Structural and Functional Analysis of a ß2-Adrenergic Receptor Complex with GRK5.

The phosphorylation of agonist-occupied G-protein-coupled receptors (GPCRs) by GPCR kinases (GRKs) functions to turn off G-protein signaling and turn on arrestin-mediated signaling. While a structural understanding of GPCR/G-protein and GPCR/arrestin complexes has emerged in recent years, the molecular architecture of a GPCR/GRK complex remains poorly defined. We used a comprehensive integrated approach of cross-linking, hydrogen-deuterium exchange mass spectrometry (MS), electron microscopy, mutagenesis, molecular dynamics simulations, and computational docking to analyze GRK5 interaction with the ß2-adrenergic receptor (ß2AR). These studies revealed a dynamic mechanism of complex formation that involves large conformational changes in the GRK5 RH/catalytic domain interface upon receptor binding. These changes facilitate contacts between intracellular loops 2 and 3 and the C terminus of the ß2AR with the GRK5 RH bundle subdomain, membrane-binding surface, and kinase catalytic cleft, respectively. These studies significantly contribute to our understanding of the mechanism by which GRKs regulate the function of activated GPCRs. PAPERCLIP.

Structure of a GRK5-Calmodulin Complex Reveals Molecular Mechanism of GRK Activation and Substrate Targeting.

The phosphorylation of G protein-coupled receptors (GPCRs) by GPCR kinases (GRKs) facilitates arrestin binding and receptor desensitization. Although this process can be regulated by Ca2+-binding proteins such as calmodulin (CaM) and recoverin, the molecular mechanisms are poorly understood. Here, we report structural, computational, and biochemical analysis of a CaM complex with GRK5, revealing how CaM shapes GRK5 response to calcium. The CaM N and C domains bind independently to two helical regions at the GRK5 N and C termini to inhibit GPCR phosphorylation, though only the C domain interaction disrupts GRK5 membrane association, thereby facilitating cytoplasmic translocation. The CaM N domain strongly activates GRK5 via ordering of the amphipathic αN-helix of GRK5 and allosteric disruption of kinase-RH domain interaction for phosphorylation of cytoplasmic GRK5 substrates. These results provide a framework for understanding how two functional effects, GRK5 activation and localization, can cooperate under control of CaM for selective substrate targeting by GRK5.

G-protein-coupled receptors signal victory.

The 2012 Nobel Prize in Chemistry has been awarded to Brian K. Kobilka and Robert J. Lefkowitz for their studies of G-protein-coupled receptors. Their pioneering work over the past 40 years has provided detailed molecular insight into the structure and function of this fundamentally important family of receptors.

The Role of G Protein-Coupled Receptors and Receptor Kinases in Pancreatic ß-Cell Function and Diabetes.

Type 2 diabetes (T2D) mellitus has emerged as a major global health concern that has accelerated in recent years due to poor diet and lifestyle. Afflicted individuals have high blood glucose levels that stem from the inability of the pancreas to make enough insulin to meet demand. Although medication can help to maintain normal blood glucose levels in individuals with chronic disease, many of these medicines are outdated, have severe side effects, and often become less efficacious over time, necessitating the need for insulin therapy. G protein-coupled receptors (GPCRs) regulate many physiologic processes, including blood glucose levels. In pancreatic ß cells, GPCRs regulate ß-cell growth, apoptosis, and insulin secretion, which are all critical in maintaining sufficient ß-cell mass and insulin output to ensure euglycemia. In recent years, new insights into the signaling of incretin receptors and other GPCRs have underscored the potential of these receptors as desirable targets in the treatment of diabetes. The signaling of these receptors is modulated by GPCR kinases (GRKs) that phosphorylate agonist-activated GPCRs, marking the receptor for arrestin binding and internalization. Interestingly, genome-wide association studies using diabetic patient cohorts link the GRKs and arrestins with T2D. Moreover, recent reports show that GRKs and arrestins expressed in the ß cell serve a critical role in the regulation of ß-cell function, including ß-cell growth and insulin secretion in both GPCR-dependent and -independent pathways. In this review, we describe recent insights into GPCR signaling and the importance of GRK function in modulating ß-cell physiology. SIGNIFICANCE STATEMENT: Pancreatic ß cells contain a diverse array of G protein-coupled receptors (GPCRs) that have been shown to improve ß-cell function and survival, yet only a handful have been successfully targeted in the treatment of diabetes. This review discusses recent advances in our understanding of ß-cell GPCR pharmacology and regulation by GPCR kinases while also highlighting the necessity of investigating islet-enriched GPCRs that have largely been unexplored to unveil novel treatment strategies.

Identification of a ß-arrestin-biased negative allosteric modulator for the ß2-adrenergic receptor.

Catecholamine-stimulated ß2-adrenergic receptor (ß2AR) signaling via the canonical Gs-adenylyl cyclase-cAMP-PKA pathway regulates numerous physiological functions, including the therapeutic effects of exogenous ß-agonists in the treatment of airway disease. ß2AR signaling is tightly regulated by GRKs and ß-arrestins, which together promote ß2AR desensitization and internalization as well as downstream signaling, often antithetical to the canonical pathway. Thus, the ability to bias ß2AR signaling toward the Gs pathway while avoiding ß-arrestin-mediated effects may provide a strategy to improve the functional consequences of ß2AR activation. Since attempts to develop Gs-biased agonists and allosteric modulators for the ß2AR have been largely unsuccessful, here we screened small molecule libraries for allosteric modulators that selectively inhibit ß-arrestin recruitment to the receptor. This screen identified several compounds that met this profile, and, of these, a difluorophenyl quinazoline (DFPQ) derivative was found to be a selective negative allosteric modulator of ß-arrestin recruitment to the ß2AR while having no effect on ß2AR coupling to Gs. DFPQ effectively inhibits agonist-promoted phosphorylation and internalization of the ß2AR and protects against the functional desensitization of ß-agonist mediated regulation in cell and tissue models. The effects of DFPQ were also specific to the ß2AR with minimal effects on the ß1AR. Modeling, mutagenesis, and medicinal chemistry studies support DFPQ derivatives binding to an intracellular membrane-facing region of the ß2AR, including residues within transmembrane domains 3 and 4 and intracellular loop 2. DFPQ thus represents a class of biased allosteric modulators that targets an allosteric site of the ß2AR.

Kinome profiling identifies MARK3 and STK10 as potential therapeutic targets in uveal melanoma.

Most uveal melanoma cases harbor activating mutations in either GNAQ or GNA11. Despite activation of the mitogen-activated protein kinase (MAPK) signaling pathway downstream of Gαq/11, there are no effective targeted kinase therapies for metastatic uveal melanoma. The human genome encodes numerous understudied kinases, also called the "dark kinome". Identifying additional kinases regulated by Gαq/11 may uncover novel therapeutic targets for uveal melanoma. In this study, we treated GNAQ-mutant uveal melanoma cell lines with a Gαq/11 inhibitor, YM-254890, and conducted a kinase signaling proteomic screen using multiplexed-kinase inhibitors followed by mass spectrometry. We observed downregulated expression and/or activity of 22 kinases. A custom siRNA screen targeting these kinases demonstrated that knockdown of microtubule affinity regulating kinase 3 (MARK3) and serine/threonine kinase 10 (STK10) significantly reduced uveal melanoma cell growth and decreased expression of cell cycle proteins. Additionally, knockdown of MARK3 but not STK10 decreased ERK1/2 phosphorylation. Analysis of RNA-sequencing and proteomic data showed that Gαq signaling regulates STK10 expression and MARK3 activity. Our findings suggest an involvement of STK10 and MARK3 in the Gαq/11 oncogenic pathway and prompt further investigation into the specific roles and targeting potential of these kinases in uveal melanoma.

Characterization of a new WHIM syndrome mutant reveals mechanistic differences in regulation of the chemokine receptor CXCR4.

WHIM syndrome is a rare immunodeficiency disorder that is characterized by warts, hypogammaglobulinemia, infections, and myelokathexis. While several gain-of-function mutations that lead to C-terminal truncations, frame shifts and point mutations in the chemokine receptor CXCR4 have been identified in WHIM syndrome patients, the functional effect of these mutations are not fully understood. Here, we report on a new WHIM syndrome mutation that results in a frame shift within the codon for Ser339 (S339fs5) and compare the properties of S339fs5 with wild-type CXCR4 and a previously identified WHIM syndrome mutant, R334X. The S339fs5 and R334X mutants exhibited significantly increased signaling compared to wild-type CXCR4 including agonist-promoted calcium flux and extracellular-signal-regulated kinase activation. This increase is at least partially due to a significant decrease in agonist-promoted phosphorylation, ß-arrestin binding, and endocytosis of S339fs5 and R334X compared with wild-type CXCR4. Interestingly, there were also significant differences in receptor degradation, with S339fs5 having a very high basal level of degradation compared with that of R334X and wild-type CXCR4. In contrast to wild-type CXCR4, both R334X and S339fs5 were largely insensitive to CXCL12-promoted degradation. Moreover, while basal and agonist-promoted degradation of wild-type CXCR4 was effectively inhibited by the CXCR4 antagonist TE-14016, this had no effect on the degradation of the WHIM mutants. Taken together, these studies identify a new WHIM syndrome mutant, CXCR4-S339fs5, which promotes enhanced signaling, reduced phosphorylation, ß-arrestin binding and endocytosis, and a very high basal rate of degradation that is not protected by antagonist treatment.

G protein-coupled receptor kinase 6 (GRK6) regulates insulin processing and secretion via effects on proinsulin conversion to insulin.

Recent studies identified a missense mutation in the gene coding for G protein-coupled receptor kinase 6 (GRK6) that segregates with type 2 diabetes (T2D). To better understand how GRK6 might be involved in T2D, we used pharmacological inhibition and genetic knockdown in the mouse ß-cell line, MIN6, to determine whether GRK6 regulates insulin dynamics. We show inhibition of GRK5 and GRK6 increased insulin secretion but reduced insulin processing while GRK6 knockdown revealed these same processing defects with reduced levels of cellular insulin. GRK6 knockdown cells also had attenuated insulin secretion but enhanced proinsulin secretion consistent with decreased processing. In support of these findings, we demonstrate GRK6 rescue experiments in knockdown cells restored insulin secretion after glucose treatment. The altered insulin profile appears to be caused by changes in the proprotein convertases, the enzymes responsible for proinsulin to insulin conversion, as GRK6 knockdown resulted in significantly reduced convertase expression and activity. To identify how the GRK6-P384S mutation found in T2D patients might affect insulin processing, we performed biochemical and cell biological assays to study the properties of the mutant. We found that while GRK6-P384S was more active than WT GRK6, it displayed a cytosolic distribution in cells compared to the normal plasma membrane localization of GRK6. Additionally, GRK6 overexpression in MIN6 cells enhanced proinsulin processing, while GRK6-P384S expression had little effect. Taken together, our data show that GRK6 regulates insulin processing and secretion in a glucose-dependent manner and provide a foundation for understanding the contribution of GRK6 to T2D.

Pepducin ICL1-9-Mediated ß2-Adrenergic Receptor-Dependent Cardiomyocyte Contractility Occurs in a Gi Protein/ROCK/PKD-Sensitive Manner.

PURPOSE: ß-Adrenergic receptors (ßAR) are essential targets for the treatment of heart failure (HF); however, chronic use of ßAR agonists as positive inotropes to increase contractility in a Gs protein-dependent manner is associated with increased mortality. Alternatively, we previously reported that allosteric modulation of ß2AR with the pepducin intracellular loop (ICL)1-9 increased cardiomyocyte contractility in a ß-arrestin (ßarr)-dependent manner, and subsequently showed that ICL1-9 activates the Ras homolog family member A (RhoA). Here, we aimed to elucidate both the proximal and downstream signaling mediators involved in the promotion of cardiomyocyte contractility in response to ICL1-9. METHODS: We measured adult mouse cardiomyocyte contractility in response to ICL1-9 or isoproterenol (ISO, as a positive control) alone or in the presence of inhibitors of various potential components of ßarr- or RhoA-dependent signaling. We also assessed the contractile effects of ICL1-9 on cardiomyocytes lacking G protein-coupled receptor (GPCR) kinase 2 (GRK2) or 5 (GRK5). RESULTS: Consistent with RhoA activation by ICL1-9, both Rho-associated protein kinase (ROCK) and protein kinase D (PKD) inhibition were able to attenuate ICL1-9-mediated contractility, as was inhibition of myosin light chain kinase (MLCK). While neither GRK2 nor GRK5 deletion impacted ICL1-9-mediated contractility, pertussis toxin attenuated the response, suggesting that ICL1-9 promotes downstream RhoA-dependent signaling in a Gi protein-dependent manner. CONCLUSION: Altogether, our study highlights a novel signaling modality that may offer a new approach to the promotion, or preservation, of cardiac contractility during HF via the allosteric regulation of ß2AR to promote Gi protein/ßarr-dependent activation of RhoA/ROCK/PKD signaling.

Emerging Paradigm of Intracellular Targeting of G Protein-Coupled Receptors.

G protein-coupled receptors (GPCRs) recognize a diverse array of extracellular stimuli, and they mediate a broad repertoire of signaling events involved in human physiology. Although the major effort on targeting GPCRs has typically been focused on their extracellular surface, a series of recent developments now unfold the possibility of targeting them from the intracellular side as well. Allosteric modulators binding to the cytoplasmic surface of GPCRs have now been described, and their structural mechanisms are elucidated by high-resolution crystal structures. Furthermore, pepducins, aptamers, and intrabodies targeting the intracellular face of GPCRs have also been successfully utilized to modulate receptor signaling. Moreover, small molecule compounds, aptamers, and synthetic intrabodies targeting ß-arrestins have also been discovered to modulate GPCR endocytosis and signaling. Here, we discuss the emerging paradigm of intracellular targeting of GPCRs, and outline the current challenges, potential opportunities, and future outlook in this particular area of GPCR biology.

PD 102807 Induces M3 mAChR-Dependent GRK-/Arrestin-Biased Signaling in Airway Smooth Muscle Cells.

G protein-coupled receptors (GPCRs) not only are turned on or off to control canonical G protein signaling but also may be fine-tuned to promote qualitative/biased signaling. Qualitative signaling by M3 muscarinic acetylcholine receptors (mAChRs) has been proposed, but its impact on physiologic systems remains unclear, and currently no biased M3 mAChR ligands have been described. Herein, we identify PD 102807 as a biased M3 ligand and delineate its signaling and function in human airway smooth muscle (ASM) cells. PD 102807 induced M3-mediated ß-arrestin recruitment but not calcium mobilization. PD 102807 inhibited methacholine (MCh)-induced calcium mobilization in (M3-expressing) ASM cells. PD 102807 induced phosphorylation of AMP-activated protein kinase (AMPK) and the downstream effector acetyl-coenzyme A carboxylase (ACC). PD 102807- induced phosphorylated (p)-AMPK levels were greatly reduced in ASM cells with minimal M3 expression and were not inhibited by the Gq inhibitor YM-254890. Induction of p-AMPK and p-ACC was inhibited by ß-arrestin 1 or GRK2/3 knockdown. Similarly, MCh induced phosphorylation of AMPK/ACC, but these effects were Gq dependent and unaffected by GRK2/3 knockdown. Consistent with the known ability of AMPK to inhibit transforming growth factor ß (TGF-ß)-mediated functions, PD 102807 inhibited TGF-ß-induced SMAD-Luc activity, sm-α-actin expression, actin stress fiber formation, and ASM cell hypercontractility. These findings reveal that PD 102807 is a biased M3 ligand that inhibits M3-transduced Gq signaling but promotes Gq protein-independent, GRK-/arrestin-dependent, M3-mediated AMPK signaling, which in turn regulates ASM phenotype and contractile function. Consequently, biased M3 ligands hold significant promise as therapeutic agents capable of exploiting the pleiotropic nature of M3 signaling.

Phenylalanine 193 in Extracellular Loop 2 of the ß 2-Adrenergic Receptor Coordinates ß-Arrestin Interaction.

G protein-coupled receptors (GPCRs) transduce a diverse variety of extracellular stimuli into intracellular signaling. These receptors are the most clinically productive drug targets at present. Despite decades of research on the signaling consequences of molecule-receptor interactions, conformational components of receptor-effector interactions remain incompletely described. The ß 2-adrenergic receptor (ß 2AR) is a prototypical and extensively studied GPCR that can provide insight into this aspect of GPCR signaling thanks to robust structural data and rich pharmacopeia. Using bioluminescence resonance energy transfer -based biosensors, second messenger assays, and biochemical techniques, we characterize the properties of ß 2AR-F193A. This single point mutation in extracellular loop 2 of the ß 2AR is sufficient to intrinsically bias the ß 2AR away from ß-arrestin interaction and demonstrates altered regulatory outcomes downstream of this functional selectivity. This study highlights the importance of extracellular control of intracellular response to stimuli and suggests a previously undescribed role for the extracellular loops of the receptor and the extracellular pocket formed by transmembrane domains 2, 3, and 7 in GPCR regulation that may contribute to biased signaling at GPCRs. SIGNIFICANCE STATEMENT: The role of extracellular G protein-coupled receptor (GPCR) domains in mediating intracellular interactions is poorly understood. We characterized the effects of extracellular loop mutations on agonist-promoted interactions of GPCRs with G protein and ß-arrestin. Our studies reveal that F193 in extracellular loop 2 in the ß2-adrenergic receptor mediates interactions with G protein and ß-arrestin with a biased loss of ß-arrestin binding. These results provide new insights on the role of the extracellular domain in differentially modulating intracellular interactions with GPCRs.

Biased signaling of the proton-sensing receptor OGR1 by benzodiazepines.

GPCRs have diverse signaling capabilities, based on their ability to assume various conformations. Moreover, it is now appreciated that certain ligands can promote distinct receptor conformations and thereby bias signaling toward a specific pathway to differentially affect cell function. The recently deorphanized G protein-coupled receptor OGR1 [ovarian cancer G protein-coupled receptor 1 ( GPR68)] exhibits diverse signaling events when stimulated by reductions in extracellular pH. We recently demonstrated airway smooth muscle cells transduce multiple signaling events, reflecting a diverse capacity to couple to multiple G proteins. Moreover, we recently discovered that the benzodiazepine lorazepam, more commonly recognized as an agonist of the γ-aminobutyric acid A (GABAA) receptor, can function as an allosteric modulator of OGR1 and, similarly, can promote multiple signaling events. In this study, we demonstrated that different benzodiazepines exhibit a range of biases for OGR1, with sulazepam selectively activating the canonical Gs of the G protein signaling pathway, in heterologous expression systems, as well as in several primary cell types. These findings highlight the potential power of biased ligand pharmacology for manipulating receptor signaling qualitatively, to preferentially activate pathways that are therapeutically beneficial.-Pera, T., Deshpande, D. A., Ippolito, M., Wang, B., Gavrila, A., Michael, J. V., Nayak, A. P., Tompkins, E., Farrell, E., Kroeze, W. K., Roth, B. L., Panettieri, R. A. Jr Benovic, J. L., An, S. S., Dulin, N. O., Penn, R. B. Biased signaling of the proton-sensing receptor OGR1 by benzodiazepines.

G protein-coupled receptor kinase 5 modifies cancer cell resistance to paclitaxel.

G protein-coupled receptor kinases (GRKs) phosphorylate the activated forms of G protein-coupled receptors (GPCRs), leading to receptor desensitization and internalization. In addition, GRKs can modify the activity of many non-GPCR-signaling pathways as well, controlling other cellular functions beyond that directly associated with a GPCR. In this report, we show that cervical cancer HeLa cells and breast cancer MDA MB 231 cells with reduced GRK5 expression display increased sensitivity to the apoptotic effects of paclitaxel (Taxol). This effect in cancer cells with low GRK5 levels could be because of blunted histone deacetylase 6 (HDAC6) activity that leads to an increase in α-tubulin acetylation levels, which augments paclitaxel sensitivity. We demonstrate that GRK5 and HDAC6 form a signaling complex in cells and in vitro. GRK5 phosphorylates HDAC6 at Ser-21 to promote its deacetylase activity. Therefore, the GRK5-HDAC6 interaction may contribute to paclitaxel resistance in cancer cells.

ß-arrestin-biased signaling through the ß2-adrenergic receptor promotes cardiomyocyte contraction.

ß-adrenergic receptors (ßARs) are critical regulators of acute cardiovascular physiology. In response to elevated catecholamine stimulation during development of congestive heart failure (CHF), chronic activation of Gs-dependent ß1AR and Gi-dependent ß2AR pathways leads to enhanced cardiomyocyte death, reduced ß1AR expression, and decreased inotropic reserve. ß-blockers act to block excessive catecholamine stimulation of ßARs to decrease cellular apoptotic signaling and normalize ß1AR expression and inotropy. Whereas these actions reduce cardiac remodeling and mortality outcomes, the effects are not sustained. Converse to G-protein-dependent signaling, ß-arrestin-dependent signaling promotes cardiomyocyte survival. Given that ß2AR expression is unaltered in CHF, a ß-arrestin-biased agonist that operates through the ß2AR represents a potentially useful therapeutic approach. Carvedilol, a currently prescribed nonselective ß-blocker, has been classified as a ß-arrestin-biased agonist that can inhibit basal signaling from ßARs and also stimulate cell survival signaling pathways. To understand the relative contribution of ß-arrestin bias to the efficacy of select ß-blockers, a specific ß-arrestin-biased pepducin for the ß2AR, intracellular loop (ICL)1-9, was used to decouple ß-arrestin-biased signaling from occupation of the orthosteric ligand-binding pocket. With similar efficacy to carvedilol, ICL1-9 was able to promote ß2AR phosphorylation, ß-arrestin recruitment, ß2AR internalization, and ß-arrestin-biased signaling. Interestingly, ICL1-9 was also able to induce ß2AR- and ß-arrestin-dependent and Ca(2+)-independent contractility in primary adult murine cardiomyocytes, whereas carvedilol had no efficacy. Thus, ICL1-9 is an effective tool to access a pharmacological profile stimulating cardioprotective signaling and inotropic effects through the ß2AR and serves as a model for the next generation of cardiovascular drug development.

G protein-coupled receptor kinase-2 (GRK-2) regulates serotonin metabolism through the monoamine oxidase AMX-2 in Caenorhabditis elegans.

G protein-coupled receptors (GPCRs) regulate many animal behaviors. GPCR signaling is mediated by agonist-promoted interactions of GPCRs with heterotrimeric G proteins, GPCR kinases (GRKs), and arrestins. To further elucidate the role of GRKs in regulating GPCR-mediated behaviors, we utilized the genetic model system Caenorhabditis elegans Our studies demonstrate that grk-2 loss-of-function strains are egg laying-defective and contain low levels of serotonin (5-HT) and high levels of the 5-HT metabolite 5-hydroxyindole acetic acid (5-HIAA). The egg laying defect could be rescued by the expression of wild type but not by catalytically inactive grk-2 or by the selective expression of grk-2 in hermaphrodite-specific neurons. The addition of 5-HT or inhibition of 5-HT metabolism also rescued the egg laying defect. Furthermore, we demonstrate that AMX-2 is the primary monoamine oxidase that metabolizes 5-HT in C. elegans, and we also found that grk-2 loss-of-function strains have abnormally high levels of AMX-2 compared with wild-type nematodes. Interestingly, GRK-2 was also found to interact with and promote the phosphorylation of AMX-2. Additional studies reveal that 5-HIAA functions to inhibit egg laying in a manner dependent on the 5-HT receptor SER-1 and the G protein GOA-1. These results demonstrate that GRK-2 modulates 5-HT metabolism by regulating AMX-2 function and that 5-HIAA may function in the SER-1 signaling pathway.

Identification of the Rac-GEF P-Rex1 as an essential mediator of ErbB signaling in breast cancer.

While the small GTPase Rac1 and its effectors are well-established mediators of mitogenic and motile signaling by tyrosine kinase receptors and have been implicated in breast tumorigenesis, little is known regarding the exchange factors (Rac-GEFs) that mediate ErbB receptor responses. Here, we identify the PIP(3)-Gßγ-dependent Rac-GEF P-Rex1 as an essential mediator of Rac1 activation, motility, cell growth, and tumorigenesis driven by ErbB receptors in breast cancer cells. Notably, activation of P-Rex1 in breast cancer cells requires the convergence of inputs from ErbB receptors and a Gßγ- and PI3Kγ-dependent pathway. Moreover, we identified the GPCR CXCR4 as a crucial mediator of P-Rex1/Rac1 activation in response to ErbB ligands. P-Rex1 is highly overexpressed in human breast cancers and their derived cell lines, particularly those with high ErbB2 and ER expression. In addition to the prognostic and therapeutic implications, our findings reveal an ErbB effector pathway that is crucial for breast cancer progression.

G Protein-Coupled Receptor Kinase 3 and Protein Kinase C Phosphorylate the Distal C-Terminal Tail of the Chemokine Receptor CXCR4 and Mediate Recruitment of ß-Arrestin.

Phosphorylation of G protein-coupled receptors (GPCRs) is a key event for cell signaling and regulation of receptor function. Previously, using tandem mass spectrometry, we identified two phosphorylation sites at the distal C-terminal tail of the chemokine receptor CXCR4, but were unable to determine which specific residues were phosphorylated. Here, we demonstrate that serines (Ser) 346 and/or 347 (Ser-346/7) of CXCR4 are phosphorylated upon stimulation with the agonist CXCL12 as well as a CXCR4 pepducin, ATI-2341. ATI-2341, a Gαißγ heterotrimer-biased CXCR4 agonist, induced more robust phosphorylation of Ser-346/7 compared with CXCL12. Knockdown of G protein-coupled receptor kinase (GRK) 2, GRK3, or GRK6 reduced CXCL12-induced phosphorylation of Ser-346/7 with GRK3 knockdown having the strongest effect, while inhibition of the conventional protein kinase C (PKC) isoforms, particularly PKCα, reduced phosphorylation of Ser-346/7 induced by either CXCL12 or ATI-2341. The loss of GRK3- or PKC-mediated phosphorylation of Ser-346/7 impaired the recruitment of ß-arrestin to CXCR4. We also found that a pseudo-substrate peptide inhibitor for PKCζ effectively inhibited CXCR4 phosphorylation and signaling, most likely by functioning as a nonspecific CXCR4 antagonist. Together, these studies demonstrate the role Ser-346/7 plays in arrestin recruitment and initiation of receptor desensitization and provide insight into the dysregulation of CXCR4 observed in patients with various forms of WHIM syndrome.

The α-Arrestin ARRDC3 Regulates the Endosomal Residence Time and Intracellular Signaling of the ß2-Adrenergic Receptor.

Arrestin domain-containing protein 3 (ARRDC3) is a member of the mammalian α-arrestin family, which is predicted to share similar tertiary structure with visual-/ß-arrestins and also contains C-terminal PPXY motifs that mediate interaction with E3 ubiquitin ligases. Recently, ARRDC3 has been proposed to play a role in regulating the trafficking of G protein-coupled receptors, although mechanistic insight into this process is lacking. Here, we focused on characterizing the role of ARRDC3 in regulating the trafficking of the ß2-adrenergic receptor (ß2AR). We find that ARRDC3 primarily localizes to EEA1-positive early endosomes and directly interacts with the ß2AR in a ligand-independent manner. Although ARRDC3 has no effect on ß2AR endocytosis or degradation, it negatively regulates ß2AR entry into SNX27-occupied endosomal tubules. This results in delayed recycling of the receptor and a concomitant increase in ß2AR-dependent endosomal signaling. Thus, ARRDC3 functions as a switch to modulate the endosomal residence time and subsequent intracellular signaling of the ß2AR.

c-Src, Insulin-Like Growth Factor I Receptor, G-Protein-Coupled Receptor Kinases and Focal Adhesion Kinase are Enriched Into Prostate Cancer Cell Exosomes.

It is well known that Src tyrosine kinase, insulin-like growth factor 1 receptor (IGF-IR), and focal adhesion kinase (FAK) play important roles in prostate cancer (PrCa) development and progression. Src, which signals through FAK in response to integrin activation, has been implicated in many aspects of tumor biology, such as cell proliferation, metastasis, and angiogenesis. Furthermore, Src signaling is known to crosstalk with IGF-IR, which also promotes angiogenesis. In this study, we demonstrate that c-Src, IGF-IR, and FAK are packaged into exosomes (Exo), c-Src in particular being highly enriched in Exo from the androgen receptor (AR)-positive cell line C4-2B and AR-negative cell lines PC3 and DU145. Furthermore, we show that the active phosphorylated form of Src (SrcpY416 ) is co-expressed in Exo with phosphorylated FAK (FAKpY861 ), a known target site of Src, which enhances proliferation and migration. We further demonstrate for the first time exosomal enrichment of G-protein-coupled receptor kinase (GRK) 5 and GRK6, both of which regulate Src and IGF-IR signaling and have been implicated in cancer. Finally, SrcpY416 and c-Src are both expressed in Exo isolated from the plasma of prostate tumor-bearing TRAMP mice, and those same mice have higher levels of exosomal c-Src than their wild-type counterparts. In summary, we provide new evidence that active signaling molecules relevant to PrCa are enriched in Exo, and this suggests that the Src signaling network may provide useful biomarkers detectable by liquid biopsy, and may contribute to PrCa progression via Exo. J. Cell. Biochem. 118: 66-73, 2017. © 2016 Wiley Periodicals, Inc.