Minireview: Constitutively Active G Protein-Coupled Receptors: Potential Mechanisms of Receptor Activation

Abstract

Mutations of G protein-coupled receptors can increase their constitutive (agonist-independent) activity. Some of these mutations have been artificially introduced by site-directed mutagenesis, others occur spontaneously in human diseases. The analysis of the constitutively active G protein-coupled receptors has provided important informations about the molecular mechanisms underlying receptor activation and drug action.     

Minireview: Differential Targeting and Retention of G Protein-Coupled Receptors in Polarized Epithelial Cells

Abstract

Localization of receptors in discrete cellular microdomains undoubtedly contributes to their interaction with particular effectors and receptor targets. For G protein-coupled receptors, virtually nothing is known about the mechanisms and structural features responsible for their targeting to and retention in varying surface domains. We have shown that the G_j/G_o-coupled α_2A-adrenergic receptor (α_2AAR) is directly targeted to the lateral subdomain of MDCK II cells. Mutational analysis has revealed that regions in or near the bilayer are likely critical for α_2AAR targeting, whereas endofacial domains contribute to α_2AAR retention on the lateral surface. Although the α_2BAR also is enriched on the lateral subdomain at steady-state, its polarization occurs after initial random delivery to both apical and basolateral surfaces followed by a selective accumulation on the lateral subdomain. The α_2CAR also is expressed on the lateral subdomain and achieves its localization via direct delivery to the basolateral surface; however, the α_2CAR also exists in an as yet not fully characterized intracellular compartment. Interestingly, another G_j/G_o-coupled receptor, the A₁ adenosine receptor, is enriched on the apical surface of MDCK II calls and achieves this localization by direct apical delivery. These findings indicate that receptor delivery to polarized surfaces is not determined by receptor coupling to a specific subpopulation of G proteins.     

On the G Protein-Coupled Receptor Neuromodulation of the Claustrum

Neurochemical Research - G protein-coupled receptors modulate the synaptic glutamate and GABA transmission of the claustrum. The work focused on the transmitter–receptor relationships in the...     

Structural Model of Ghrelin Bound to its G Protein-Coupled Receptor

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Deorphanization of Dresden G Protein-Coupled Receptor for an Odorant Receptor

Dresden G protein-coupled receptor (D-GPCR) is one of orphan G protein-coupled receptors (GPCR). Here we report the identification of the ligands and the characterization of D-GPCR. We investigated over 5000 compounds to evoke the response mediated by D-GPCR and identified 3-methyl-valeric acid and 4-methyl-valeric acid as agonists using a cAMP assay. It is of interest that they dramatically enhanced the intracellular cAMP accumulation and the CRE-luciferase activity in CHO-K1 cells and HEK293 cells expressing the chimeric protein of D-GPCR with a rhodopsin-tag at its N-terminus. Our results established new characteristics of D-GPCR as an olfactory receptor. First, agonists of D-GPCR belong to odorants. Second, D-GPCR mRNA is expressed in the olfactory bulb. In addition, D-GPCR was reported to have similar sequences and its genome locus nearby other olfactory receptors. These results suggest D-GPCR is an olfactory receptor.     

Thematic Minireview Series: New Directions in G Protein-coupled Receptor Pharmacology

Over the past half-century, The Journal of Biological Chemistry has been the venue for many landmark publications on the topic of G protein-coupled receptors (GPCRs, also known as seven-transmembrane receptors). The GPCR superfamily in humans is composed of about 800 members, and is the target of about one-third of all pharmaceuticals. Most of these drugs target a very small subset of GPCRs, and do so by mimicking or competing with endogenous hormones and neurotransmitters. This thematic minireview series examines some emerging trends in GPCR drug discovery. The first article describes efforts to systematically interrogate the human “GPCR-ome,” including more than 150 uncharacterized “orphan” receptors. The second article describes recent efforts to target alternative receptor binding sites with drugs that act as allosteric modulators of orthosteric ligands. The third article describes how the recent expansion of GPCR structures is providing new opportunities for computer-guided drug discovery. Collectively, these three articles provide a roadmap for the most important emerging trends in GPCR pharmacology.     

Mechanisms of Lipid Scrambling by the G Protein-Coupled Receptor Opsin

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Internalization of G Protein-Coupled Receptors in Single Olfactory Receptor Neurons

Abstract : Desensitization of many G protein-coupled receptors after ligand binding generally involves phosphorylation of the receptors and internalization of the ligandbound, phosphorylated receptors by a clathrin-mediated endocytic pathway. Olfactory receptor neurons from the channel catfish ( Ictalurus punctatus ) express the G protein-coupled odorant receptors and metabotropic glutamate receptors. To determine whether a clathrin-dependent receptor internalization pathway exists in olfactory receptor neurons, western blotting and immunocytochemistry were used to identify and localize clathrin and dynamin in isolated olfactory neurons. Clathrin and dynamin immunoreactivity was found in the cell bodies, dendrites, and dendritic knobs of the neurons. Using the activity-dependent fluorescent dye FM1-43 to monitor receptor internalization, we show that single olfactory neurons stimulated with the odorant amino acid l -glumate internalized the dye. Odorant-stimulated neurons showed a consistent pattern of internalized FM1-43 fluorescence localized in the cell bodies and dendritic knobs. Odorant-stimulated internalization was unaffected by the caveolae activator okadaic acid and was significantly decreased by a metabotropic glutamate receptor antagonist, suggesting that a functional, clathrindependent, receptor-mediated internalization pathway exists in olfactory receptor neurons.     

Regulation of G Protein-Coupled Receptor Signaling by A-Kinase Anchoring Proteins

Specificity of transduction events is controlled at the molecular level by scaffold, anchoring, and adaptor proteins, which position signaling enzymes at proper subcellular localization. This allows their efficient catalytic activation and accurate substrate selection. A-kinase anchoring proteins (AKAPs) are group of functionally related proteins that compartmentalize the cAMP-dependent protein kinase (PKA) and other signaling enyzmes at precise subcellular sites in close proximity to their physiological substrate(s) and favor specific phosphorylation events. Recent evidence suggests that AKAP transduction complexes play a key role in regulating G protein-coupled receptor (GPCR) signaling. Regulation can occur at multiple levels because AKAPs have been shown both to directly modulate GPCR function and to act as downstream effectors of GPCR signaling. In this minireview, we focus on the molecular mechanisms through which AKAP-signaling complexes modulate GPCR transduction cascades.     

Pro-Aging Effects of Glucose Signaling through a G Protein-Coupled Glucose Receptor in Fission Yeast

Glucose is the preferred carbon and energy source in prokaryotes, unicellular eukaryotes, and metazoans. However, excess of glucose has been associated with several diseases, including diabetes and the less understood process of aging. On the contrary, limiting glucose (i.e., calorie restriction) slows aging and age-related diseases in most species. Understanding the mechanism by which glucose limits life span is therefore important for any attempt to control aging and age-related diseases. Here, we use the yeast Schizosaccharomyces pombe as a model to study the regulation of chronological life span by glucose. Growth of S. pombe at a reduced concentration of glucose increased life span and oxidative stress resistance as reported before for many other organisms. Surprisingly, loss of the Git3 glucose receptor, a G protein-coupled receptor, also increased life span in conditions where glucose consumption was not affected. These results suggest a role for glucose-signaling pathways in life span regulation. In agreement, constitutive activation of the Gα subunit acting downstream of Git3 accelerated aging in S. pombe and inhibited the effects of calorie restriction. A similar pro-aging effect of glucose was documented in mutants of hexokinase, which cannot metabolize glucose and, therefore, are exposed to constitutive glucose signaling. The pro-aging effect of glucose signaling on life span correlated with an increase in reactive oxygen species and a decrease in oxidative stress resistance and respiration rate. Likewise, the anti-aging effect of both calorie restriction and the Δgit3 mutation was accompanied by increased respiration and lower reactive oxygen species production. Altogether, our data suggest an important role for glucose signaling through the Git3/PKA pathway to regulate S. pombe life span.     

Desensitization of the δ-Opioid Receptor Correlates with Its Phosphorylation in SK-N-BE Cells: Involvement of a G Protein-Coupled Receptor Kinase

Abstract: Phosphorylation of G protein-coupled receptors is considered an important step during their desensitization. In SK-N-BE cells, recently presented as a pertinent model for the studies of the human δ-opioid receptor, pretreatment with the opioid agonist etorphine increased time-dependently the rate of phosphorylation of a 51-kDa membrane protein. Immunological characterization of this protein with an antibody, raised against the amino-terminal region of the cloned human δ-opioid receptor, revealed that it corresponded to the δ-opioid receptor. During prolonged treatment with etorphine, phosphorylation increased as early as 15 min to reach a maximum within 1 h. Phosphorylation and desensitization of adenylyl cyclase inhibition paralleled closely and okadaic acid inhibited the resensitization, a result strongly suggesting that phosphorylation of the δ-opioid receptor plays a prominent role in its rapid desensitization. The increase in phosphorylation of the δ-opioid receptor, as well as its desensitization, was not affected by H7, an inhibitor of protein kinase A and protein kinase C, but was drastically reduced by heparin or Zn²⁺, known to act as G protein-coupled receptor kinase (GRK) inhibitors. These results are the first to show, on endogenously expressed human δ-opioid receptor, that a close link exists between receptor phosphorylation and agonist-promoted desensitization and that desensitization involves a GRK.     

孤儿G蛋白偶联受体hGPCRc的亚细胞定位及组织分布

利用相关生物信息学软件,对从人结肠组织克隆所得某一孤儿G蛋白偶联受体(orphanGprotein_coupledreceptors ,oGPCRs)成员hGPCRc的氨基酸序列进行分析显示,hGPCRc对应的氨基酸序列组成了七个跨膜区段的结构域,具备GPCR的结构特征;然后,将hGPCRc之cDNA与绿色荧光载体pEGFP-N₁ 构建GFP_hGPCRc表达载体,以空白质粒pEGFP-N₁ 作对照,转染CHO-K₁ 细胞,在激光扫描共聚焦显微镜下观察到空白质粒pEGFP-N₁ 转染的细胞表达了GFP并均匀分布于整个细胞,而GFP_hGPCRc转染的细胞观察到荧光清晰聚集于细胞膜和各细胞器质膜上,因而hGPCRc蛋白定位于膜上并稳定表达,与软件分析结果相一致;最后,以RT_PCR检测hGPCRc在2 0周龄胎儿重要组织器官及部分成人组织中的表达情况,结果显示hGPCRc在人心、肾、小脑及结肠等组织均有表达,但在肝、大脑、小肠及肌肉等组织里未检测到表达。该表达谱对于进一步认识hGPCRc在胚胎发育中的作用及生理功能提供了线索。     

GGA3 Interacts with a G Protein-Coupled Receptor and Modulates Its Cell Surface Export

Molecular mechanisms governing the anterograde trafficking of nascent G protein-coupled receptors (GPCRs) are poorly understood. Here, we have studied the regulation of cell surface transport of α₂-adrenergic receptors (α₂-ARs) by GGA3 (Golgi-localized, γ-adaptin ear domain homology, ADP ribosylation factor-binding protein 3), a multidomain clathrin adaptor protein that sorts cargo proteins at the trans-Golgi network (TGN) to the endosome/lysosome pathway. By using an inducible system, we demonstrated that GGA3 knockdown significantly inhibited the cell surface expression of newly synthesized α_2B-AR without altering overall receptor synthesis and internalization. The receptors were arrested in the TGN. Furthermore, GGA3 knockdown attenuated α_2B-AR-mediated signaling, including extracellular signal-regulated kinase 1/2 (ERK1/2) activation and cyclic AMP (cAMP) inhibition. More interestingly, GGA3 physically interacted with α_2B-AR, and the interaction sites were identified as the triple Arg motif in the third intracellular loop of the receptor and the acidic motif EDWE in the VHS domain of GGA3. In contrast, α_2A-AR did not interact with GGA3 and its cell surface export and signaling were not affected by GGA3 knockdown. These data reveal a novel function of GGA3 in export trafficking of a GPCR that is mediated via a specific interaction with the receptor.     

Biochemical and Cellular Specificity of Peptide Inhibitors of G Protein-Coupled Receptor Kinases

Identifying novel allosteric inhibitors of G protein-coupled receptor kinases (GRKs) would be of considerable use in limiting both the extent of desensitization of GPCRs as well as downstream positive regulation through GRKs. Several peptides have previously been identified as inhibitors of specific GRKs, but to date there have been few comparisons of the selectivities of these materials on the seven GRKs, modifications to allow cell penetration, or off-target activities. The goal of this study was to determine if a panel of peptides mimicking domains on either GPCRs or GRKs would exhibit selective inhibition of GRKs 2, 5, 6 and 7 phosphorylation of rhodopsin. Peptides included sequences from GRK5; helices 3, 9, and 10 (α3, α9, and α10) in the RH domain, and the N-terminal peptide (N-Ter), as well as the intracellular loop 1 (iL1) of the β2-adrenergic receptor (β2AR), and the G_α transducin C-tail (TCT). While some selectivity for individual GRKs was found, overall selectivity was limited and often not reflective of structural predictions. Off-target effects were probed by determining peptide inhibition of adenylyl cyclase (AC) and PKA, and while peptides had no effect on AC activity, N-Ter, iL1, and α10 were potent inhibitors of PKA. To probe inhibition of GRK activity in intact cells, we synthesized TAT-tagged peptides, and found that TAT-α9-R169A and TAT–TCT inhibited isoproterenol-stimulated GRK phosphorylation of the β2AR; however, the TAT peptides also inhibited isoproterenol and forskolin stimulation of AC activity. Our findings demonstrate potent peptide inhibition of GRK activities in vitro, highlight the differences in the environments of biochemical and cell-based assays, and illustrate the care that must be exercised in interpreting results of either assay alone.     

Molecular Evolution of CXCR1, a G Protein-Coupled Receptor Involved in Signal Transduction of Neutrophils

Human neutrophils are a type of white blood cell, which forms an early line of defense against bacterial infections. Neutrophils are highly responsive to the chemokine, interleukin-8 (IL-8) due to the abundant distribution of CXCR1, one of the IL-8 receptors on the neutrophil cell surface. As a member of the GPCR family, CXCR1 plays a crucial role in the IL-8 signal transduction pathway in neutrophils. We sequenced the complete coding region of the CXCR1 gene in worldwide human populations and five representative nonhuman primate species. Our results indicate accelerated protein evolution in the human lineage, which was likely caused by Darwinian positive selection. The sliding window analysis and the codon-based neutrality test identified signatures of positive selection at the N-terminal ligand/receptor recognition domain of human CXCR1. [Reviewing Editor: Dr. Manyuan Long] The GenBank accession numbers of sequences reported herein are AY916760–AY916773.