against GRK coupling, rather than against ß-arrestin. Together, these findings highlight the importance of GRKs in modulating the biased agonism of GPCRs.

Manifold roles of ß-arrestins in GPCR signaling elucidated with siRNA and CRISPR/Cas9.

G protein-coupled receptors (GPCRs) use diverse mechanisms to regulate the mitogen-activated protein kinases ERK1/2. ß-Arrestins (ßArr1/2) are ubiquitous inhibitors of G protein signaling, promoting GPCR desensitization and internalization and serving as scaffolds for ERK1/2 activation. Studies using CRISPR/Cas9 to delete ßArr1/2 and G proteins have cast doubt on the role of ß-arrestins in activating specific pools of ERK1/2. We compared the effects of siRNA-mediated knockdown of ßArr1/2 and reconstitution with ßArr1/2 in three different parental and CRISPR-derived ßArr1/2 knockout HEK293 cell pairs to assess the effect of ßArr1/2 deletion on ERK1/2 activation by four Gs-coupled GPCRs. In all parental lines with all receptors, ERK1/2 stimulation was reduced by siRNAs specific for ßArr2 or ßArr1/2. In contrast, variable effects were observed with CRISPR-derived cell lines both between different lines and with activation of different receptors. For ß2 adrenergic receptors (ß2ARs) and ß1ARs, ßArr1/2 deletion increased, decreased, or had no effect on isoproterenol-stimulated ERK1/2 activation in different CRISPR clones. ERK1/2 activation by the vasopressin V2 and follicle-stimulating hormone receptors was reduced in these cells but was enhanced by reconstitution with ßArr1/2. Loss of desensitization and receptor internalization in CRISPR ßArr1/2 knockout cells caused ß2AR-mediated stimulation of ERK1/2 to become more dependent on G proteins, which was reversed by reintroducing ßArr1/2. These data suggest that ßArr1/2 function as a regulatory hub, determining the balance between mechanistically different pathways that result in activation of ERK1/2, and caution against extrapolating results obtained from ßArr1/2- or G protein-deleted cells to GPCR behavior in native systems.