The ß2-adrenergic receptor associates with CXCR4 multimers in human cancer cells.

While the existence and functional role of class C G-protein-coupled receptors (GPCR) dimers is well established, there is still a lack of consensus regarding class A and B GPCR multimerization. This lack of consensus is largely due to the inherent challenges of demonstrating the presence of multimeric receptor complexes in a physiologically relevant cellular context. The C-X-C motif chemokine receptor 4 (CXCR4) is a class A GPCR that is a promising target of anticancer therapy. Here, we investigated the potential of CXCR4 to form multimeric complexes with other GPCRs and characterized the relative size of the complexes in a live-cell environment. Using a bimolecular fluorescence complementation (BiFC) assay, we identified the ß2 adrenergic receptor (ß2AR) as an interaction partner. To investigate the molecular scale details of CXCR4-ß2AR interactions, we used a time-resolved fluorescence spectroscopy method called pulsed-interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS). PIE-FCCS can resolve membrane protein density, diffusion, and multimerization state in live cells at physiological expression levels. We probed CXCR4 and ß2AR homo- and heteromultimerization in model cell lines and found that CXCR4 assembles into multimeric complexes larger than dimers in MDA-MB-231 human breast cancer cells and in HCC4006 human lung cancer cells. We also found that ß2AR associates with CXCR4 multimers in MDA-MB-231 and HCC4006 cells to a higher degree than in COS-7 and CHO cells and in a ligand-dependent manner. These results suggest that CXCR4-ß2AR heteromers are present in human cancer cells and that GPCR multimerization is significantly affected by the plasma membrane environment.

Functional Rescue of Inactivating Mutations of the Human Neurokinin 3 Receptor Using Pharmacological Chaperones.

G protein-coupled receptors (GPCRs) facilitate the majority of signal transductions across cell membranes in humans, with numerous diseases attributed to inactivating GPCR mutations. Many of these mutations result in misfolding during nascent receptor synthesis in the endoplasmic reticulum (ER), resulting in intracellular retention and degradation. Pharmacological chaperones (PCs) are cell-permeant small molecules that can interact with misfolded receptors in the ER and stabilise/rescue their folding to promote ER exit and trafficking to the cell membrane. The neurokinin 3 receptor (NK3R) plays a pivotal role in the hypothalamic-pituitary-gonadal reproductive axis. We sought to determine whether NK3R missense mutations result in a loss of cell surface receptor expression and, if so, whether a cell-permeant small molecule NK3R antagonist could be repurposed as a PC to restore function to these mutants. Quantitation of cell surface expression levels of seven mutant NK3Rs identified in hypogonadal patients indicated that five had severely impaired cell surface expression. A small molecule NK3R antagonist, M8, increased cell surface expression in four of these five and resulted in post-translational receptor processing in a manner analogous to the wild type. Importantly, there was a significant improvement in receptor activation in response to neurokinin B (NKB) for all four receptors following their rescue with M8. This demonstrates that M8 may have potential for therapeutic development in the treatment of hypogonadal patients harbouring NK3R mutations. The repurposing of existing small molecule GPCR modulators as PCs represents a novel and therapeutically viable option for the treatment of disorders attributed to mutations in GPCRs that cause intracellular retention.

Trafficking-defective mutant PROKR2 cycles between endoplasmic reticulum and Golgi to attenuate endoplasmic reticulum stress.

G protein-coupled receptors (GPCRs) play crucial roles in numerous physiological and pathological processes. Mutations in GPCRs that result in loss of function or alterations in signaling can lead to inherited or acquired diseases. Herein, studying prokineticin receptor 2 (PROKR2), we initially identify distinct interactomes for wild-type (WT) versus a mutant (P290S) PROKR2 that causes hypogonadotropic hypogonadism. We then find that both the WT and mutant PROKR2 are targeted for endoplasmic reticulum (ER)-associated degradation, but the mutant is degraded to a greater extent. Further analysis revealed that both forms can also leave the ER to reach the Golgi. However, whereas most of the WT is further transported to the cell surface, most of the mutant is retrieved to the ER. Thus, the post-ER itinerary plays an important role in distinguishing the ultimate fate of the WT versus the mutant. We have further discovered that this post-ER itinerary reduces ER stress induced by the mutant PROKR2. Moreover, we extend the core findings to another model GPCR. Our findings advance the understanding of disease pathogenesis induced by a mutation at a key residue that is conserved across many GPCRs and thus contributes to a fundamental understanding of the diverse mechanisms used by cellular quality control to accommodate misfolded proteins.

Connecting nutritional deprivation and pubertal inhibition via GRK2-mediated repression of kisspeptin actions in GnRH neurons.

BACKGROUND: Perturbations in the timing of puberty, with potential adverse consequences in later health, are increasingly common. The underlying neurohormonal mechanisms are unfolded, but nutritional alterations are key contributors. Efforts to unveil the basis of normal puberty and its metabolic control have focused on mechanisms controlling expression of Kiss1, the gene encoding the puberty-activating neuropeptide, kisspeptin. However, other regulatory phenomena remain ill-defined. Here, we address the putative role of the G protein-coupled-receptor kinase-2, GRK2, in GnRH neurons, as modulator of pubertal timing via repression of the actions of kisspeptin, in normal maturation and conditions of nutritional deficiency. METHODS: Hypothalamic RNA and protein expression analyses were conducted in maturing female rats. Pharmacological studies involved central administration of GRK2 inhibitor, ßARK1-I, and assessment of gonadotropin responses to kisspeptin or phenotypic and hormonal markers of puberty, under normal nutrition or early subnutrition in female rats. In addition, a mouse line with selective ablation of GRK2 in GnRH neurons, aka G-GRKO, was generated, in which hormonal responses to kisspeptin and puberty onset were monitored, in normal conditions and after nutritional deprivation. RESULTS: Hypothalamic GRK2 expression increased along postnatal maturation in female rats, especially in the preoptic area, where most GnRH neurons reside, but decreased during the juvenile-to-pubertal transition. Blockade of GRK2 activity enhanced Ca+2 responses to kisspeptin in vitro, while central inhibition of GRK2 in vivo augmented gonadotropin responses to kisspeptin and advanced puberty onset. Postnatal undernutrition increased hypothalamic GRK2 expression and delayed puberty onset, the latter being partially reversed by central GRK2 inhibition. Conditional ablation of GRK2 in GnRH neurons enhanced gonadotropin responses to kisspeptin, accelerated puberty onset, and increased LH pulse frequency, while partially prevented the negative impact of subnutrition on pubertal timing and LH pulsatility in mice. CONCLUSIONS: Our data disclose a novel pathway whereby GRK2 negatively regulates kisspeptin actions in GnRH neurons, as major regulatory mechanism for tuning pubertal timing in nutritionally-compromised conditions.

MKRN3 inhibits the reproductive axis through actions in kisspeptin-expressing neurons.

The identification of loss-of-function mutations in MKRN3 in patients with central precocious puberty in association with the decrease in MKRN3 expression in the medial basal hypothalamus of mice before the initiation of reproductive maturation suggests that MKRN3 is acting as a brake on gonadotropin-releasing hormone (GnRH) secretion during childhood. In the current study, we investigated the mechanism by which MKRN3 prevents premature manifestation of the pubertal process. We showed that, as in mice, MKRN3 expression is high in the hypothalamus of rats and nonhuman primates early in life, decreases as puberty approaches, and is independent of sex steroid hormones. We demonstrated that Mkrn3 is expressed in Kiss1 neurons of the mouse hypothalamic arcuate nucleus and that MKRN3 repressed promoter activity of human KISS1 and TAC3, 2 key stimulators of GnRH secretion. We further showed that MKRN3 has ubiquitinase activity, that this activity is reduced by MKRN3 mutations affecting the RING finger domain, and that these mutations compromised the ability of MKRN3 to repress KISS1 and TAC3 promoter activity. These results indicate that MKRN3 acts to prevent puberty initiation, at least in part, by repressing KISS1 and TAC3 transcription and that this action may involve an MKRN3-directed ubiquitination-mediated mechanism.

Tac1 Signaling Is Required for Sexual Maturation and Responsiveness of GnRH Neurons to Kisspeptin in the Male Mouse.

The tachykinins substance P (SP) and neurokinin A (Tac1) have emerged as novel regulators of kisspeptin/GnRH release. Recently, we documented that SP modulates reproductive function in the female mouse. Here, we extended this characterization to the male mouse. Tac1-/- male mice showed delayed puberty onset. They also presented significantly decreased expression levels of Pdyn (dynorphin) and Nos1 (nitric oxide synthase) in the mediobasal hypothalamus and elevated Gnrh1 levels. Unexpectedly, the response of Tac1-/- mice to central kisspeptin or senktide (neurokinin B receptor-agonist) administration was significantly decreased compared with controls, despite the preserved ability of GnRH neurons to stimulate luteinizing hormone release as demonstrated by central N-methyl-D-aspartate receptor administration, suggesting a deficit at the GnRH neuron level. Importantly, we demonstrated that kisspeptin receptor and SP receptor (NK1R) heterodimerize, indicating that changes in the SP tone could alter the responsiveness of GnRH neurons to kisspeptin. Finally, electrophysiological recordings from arcuate Kiss1 neurons showed that, although virtually all Kiss1 neurons responded to NKB and senktide, only half responded to an NK1R agonist and none to the neurokinin A receptor agonist at a 1-µM dose. In summary, we provide compelling evidence for a role of Tac1 in the control of reproductive function in the male mouse, suggesting a predominant central action that may involve a change in the balance of neural factors that control GnRH expression.

Monitoring G protein-coupled receptor activation using an adenovirus-based ß-arrestin bimolecular fluorescence complementation assay.

G protein-coupled receptors (GPCRs) are the largest family of cell-surface receptors and are involved in a variety of pathological conditions including cancer and cardiovascular, metabolic, neurological, and autoimmune diseases. GPCRs are being intensively investigated as targets for therapeutic intervention, and the ß-arrestin recruitment assay has become a popular tool for analyzing GPCR activation. Here, we report a high-throughput method for cloning GPCR cDNAs into adenoviral bimolecular fluorescence complementation (BiFC) vectors and performing the ß-arrestin BiFC assay in cells transduced with recombinant adenoviruses. An analysis of the activation of somatostatin receptor 2 (SSTR2) with the adenovirus-based ß-arrestin BiFC assay showed that the assay is suitable for quantifying SSTR2 activation in response to specific agonists or antagonists. Furthermore, the adenovirus-based ß-arrestin BiFC assay was able to detect the activation of a broad range of GPCRs. Collectively, our data indicate that the adenovirus-based ß-arrestin BiFC assay can serve as a simple and universal platform for studying GPCR activation and thus will be useful for high-throughput screening of drugs that target GPCRs.

AdHTS: a high-throughput system for generating recombinant adenoviruses.

The need for efficient high-throughput gene delivery system for mammalian cells is rapidly increasing with the growing request for functional genomics studies and drug discoveries in various physiologically relevant systems. However, plasmid-based gene delivery has limitations in transfection efficiency and available cell types. Viral vectors have great advantages over plasmid-based vectors, but construction of recombinant viruses remains to be a big hurdle for high-throughput applications. Here we demonstrate a rapid and simple high-throughput system for constructing recombinant adenoviruses which have been used as efficient gene delivery tools in mammalian systems in vitro and in vivo. By combining Gateway-based site-specific recombination with Terminal protein-coupled adenovirus vector, the adenovirus high-throughput system (AdHTS) generates multiple recombinant adenoviruses in 96-well plates simultaneously without the need for additional cloning or recombination in bacteria or mammalian cells. The AdHTS allows rapid and robust cloning and expression of genes in mammalian cells by removing shuttle vector construction, bacterial transformation, or selection and by minimizing effort in plaque isolation. By shortening the time required to convert whole cDNA library into desired viral vector constructs, the AdHTS would greatly facilitate functional genomics and proteomics studies in various mammalian systems.

Redox regulation of the tumor suppressor PTEN by glutaredoxin 5 and Ycp4.

Human PTEN (phosphatase and tensin homolog deleted on chromosome 10; a phosphatidylinositol 3-phosphatase) expressed in Saccharomyces cerevisiae was oxidized in a time- and H(2)O(2)-concentration-dependent manner. Oxidized hPTEN was reduced by cellular reductants as in human cells. The reduction rate of oxidized hPTEN was monitored in S. cerevisiae mutants in which the genes involved in redox homeostasis had been disrupted. Reduction of hPTEN was delayed in each of S. cerevisiae grx5Δ and ycp4Δ mutants. Expression of Grx5 and Ycp4 in each of the mutants rescued the reduction rate of oxidized hPTEN. Furthermore, an in vitro assay revealed that the human Grx5/GSH system efficiently catalyzed the reduction of oxidized hPTEN. These results suggest that the reduction of oxidized hPTEN is regulated by Grx5 and Ycp4.

Redox regulation of the tumor suppressor PTEN by glutathione.

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) expressed in Saccharomyces cerevisiae was reversibly oxidized by hydrogen peroxide and reduced by cellular reductants. Reduction of hPTEN was delayed in each of S. cerevisiae gsh1Delta and gsh2Delta mutants. Expression of gamma-glutamylcysteine synthetase Gsh1 in the gsh1Delta mutant rescued regeneration rate of hPTEN. Oxidized hPTEN was reduced by glutathione in a concentration- and time-dependent manner. Glutathionylated PTEN was detected. Incubation of 293T cells with BSO and knockdown expression of GCLc in HeLa cells by siRNA resulted in the delay of reduction of oxidized PTEN. Also, in HeLa cells transfected with GCLc siRNA, stimulation with epidermal growth factor resulted in the increase of oxidized PTEN and phosphorylation of Akt. These results suggest that the reduction of oxidized hPTEN is mediated by glutathione.

Quantitative proteomic analysis of ribosomal protein L35b mutant of Saccharomyces cerevisiae.

Recent studies have revealed that in higher eukaryotes, several ribosomal proteins are involved in some pathological events or developmental defects, indicating that ribosomal proteins perform unconventional functions other than protein biosynthesis. To obtain an insight into the novel roles of ribosomal proteins, we aimed to analyze the changes in proteome expression in ribosomal protein mutants by using Saccharomyces cerevisiae as a model system. We introduced the rpl35bDelta mutation into the 4159 green fluorescent protein (GFP)-tagged yeast strains by using the synthetic genetic array (SGA) method, and performed quantitative proteomic analysis by using a multilabel microplate reader and flow cytometer. We identified 22 upregulated and 20 downregulated proteins in the rpl35bDelta mutant. These proteins were primarily classified into the Gene Ontology (GO) categories of cellular biosynthetic process, translation, protein or nucleotide metabolic process, cell wall organization and biogenesis, and hyperosmotic response. We also investigated the correlation between the mRNA and protein levels of the identified proteins. Our results show that a ribosomal protein mutation can lead to perturbation in the expression of several proteins, including some other ribosomal proteins. Furthermore, our approach of combining a library of GFP-tagged yeast strains and the SGA method provides an effective and highly sensitive method for dynamic analysis of the effects of various mutations on proteome expression.

D-Erythroascorbic acid activates cyanide-resistant respiration in Candida albicans.

Higher plants, protists and fungi possess cyanide-resistant respiratory pathway, which is mediated by alternative oxidase (AOX). The activity of AOX has been found to be dependent on several regulatory mechanisms including gene expression and posttranslational regulation. In the present study, we report that the presence of cyanide in culture medium remarkably retarded the growth of alo1/alo1 mutant of Candida albicans, which lacks d-arabinono-1,4-lactone oxidase (ALO) that catalyzes the final step of d-erythroascorbic acid (EASC) biosynthesis. Measurement of respiratory activity and Western blot analysis revealed that increase in the intracellular EASC level induces the expression of AOX in C. albicans. AOX could still be induced by antimycin A, a respiratory inhibitor, in the absence of EASC, suggesting that several factors may act in parallel pathways to induce the expression of AOX. Taken together, our results suggest that EASC plays important roles in activation of cyanide-resistant respiration in C. albicans.