Distinctive Activation Mechanism for Angiotensin Receptor Revealed by a Synthetic Nanobody.

The angiotensin II (AngII) type 1 receptor (AT1R) is a critical regulator of cardiovascular and renal function and is an important model for studies of G-protein-coupled receptor (GPCR) signaling. By stabilizing the receptor with a single-domain antibody fragment ("nanobody") discovered using a synthetic yeast-displayed library, we determined the crystal structure of active-state human AT1R bound to an AngII analog with partial agonist activity. The nanobody binds to the receptor's intracellular transducer pocket, stabilizing the large conformational changes characteristic of activated GPCRs. The peptide engages the AT1R through an extensive interface spanning from the receptor core to its extracellular face and N terminus, remodeling the ligand-binding cavity. Remarkably, the mechanism used to propagate conformational changes through the receptor diverges from other GPCRs at several key sites, highlighting the diversity of allosteric mechanisms among GPCRs. Our structure provides insight into how AngII and its analogs stimulate full or biased signaling, respectively.

Angiotensin Analogs with Divergent Bias Stabilize Distinct Receptor Conformations.

"Biased" G protein-coupled receptor (GPCR) agonists preferentially activate pathways mediated by G proteins or ß-arrestins. Here, we use double electron-electron resonance spectroscopy to probe the changes that ligands induce in the conformational distribution of the angiotensin II type I receptor. Monitoring distances between 10 pairs of nitroxide labels distributed across the intracellular regions enabled mapping of four underlying sets of conformations. Ligands from different functional classes have distinct, characteristic effects on the conformational heterogeneity of the receptor. Compared to angiotensin II, the endogenous agonist, agonists with enhanced Gq coupling more strongly stabilize an "open" conformation with an accessible transducer-binding site. ß-arrestin-biased agonists deficient in Gq coupling do not stabilize this open conformation but instead favor two more occluded conformations. These data suggest a structural mechanism for biased ligand action at the angiotensin receptor that can be exploited to rationally design GPCR-targeting drugs with greater specificity of action.

Multidimensional Tracking of GPCR Signaling via Peroxidase-Catalyzed Proximity Labeling.

G-protein-coupled receptors (GPCRs) play critical roles in regulating physiological processes ranging from neurotransmission to cardiovascular function. Current methods for tracking GPCR signaling suffer from low throughput, modification or overexpression of effector proteins, and low temporal resolution. Here, we show that peroxidase-catalyzed proximity labeling can be combined with isobaric tagging and mass spectrometry to enable quantitative, time-resolved measurement of GPCR agonist response in living cells. Using this technique, termed "GPCR-APEX," we track activation and internalization of the angiotensin II type 1 receptor and the ß2 adrenoceptor. These receptors co-localize with a variety of G proteins even before receptor activation, and activated receptors are largely sequestered from G proteins upon internalization. Additionally, the two receptors show differing internalization kinetics, and we identify the membrane protein LMBRD2 as a potential regulator of ß2 adrenoceptor signaling, underscoring the value of a dynamic view of receptor function.

Antibodies expand the scope of angiotensin receptor pharmacology.

G-protein-coupled receptors (GPCRs) are key regulators of human physiology and are the targets of many small-molecule research compounds and therapeutic drugs. While most of these ligands bind to their target GPCR with high affinity, selectivity is often limited at the receptor, tissue and cellular levels. Antibodies have the potential to address these limitations but their properties as GPCR ligands remain poorly characterized. Here, using protein engineering, pharmacological assays and structural studies, we develop maternally selective heavy-chain-only antibody ('nanobody') antagonists against the angiotensin II type I receptor and uncover the unusual molecular basis of their receptor antagonism. We further show that our nanobodies can simultaneously bind to angiotensin II type I receptor with specific small-molecule antagonists and demonstrate that ligand selectivity can be readily tuned. Our work illustrates that antibody fragments can exhibit rich and evolvable pharmacology, attesting to their potential as next-generation GPCR modulators.

Structure of the M2 muscarinic receptor-ß-arrestin complex in a lipid nanodisc.

After activation by an agonist, G-protein-coupled receptors (GPCRs) recruit ß-arrestin, which desensitizes heterotrimeric G-protein signalling and promotes receptor endocytosis1. Additionally, ß-arrestin directly regulates many cell signalling pathways that can induce cellular responses distinct from that of G proteins2. In contrast to G proteins, for which there are many high-resolution structures in complex with GPCRs, the molecular mechanisms underlying the interaction of ß-arrestin with GPCRs are much less understood. Here we present a cryo-electron microscopy structure of ß-arrestin 1 (ßarr1) in complex with M2 muscarinic receptor (M2R) reconstituted in lipid nanodiscs. The M2R-ßarr1 complex displays a multimodal network of flexible interactions, including binding of the N domain of ßarr1 to phosphorylated receptor residues and insertion of the finger loop of ßarr1 into the M2R seven-transmembrane bundle, which adopts a conformation similar to that in the M2R-heterotrimeric Go protein complex3. Moreover, the cryo-electron microscopy map reveals that the C-edge of ßarr1 engages the lipid bilayer. Through atomistic simulations and biophysical, biochemical and cellular assays, we show that the C-edge is critical for stable complex formation, ßarr1 recruitment, receptor internalization, and desensitization of G-protein activation. Taken together, these data suggest that the cooperative interactions of ß-arrestin with both the receptor and the phospholipid bilayer contribute to its functional versatility.

Synthetic nanobodies as angiotensin receptor blockers.

There is considerable interest in developing antibodies as functional modulators of G protein-coupled receptor (GPCR) signaling for both therapeutic and research applications. However, there are few antibody ligands targeting GPCRs outside of the chemokine receptor group. GPCRs are challenging targets for conventional antibody discovery methods, as many are highly conserved across species, are biochemically unstable upon purification, and possess deeply buried ligand-binding sites. Here, we describe a selection methodology to enrich for functionally modulatory antibodies using a yeast-displayed library of synthetic camelid antibody fragments called "nanobodies." Using this platform, we discovered multiple nanobodies that act as antagonists of the angiotensin II type 1 receptor (AT1R). Following angiotensin II infusion in mice, we found that an affinity matured nanobody antagonist has comparable antihypertensive activity to the angiotensin receptor blocker (ARB) losartan. The unique pharmacology and restricted biodistribution of nanobody antagonists may provide a path for treating hypertensive disorders when small-molecule drugs targeting the AT1R are contraindicated, for example, in pregnancy.

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.

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.

Detergent- and phospholipid-based reconstitution systems have differential effects on constitutive activity of G-protein-coupled receptors.

A hallmark of G-protein-coupled receptors (GPCRs) is the conversion of external stimuli into specific cellular responses. In this tightly-regulated process, extracellular ligand binding by GPCRs promotes specific conformational changes within the seven transmembrane helices, leading to the coupling and activation of intracellular "transducer" proteins, such as heterotrimeric G proteins. Much of our understanding of the molecular mechanisms that govern GPCR activation is derived from experiments with purified receptors reconstituted in detergent micelles. To elucidate the influence of the phospholipid bilayer on GPCR activation, here we interrogated the functional, pharmacological, and biophysical properties of a GPCR, the ß2-adrenergic receptor (ß2AR), in high-density lipoprotein (HDL) particles. Compared with detergent-reconstituted ß2AR, the ß2AR in HDL particles had greatly enhanced levels of basal (constitutive) activity and displayed increased sensitivity to agonist activation, as assessed by activation of heterotrimeric G protein and allosteric coupling between the ligand-binding and transducer-binding pockets. Using 19F NMR spectroscopy, we directly linked these functional differences in detergent- and HDL-reconstituted ß2AR to a change in the equilibrium between inactive and active receptor states. The contrast between the low levels of ß2AR constitutive activity in cells and the high constitutive activity observed in an isolated phospholipid bilayer indicates that ß2AR basal activity depends on the reconstitution system and further suggests that various cellular mechanisms suppress ß2AR basal activity physiologically. Our findings provide critical additional insights into GPCR activation and reveal how dramatically reconstitution systems can impact membrane protein function.

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.

Structure of active ß-arrestin-1 bound to a G-protein-coupled receptor phosphopeptide.

The functions of G-protein-coupled receptors (GPCRs) are primarily mediated and modulated by three families of proteins: the heterotrimeric G proteins, the G-protein-coupled receptor kinases (GRKs) and the arrestins. G proteins mediate activation of second-messenger-generating enzymes and other effectors, GRKs phosphorylate activated receptors, and arrestins subsequently bind phosphorylated receptors and cause receptor desensitization. Arrestins activated by interaction with phosphorylated receptors can also mediate G-protein-independent signalling by serving as adaptors to link receptors to numerous signalling pathways. Despite their central role in regulation and signalling of GPCRs, a structural understanding of ß-arrestin activation and interaction with GPCRs is still lacking. Here we report the crystal structure of ß-arrestin-1 (also called arrestin-2) in complex with a fully phosphorylated 29-amino-acid carboxy-terminal peptide derived from the human V2 vasopressin receptor (V2Rpp). This peptide has previously been shown to functionally and conformationally activate ß-arrestin-1 (ref. 5). To capture this active conformation, we used a conformationally selective synthetic antibody fragment (Fab30) that recognizes the phosphopeptide-activated state of ß-arrestin-1. The structure of the ß-arrestin-1-V2Rpp-Fab30 complex shows marked conformational differences in ß-arrestin-1 compared to its inactive conformation. These include rotation of the amino- and carboxy-terminal domains relative to each other, and a major reorientation of the 'lariat loop' implicated in maintaining the inactive state of ß-arrestin-1. These results reveal, at high resolution, a receptor-interacting interface on ß-arrestin, and they indicate a potentially general molecular mechanism for activation of these multifunctional signalling and regulatory proteins.

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.

Regulation of ß2-adrenergic receptor function by conformationally selective single-domain intrabodies.

The biologic activity induced by ligand binding to orthosteric or allosteric sites on a G protein-coupled receptor (GPCR) is mediated by stabilization of specific receptor conformations. In the case of the ß2 adrenergic receptor, these ligands are generally small-molecule agonists or antagonists. However, a monomeric single-domain antibody (nanobody) from the Camelid family was recently found to allosterically bind and stabilize an active conformation of the ß2-adrenergic receptor (ß2AR). Here, we set out to study the functional interaction of 18 related nanobodies with the ß2AR to investigate their roles as novel tools for studying GPCR biology. Our studies revealed several sequence-related nanobody families with preferences for active (agonist-occupied) or inactive (antagonist-occupied) receptors. Flow cytometry analysis indicates that all nanobodies bind to epitopes displayed on the intracellular receptor surface; therefore, we transiently expressed them intracellularly as "intrabodies" to test their effects on ß2AR-dependent signaling. Conformational specificity was preserved after intrabody conversion as demonstrated by the ability for the intracellularly expressed nanobodies to selectively bind agonist- or antagonist-occupied receptors. When expressed as intrabodies, they inhibited G protein activation (cyclic AMP accumulation), G protein-coupled receptor kinase (GRK)-mediated receptor phosphorylation, ß-arrestin recruitment, and receptor internalization to varying extents. These functional effects were likely due to either steric blockade of downstream effector (Gs, ß-arrestin, GRK) interactions or stabilization of specific receptor conformations which do not support effector coupling. Together, these findings strongly implicate nanobody-derived intrabodies as novel tools to study GPCR biology.

Nuclear RhoA signaling regulates MRTF-dependent SMC-specific transcription.

We have previously shown that RhoA-mediated actin polymerization stimulates smooth muscle cell (SMC)-specific transcription by regulating the nuclear localization of the myocardin-related transcription factors (MRTFs). On the basis of the recent demonstration that nuclear G-actin regulates MRTF nuclear export and observations from our laboratory and others that the RhoA effector, mDia2, shuttles between the nucleus and cytoplasm, we investigated whether nuclear RhoA signaling plays a role in regulating MRTF activity. We identified sequences that control mDia2 nuclear-cytoplasmic shuttling and used mDia2 variants to demonstrate that the ability of mDia2 to fully stimulate MRTF nuclear accumulation and SMC-specific gene transcription was dependent on its localization to the nucleus. To test whether RhoA signaling promotes nuclear actin polymerization, we established a fluorescence recovery after photobleaching (FRAP)-based assay to measure green fluorescent protein-actin diffusion in the nuclear compartment. Nuclear actin FRAP was delayed in cells expressing nuclear-targeted constitutively active mDia1 and mDia2 variants and in cells treated with the polymerization inducer, jasplakinolide. In contrast, FRAP was enhanced in cells expressing a nuclear-targeted variant of mDia that inhibits both mDia1 and mDia2. Treatment of 10T1/2 cells with sphingosine 1-phosphate induced RhoA activity in the nucleus and forced nuclear localization of RhoA or the Rho-specific guanine nucleotide exchange factor (GEF), leukemia-associated RhoGEF, enhanced the ability of these proteins to stimulate MRTF activity. Taken together, these data support the emerging idea that RhoA-dependent nuclear actin polymerization has important effects on transcription and nuclear structure.

Antibodies Expand the Scope of Angiotensin Receptor Pharmacology.

G protein-coupled receptors (GPCRs) are key regulators of human physiology and are the targets of many small molecule research compounds and therapeutic drugs. While most of these ligands bind to their target GPCR with high affinity, selectivity is often limited at the receptor, tissue, and cellular level. Antibodies have the potential to address these limitations but their properties as GPCR ligands remain poorly characterized. Here, using protein engineering, pharmacological assays, and structural studies, we develop maternally selective heavy chain-only antibody ("nanobody") antagonists against the angiotensin II type I receptor (AT1R) and uncover the unusual molecular basis of their receptor antagonism. We further show that our nanobodies can simultaneously bind to AT1R with specific small-molecule antagonists and demonstrate that ligand selectivity can be readily tuned. Our work illustrates that antibody fragments can exhibit rich and evolvable pharmacology, attesting to their potential as next-generation GPCR modulators.

Formin homology domain-containing protein 1 regulates smooth muscle cell phenotype.

OBJECTIVE: Our goal was to test whether formin homology protein 1 (FHOD1) plays a significant role in the regulation of smooth muscle cell (SMC) differentiation and, if so, whether Rho kinase (ROCK)-dependent phosphorylation in the diaphanous autoinhibitory domain is an important signaling mechanism that controls FHOD1 activity in SMC. METHODS AND RESULTS: FHOD1 is highly expressed in aortic SMCs and in tissues with a significant SMC component. Exogenous expression of constitutively active FHOD1, but not wild-type, strongly activated SMC-specific gene expression in 10T1/2 cells. Treatment of SMC with the RhoA activator sphingosine-1-phosphate increased FHOD1 phosphorylation at Thr1141, and this effect was completely prevented by inhibition of ROCK with Y-27632. Phosphomimetic mutations to ROCK target residues enhanced FHOD1 activity, suggesting that phosphorylation interferes with FHOD1 autoinhibition. Importantly, knockdown of FHOD1 in SMC strongly inhibited sphingosine-1-phosphate-dependent increases in SMC differentiation marker gene expression and actin polymerization, suggesting that FHOD1 plays a major role in RhoA-dependent signaling in SMC. CONCLUSIONS: Our results indicate that FHOD1 is a critical regulator of SMC phenotype and is regulated by ROCK-dependent phosphorylation. Thus, additional studies on the role of FHOD1 during development and the progression of cardiovascular disease will be important.

Enhancement of mDia2 activity by Rho-kinase-dependent phosphorylation of the diaphanous autoregulatory domain.

It is clear that RhoA activates the DRF (diaphanous-related formin) mDia2 by disrupting the molecular interaction between the DAD (diaphanous autoregulatory domain) and the DID (diaphanous inhibitory domain). Previous studies indicate that a basic motif within the DAD contributes to mDia2 auto-inhibition, and results shown in the present study suggest these residues bind a conserved acidic region within the DID. Furthermore, we demonstrate that mDia2 is phosphorylated by ROCK (Rho-kinase) at two conserved residues (Thr(1061) and Ser(1070)) just C-terminal to the DAD basic region. Phosphomimetic mutations to these residues in the context of the full-length molecule enhanced mDia2 activity as measured by increased actin polymerization, SRF (serum response factor)-dependent smooth muscle-specific gene transcription, and nuclear localization of myocardin-related transcription factor B. Biochemical and functional data indicate that the T1061E/S1070E mutation significantly inhibited the ability of DAD to interact with DID and enhanced mDia2 activation by RhoA. Taken together, the results of the present study indicate that ROCK-dependent phosphorylation of the mDia2 DAD is an important determinant of mDia2 activity and that this signalling mechanism affects actin polymerization and smooth muscle cell-specific gene expression.