Structural basis of CBP/p300 recruitment by the microphthalmia-associated transcription factor.

The microphthalmia-associated transcription factor (MITF) is a master regulator of the melanocyte cell lineage. Aberrant MITF activity can lead to multiple malignancies including skin cancer, where it modulates the progression and invasiveness of melanoma. MITF-regulated gene expression requires recruitment of the transcriptional co-regulator CBP/p300, but details of this process are not fully defined. In this study, we investigate the structural and functional interaction between the MITF N-terminal transactivation domain (MITFTAD) and CBP/p300. Using pulldown assays and nuclear magnetic resonance spectroscopy we determined that MITFTAD is intrinsically disordered and binds to the TAZ1 and TAZ2 domains of CBP/p300 with moderate affinity. The solution-state structure of the MITFTAD:TAZ2 complex reveals that MITF interacts with a hydrophobic surface of TAZ2, while remaining somewhat dynamic. Peptide array and mutagenesis experiments determined that an acidic motif is integral to the MITFTAD:TAZ2 interaction and is necessary for transcriptional activity of MITF. Peptides that bind to the same surface of TAZ2 as MITFTAD, such as the adenoviral protein E1A, are capable of displacing MITF from TAZ2 and inhibiting transactivation. These findings provide insight into co-activator recruitment by MITF that are fundamental to our understanding of MITF targeted gene regulation and melanoma biology.

Anticancer properties of cannabidiol and Δ9-tetrahydrocannabinol and synergistic effects with gemcitabine and cisplatin in bladder cancer cell lines.

INTRODUCTION: With the legalization of cannabis in multiple jurisdictions throughout the world, a larger proportion of the population consumes cannabis. Several studies have demonstrated anti-tumor effects of components present in cannabis in different models. Unfortunately, little is known about the potential anti-tumoral effects of cannabinoids in bladder cancer and how cannabinoids could potentially synergize with chemotherapeutic agents. Our study aims to identify whether a combination of cannabinoids, like cannabidiol and Δ9-tetrahydrocannabinol, with agents commonly used to treat bladder cancer, such as gemcitabine and cisplatin, can produce desirable synergistic effects. We also evaluated if co-treatment with different cannabinoids resulted in synergistic effects. METHODS: We generated concentration curves with several drugs, including several cannabinoids, to identify the range at which they could exert anti-tumor effects in bladder cancer cell lines. We tested the cytotoxic effects of gemcitabine (up to 100 nM), cisplatin (up to 100 µM), and cannabinoids (up to 10 µM) in T24 and TCCSUP cells. We also evaluated the activation of the apoptotic cascade and whether cannabinoids have the ability to reduce invasion in T24 cells. RESULTS: Cannabidiol, Δ9-tetrahydrocannabinol, cannabichromene, and cannabivarin reduce cell viability of bladder cancer cell lines, and their combination with gemcitabine or cisplatin may induce differential responses, from antagonistic to additive and synergistic effects, depending on the concentrations used. Cannabidiol and Δ9-tetrahydrocannabinol were also shown to induce apoptosis via caspase-3 cleavage and reduce invasion in a Matrigel assay. Cannabidiol and Δ9-tetrahydrocannabinol also display synergistic properties with other cannabinoids like cannabichromene or cannabivarin, although individual cannabinoids may be sufficient to reduce cell viability of bladder cancer cell lines. DISCUSSION: Our results indicate that cannabinoids can reduce human bladder transitional cell carcinoma cell viability, and that they can potentially exert synergistic effects when combined with other agents. Our in vitro results will form the basis for future studies in vivo and in clinical trials for the development of new therapies that could be beneficial for the treatment of bladder cancer in the future.

ß-Catenin interacts with the TAZ1 and TAZ2 domains of CBP/p300 to activate gene transcription.

The transcriptional co-regulator ß-catenin is a critical member of the canonical Wnt signaling pathway, which plays an important role in regulating cell fate. Deregulation of the Wnt/ß-catenin pathway is characteristic in the development of major types of cancer, where accumulation of ß-catenin promotes cancer cell proliferation and renewal. ß-catenin gene expression is facilitated through recruitment of co-activators such as histone acetyltransferases CBP/p300; however, the mechanism of their interaction is not fully understood. Here we investigate the interaction between the C-terminal transactivation domain of ß-catenin and CBP/p300. Using a combination of pulldown assays, isothermal titration calorimetry, and nuclear resonance spectroscopy we determine the disordered C-terminal region of ß-catenin binds promiscuously to the TAZ1 and TAZ2 domains of CBP/p300. We then map the interaction site of the C-terminal ß-catenin transactivation domain onto TAZ1 and TAZ2 using chemical-shift perturbation studies. Luciferase-based gene reporter assays indicate Asp750-Leu781 is critical to ß-catenin gene activation, and mutagenesis revealed that acidic and hydrophobic residues within this region are necessary to maintain TAZ1 binding. These results outline a mechanism of Wnt/ß-catenin gene regulation that underlies cell development and provides a framework to develop methods to block ß-catenin dependent signaling.

Anti-cancer properties of cannflavin A and potential synergistic effects with gemcitabine, cisplatin, and cannabinoids in bladder cancer.

INTRODUCTION: Several studies have shown anti-tumor effects of components present in cannabis in different models. Unfortunately, little is known about the potential anti-tumoral effects of most compounds present in cannabis in bladder cancer and how these compounds could potentially positively or negatively impact the actions of chemotherapeutic agents. Our study aims to evaluate the effects of a compound found in Cannabis sativa that has not been extensively studied to date, cannflavin A, in bladder cancer cell lines. We aimed to identify whether cannflavin A co-treatment with agents commonly used to treat bladder cancer, such as gemcitabine and cisplatin, is able to produce synergistic effects. We also evaluated whether co-treatment of cannflavin A with various cannabinoids could produce synergistic effects. METHODS: Two transitional cell carcinoma cell lines were used to assess the cytotoxic effects of the flavonoid cannflavin A up to 100 µM. We tested the potential synergistic cytotoxic effects of cannflavin A with gemcitabine (up to 100 nM), cisplatin (up to 100 µM), and cannabinoids (up to 10 µM). We also evaluated the activation of the apoptotic cascade using annexin V and whether cannflavin A has the ability to reduce invasion using a Matrigel assay. RESULTS: Cell viability of bladder cancer cell lines was affected in a concentration-dependent fashion in response to cannflavin A, and its combination with gemcitabine or cisplatin induced differential responses-from antagonistic to additive-and synergism was also observed in some instances, depending on the concentrations and drugs used. Cannflavin A also activated apoptosis via caspase 3 cleavage and was able to reduce invasion by 50%. Interestingly, cannflavin A displayed synergistic properties with other cannabinoids like Δ9-tetrahydrocannabinol, cannabidiol, cannabichromene, and cannabivarin in the bladder cancer cell lines. DISCUSSION: Our results indicate that compounds from Cannabis sativa other than cannabinoids, like the flavonoid cannflavin A, can be cytotoxic to human bladder transitional carcinoma cells and that this compound can exert synergistic effects when combined with other agents. In vivo studies will be needed to confirm the activity of cannflavin A as a potential agent for bladder cancer treatment.

Anti-cancer potential of cannabis terpenes in a Taxol-resistant model of breast cancer.

Chemotherapeutic resistance can limit breast cancer outcomes; therefore, the exploration of novel therapeutic options is warranted. Isolated compounds found in cannabis have previously been shown to exhibit anti-cancer effects, but little is known about their effects in resistant breast cancer. Our study aimed to evaluate the effects of terpenes found in cannabis in in vitro chemotherapy-resistant model of breast cancer. We aimed to identify whether five terpenes found in cannabis produced anti-cancer effects, and whether their effects were improved upon co-treatment with cannabinoids and flavonoids also found in cannabis. Nerolidol and ß-caryophyllene produced the greatest cytotoxic effects, activated the apoptotic cascade, and reduced cellular invasion. Combinations with the flavonoid kaempferol potentiated the cytotoxic effects of ocimene, terpinolene, and ß-myrcene. Combinations of nerolidol and Δ9-tetrahydrocannabinol or cannabidiol produced variable responses ranging from antagonism and additivity to synergy, depending on concentrations used. Our results indicate that cannabis terpenes, alone or combined with cannabinoids and flavonoids, produced anti-cancer effects in chemotherapy-resistant breast cancer cell lines. This study is a first step in the identification of compounds that could have therapeutic potential in the treatment of resistant breast cancer.

Polarity scaffolds signaling in epithelial cell permeability.

As an integral part of the innate immune system, the epithelial membrane is exposed to an array of insults that may trigger an immune response. One of the immune system's main functions is to regulate the level of communications between the mucosa and the lumen of various tissues. While it is clear that inhaled or ingested substances, or microorganisms may induce changes that affect the epithelial barrier in various ways, the proteins involved in the signaling cascades and physiological events leading to the regulation and maintenance of the barrier are not always well characterized. We review here some of the signaling components involved in regulating the barrier's paracellular permeability, and their potential effects on the activation of an immune response. While an effective immune response must be launched against pathogenic insults, tolerance must also be maintained for non-pathogenic antigens such as those in the commensal flora or for endogenous metabolites. Along with other members of the innate and adaptive immunity, the endocannabinoid system also plays an instrumental role in maintaining the balance between inflammation and tolerance. We discuss the potential effects of endo- and phytocannabinoids on epithelial permeability and how the dysregulation of this system could be involved in diseases and targeted for therapy.

Anti-Cancer Potential of Cannabinoids, Terpenes, and Flavonoids Present in Cannabis.

In recent years, and even more since its legalization in several jurisdictions, cannabis and the endocannabinoid system have received an increasing amount of interest related to their potential exploitation in clinical settings. Cannabinoids have been suggested and shown to be effective in the treatment of various conditions. In cancer, the endocannabinoid system is altered in numerous types of tumours and can relate to cancer prognosis and disease outcome. Additionally, cannabinoids display anticancer effects in several models by suppressing the proliferation, migration and/or invasion of cancer cells, as well as tumour angiogenesis. However, the therapeutic use of cannabinoids is currently limited to the treatment of symptoms and pain associated with chemotherapy, while their potential use as cytotoxic drugs in chemotherapy still requires validation in patients. Along with cannabinoids, cannabis contains several other compounds that have also been shown to exert anti-tumorigenic actions. The potential anti-cancer effects of cannabinoids, terpenes and flavonoids, present in cannabis, are explored in this literature review.

Antitumor Activity of Abnormal Cannabidiol and Its Analog O-1602 in Taxol-Resistant Preclinical Models of Breast Cancer.

Cannabinoids exhibit anti-inflammatory and antitumorigenic properties. Contrary to most cannabinoids present in the Cannabis plant, some, such as O-1602 and abnormal cannabidiol, have no or only little affinity to the CB1 or CB2 cannabinoid receptors and instead exert their effects through other receptors. Here, we investigated whether the synthetic regioisomers of cannabidiol, abnormal cannabidiol, and a closely related compound, O-1602, display antitumorigenic effects in cellular models of breast cancer and whether it could reduce tumorigenesis in vivo. Several studies have shown the effects of cannabinoids on chemotherapy-sensitive breast cancer cell lines, but less is known about the antitumorigenic effects of cannabinoids in chemotherapy-resistant cell lines. Paclitaxel-resistant MDA-MB-231 and MCF-7 breast cancer cell lines were used to study the effect of O-1602 and abnormal cannabidiol on viability, apoptosis, and migration. The effects of O-1602 and abnormal cannabidiol on cell viability were completely blocked by the combination of GPR55 and GPR18-specific siRNAs. Both O-1602 and abnormal cannabidiol decreased viability in paclitaxel-resistant breast cancer cells in a concentration-dependent manner through induction of apoptosis. The effect of these cannabinoids on tumor growth in vivo was studied in a zebrafish xenograft model. In this model, treatment with O-1602 and abnormal cannabidiol (2 µM) significantly reduced tumor growth. Our results suggest that atypical cannabinoids, like O-1602 and abnormal cannabidiol, exert antitumorigenic effects on paclitaxel-resistant breast cancer cells. Due to their lack of central sedation and psychoactive effects, these atypical cannabinoids could represent new leads for the development of additional anticancer treatments when resistance to conventional chemotherapy occurs during the treatment of breast and possibly other cancers.

Bitter taste receptors are expressed in human epithelial ovarian and prostate cancers cells and noscapine stimulation impacts cell survival.

Bitter taste receptors (Tas2Rs) are a subfamily of G-protein coupled receptors expressed not only in the oral cavity but also in several extra-oral tissues and disease states. Several natural bitter compounds from plants, such as bitter melon extract and noscapine, have displayed anti-cancer effects against various cancer types. In this study, we examined the prevalence of Tas2R subtype expression in several epithelial ovarian or prostate cancer cell lines, and the functionality of Tas2R14 was determined. qPCR analysis of five TAS2Rs demonstrated that mRNA expression often varies greatly in cancer cells in comparison to normal tissue. Using receptor-specific siRNAs, we also demonstrated that noscapine stimulation of ovarian cancer cells increased apoptosis in ovarian cancer cells in a receptor-dependent, but ROS-independent manner. This study furthers our understanding of the function of Tas2Rs in ovarian cancer by demonstrating that their activation has an impact on cell survival.

Bioactivity of the putative apelin proprotein expands the repertoire of apelin receptor ligands.

BACKGROUND: Apelin is a peptide ligand for a class A G-protein coupled receptor called the apelin receptor (AR or APJ) that regulates angiogenesis, the adipoinsular axis, and cardiovascular functions. Apelin has been shown to be bioactive as 13, 17, and 36 amino acid isoforms, C-terminal fragments of the putatively inactive 55-residue proprotein (proapelin or apelin-55). Although intracellular proprotein processing has been proposed, isolation of apelin-55 from colostrum and milk demonstrates potential for secretion prior to processing and the possibility of proapelin-AR interaction. METHODS: Apelin isoform activity and potency were compared by an In-Cell Western™ assay for ERK phosphorylation using a stably AR-transfected HEK293A cell line. Conformational comparison of apelin isoforms was carried out by circular dichroism and heteronuclear solution-state nuclear magnetic resonance spectroscopy. RESULTS: Apelin-55 is shown to activate the AR, with similar maximum ERK phophorylation response and potency to the shorter isoforms except for apelin-13, which exhibited a greater potency. Correlating to this shared activity, highly similar conformations are exhibited in all apelin isoforms for the shared C-terminal region responsible for receptor binding and activation. CONCLUSIONS: AR activation by all apelin isoforms likely hinges upon shared conformation and dynamics in the C-terminus, with apelin-55 providing an alternative bioactive isoform despite the addition of 19N-terminal residues relative to apelin-36. GENERAL SIGNIFICANCE: Beyond providing novel insight into the physiology of this system, re-annotation of proapelin to the bioactive apelin-55 isoform adds to the molecular toolkit for dissection of apelin-AR interactions and expands the repertoire of therapeutic targets for the apelinergic system.

Differential Contribution of Transmembrane Domains IV, V, VI, and VII to Human Angiotensin II Type 1 Receptor Homomer Formation.

G protein-coupled receptors (GPCRs) play an important role in drug therapy and represent one of the largest families of drug targets. The angiotensin II type 1 receptor (AT1R) is notable as it has a central role in the treatment of cardiovascular disease. Blockade of AT1R signaling has been shown to alleviate hypertension and improve outcomes in patients with heart failure. Despite this, it has become apparent that our initial understanding of AT1R signaling is oversimplified. There is considerable evidence to suggest that AT1R signaling is highly modified in the presence of receptor-receptor interactions, but there is very little structural data available to explain this phenomenon even with the recent elucidation of the AT1R crystal structure. The current study investigates the involvement of transmembrane domains in AT1R homomer assembly with the goal of identifying hydrophobic interfaces that contribute to receptor-receptor affinity. A recently published crystal structure of the AT1R was used to guide site-directed mutagenesis of outward-facing hydrophobic residues within the transmembrane region of the AT1R. Bioluminescence resonance energy transfer was employed to analyze how receptor mutation affects the assembly of AT1R homomers with a specific focus on hydrophobic residues. Mutations within transmembrane domains IV, V, VI, and VII had no effect on angiotensin-mediated ß-arrestin1 recruitment; however, they exhibited differential effects on the assembly of AT1R into oligomeric complexes. Our results demonstrate the importance of hydrophobic amino acids at the AT1R transmembrane interface and provide the first glimpse of the requirements for AT1R complex assembly.

Superoxide Dismutase 1 Regulation of CXCR4-Mediated Signaling in Prostate Cancer Cells is Dependent on Cellular Oxidative State.

BACKGROUND/AIMS: CXCL12, acting via one of its G protein-coupled receptors, CXCR4, is a chemoattractant for a broad range of cell types, including several types of cancer cells. Elevated expression of CXCR4, and its ligand CXCL12, play important roles in promoting cancer metastasis. Cancer cells have the potential for rapid and unlimited growth in an area that may have restricted blood supply, as oxidative stress is a common feature of solid tumors. Recent studies have reported that enhanced expression of cytosolic superoxide dismutase (SOD1), a critical enzyme responsible for regulation of superoxide radicals, may increase the aggressive and invasive potential of malignant cells in some cancers. METHODS: We used a variety of biochemical approaches and a prostate cancer cell line to study the effects of SOD1 on CXCR4 signaling. RESULTS: Here, we report a direct interaction between SOD1 and CXCR4. We showed that SOD1 interacts directly with the first intracellular loop (ICL1) of CXCR4 and that the CXCL12/CXCR4-mediated regulation of AKT activation, apoptosis and cell migration in prostate cancer (PCa) cells is differentially modulated under normal versus hypoxic conditions when SOD1 is present. CONCLUSIONS: This study highlights a potential new regulatory mechanism by which a sensor of the oxidative environment could directly regulate signal transduction of a receptor involved in cancer cell survival and migration.

Regulation of GPCR Anterograde Trafficking by Molecular Chaperones and Motifs.

G protein-coupled receptors (GPCRs) make up a superfamily of integral membrane proteins that respond to a wide variety of extracellular stimuli, giving them an important role in cell function and survival. They have also proven to be valuable targets in the fight against various diseases. As such, GPCR signal regulation has received considerable attention over the last few decades. With the amplitude of signaling being determined in large part by receptor density at the plasma membrane, several endogenous mechanisms for modulating GPCR expression at the cell surface have come to light. It has been shown that cell surface expression is determined by both exocytic and endocytic processes. However, the body of knowledge surrounding GPCR trafficking from the endoplasmic reticulum to the plasma membrane, commonly known as anterograde trafficking, has considerable room for growth. We focus here on the current paradigms of anterograde GPCR trafficking. We will discuss the regulatory role of both the general and "nonclassical private" chaperone systems in GPCR trafficking as well as conserved motifs that serve as modulators of GPCR export from the endoplasmic reticulum and Golgi apparatus. Together, these topics summarize some of the known mechanisms by which the cell regulates anterograde GPCR trafficking.

The apelin receptor: physiology, pathology, cell signalling, and ligand modulation of a peptide-activated class A GPCR.

The apelin receptor (AR or APJ) is a class A (rhodopsin-like) G-protein-coupled receptor with wide distribution throughout the human body. Activation of the AR by its cognate peptide ligand, apelin, induces diverse physiological effects including vasoconstriction and dilation, strengthening of heart muscle contractility, angiogenesis, and regulation of energy metabolism and fluid homeostasis. Recently, another endogenous peptidic activator of the AR, Toddler/ELABELA, was identified as having a crucial role in zebrafish (Danio rerio) embryonic development. The AR is also implicated in pathologies including cardiovascular disease, diabetes, obesity, and cancer, making it a promising therapeutic target. Despite its established importance, the precise roles of AR signalling remain poorly understood. Moreover, little is known about the mechanisms of peptide-AR activation. Additional complexity arises from modulation of the AR by 2 endogenous peptide ligands, both with multiple bioactive isoforms of variable length and distribution. The various apelin and Toddler/ELABELA isoforms may also produce distinct cellular effects. Further complexity arises through formation of functionally distinct heterodimers between the AR and other G-protein-coupled receptors. This minireview outlines key (patho)physiological actions of the AR, addresses what is known about signal transduction downstream of AR activation, and concludes by discussing unique properties of the endogenous peptidic ligands of the AR.

Type 1 cannabinoid receptor ligands display functional selectivity in a cell culture model of striatal medium spiny projection neurons.

Modulation of type 1 cannabinoid receptor (CB1) activity has been touted as a potential means of treating addiction, anxiety, depression, and neurodegeneration. Different agonists of CB1 are known to evoke varied responses in vivo. Functional selectivity is the ligand-specific activation of certain signal transduction pathways at a receptor that can signal through multiple pathways. To understand cannabinoid-specific functional selectivity, different groups have examined the effect of individual cannabinoids on various signaling pathways in heterologous expression systems. In the current study, we compared the functional selectivity of six cannabinoids, including two endocannabinoids (2-arachidonyl glycerol (2-AG) and anandamide (AEA)), two synthetic cannabinoids (WIN55,212-2 and CP55,940), and two phytocannabinoids (cannabidiol (CBD) and Δ(9)-tetrahydrocannabinol (THC)) on arrestin2-, Gα(i/o)-, Gßγ-, Gα(s)-, and Gα(q)-mediated intracellular signaling in the mouse STHdh(Q7/Q7) cell culture model of striatal medium spiny projection neurons that endogenously express CB1. In this system, 2-AG, THC, and CP55,940 were more potent mediators of arrestin2 recruitment than other cannabinoids tested. 2-AG, AEA, and WIN55,212-2, enhanced Gα(i/o) and Gßγ signaling, with 2-AG and AEA treatment leading to increased total CB1 levels. 2-AG, AEA, THC, and WIN55,212-2 also activated Gα(q)-dependent pathways. CP55,940 and CBD both signaled through Gα(s). CP55,940, but not CBD, activated downstream Gα(s) pathways via CB1 targets. THC and CP55,940 promoted CB1 internalization and decreased CB1 protein levels over an 18-h period. These data demonstrate that individual cannabinoids display functional selectivity at CB1 leading to activation of distinct signaling pathways. To effectively match cannabinoids with therapeutic goals, these compounds must be screened for their signaling bias.

Anterograde trafficking of CXCR4 and CCR2 receptors in a prostate cancer cell line.

BACKGROUND: Most prostate cancer-related deaths result from metastasis. CXCR4 and CCR2 are known to govern cellular processes resulting in cell migration, proliferation and survival. These receptors are expressed at low levels on normal prostate cells and are highly expressed on malignant and metastatic prostate cancer cells. Signaling of these receptors is relatively well understood, but processes governing their expression at the cell membrane are not. PC3 prostate cancer cells were used to demonstrate the importance of various Rab GTPases on cell surface expression and signaling of CXCR4 and CCR2, along with the CXCR4/CCR2 heterodimer. METHODS: PC3 prostate cancer cells were transfected with select Rab GTPase wild-type and dominant negative constructs. Effects of each Rab GTPase on endogenous cell surface expression of the individual receptors, along with the overexpressed CXCR4/CCR2 heterodimer, were determined by biotin-streptavidin cell surface assays. These results were corroborated by assessing signal transduction, measured by focal adhesion kinase (FAK) activation. CONCLUSION: Rab GTPases required for cell surface expression and signal transduction of CXCR4 or CCR2 differ from those required for the CXCR4/CCR2 heterodimer. Determining trafficking regulators of two key receptors involved in the metastatic transition may identify new targets to restrict expression of chemokine receptors employed during metastasis.

Structural features of the apelin receptor N-terminal tail and first transmembrane segment implicated in ligand binding and receptor trafficking.

G-protein coupled receptors (GPCRs) comprise a large family of membrane proteins with rich functional diversity. Signaling through the apelin receptor (AR or APJ) influences the cardiovascular system, central nervous system and glucose regulation. Pathophysiological involvement of apelin has been shown in atherosclerosis, cancer, human immunodeficiency virus-1 (HIV-1) infection and obesity. Here, we present the high-resolution nuclear magnetic resonance (NMR) spectroscopy-based structure of the N-terminus and first transmembrane (TM) segment of AR (residues 1-55, AR55) in dodecylphosphocholine micelles. AR55 consists of two disrupted helices, spanning residues D14-K25 and A29-R55(1.59). Molecular dynamics (MD) simulations of AR built from a hybrid of experimental NMR and homology model-based restraints allowed validation of the AR55 structure in the context of the full-length receptor in a hydrated bilayer. AR55 structural features were functionally probed using mutagenesis in full-length AR through monitoring of apelin-induced extracellular signal-regulated kinase (ERK) phosphorylation in transiently transfected human embryonic kidney (HEK) 293A cells. Residues E20 and D23 form an extracellular anionic face and interact with lipid headgroups during MD simulations in the absence of ligand, producing an ideal binding site for a cationic apelin ligand proximal to the membrane-water interface, lending credence to membrane-catalyzed apelin-AR binding. In the TM region of AR55, N46(1.50) is central to a disruption in helical character. G42(1.46), G45(1.49) and N46(1.50), which are all involved in the TM helical disruption, are essential for proper trafficking of AR. In summary, we introduce a new correlative NMR spectroscopy and computational biochemistry methodology and demonstrate its utility in providing some of the first high-resolution structural information for a peptide-activated GPCR TM domain.

G protein-coupled receptor dimers: look like their parents, but act like teenagers!

G protein-coupled receptors (GPCRs) represent the largest group of cell surface receptors and an important pharmacological target. Though originally thought to act in a one receptor-one effector fashion, it is now known that these receptors are capable of oligomerization and can function as dimers or higher order oligomers in native tissue. They do not only assemble with identical receptors as homodimers, but also associate with different GPCRs to form heterodimers. We discuss here how heterodimeric GPCRs can assemble, traffic and signal in a manner distinct from their individual receptor components or from homodimers. These receptor pairs are also demonstrated to be regulated by different chaperones, Rabs and scaffolding proteins, further emphasizing their potential as unique targets. We believe in the importance of investigating each GPCR heterodimer as an individual signaling complex, as they appear to act differently from each monomer constituting them. Just as teenagers may resemble their parents and share their genetic makeup, they can still act in a manner that is entirely unique!

Role of chaperones in G protein coupled receptor signaling complex assembly.

G protein coupled receptors are involved in highly efficient and specific activation of signaling pathways. Yet, we do not fully understand the processes required to assemble the different partners of the GPCR signaling complex. In order to address this issue, we need to understand how receptors and their signaling -partners are synthesized, folded and regulated during quality control steps in order to generate functional proteins. Several molecular chaperones are involved in this process for most proteins, including GPCRs. Several membrane proteins require the assembly of different subunits to be functional. In recent years, GPCRs have been shown to form oligomers, which could be interpreted as subunits of a larger complex. Yet, those oligomers would not be functional without the association of other signaling partners; thus, there is a requirement for the specific assembly of the -different partners. In this chapter, we will cover some aspects of the current knowledge about how chaperones are involved in both the formation of GPCR oligomers and in the assembly of the receptors with their signaling complex components.

Rescue of internalization-defective platelet-activating factor receptor function by EBP50/NHERF1.

Platelet-activating factor (PAF) is a potent phospholipid mediator involved in specific disease states such as allergic asthma, atherosclerosis and psoriasis. The human PAF receptor (PAFR) is a member of the G protein-coupled receptor (GPCR) family. Following PAF stimulation, cells become rapidly desensitized; this refractory state can be maintained for hours and is dependent on PAFR phosphorylation, internalization and trafficking. EBP50/NHERF1 has been found to interact with a variety of proteins and these interactions are involved in a growing range of functions including the assembly of signalling complexes, receptor recycling and transport of proteins to the cell surface. Crucial roles of EBP50 in GPCR physiology include its involvement in internalization, recycling, and downregulation. We were interested in identifying the role of EBP50 in PAFR trafficking. Our results showed that EBP50 binds the PAFR in its basal state, while stimulation decreased the ratio of interaction between the two proteins. We also demonstrated that EBP50 could bind PAFR via its PDZ 2 domain. In addition, we studied the role of EBP50 in various functions of the PAFR such as PAF-induced inositol phosphate accumulation and receptor internalization: EBP50 decreased the WT PAFR response and rescued the function of internalization-deficient mutant receptors, as previously described for the arrestins and the GRKs. These results suggest new roles for EBP50, some of which could help understanding the complex formation after receptor activation.