G protein coupled pattern recognition receptors expressed in neutrophils: Recognition,activation/modulation,signaling and receptor regulated functions

Neutrophils, the most abundant white blood cell in human blood, express receptors that recognize damage/microbial associated pattern molecules of importance for cell recruitment to sites of inflammation. Many of these receptors belong to the family of G protein coupled receptors (GPCRs). These receptor-proteins span the plasma membrane in expressing cells seven times and the down-stream signaling rely in most cases on an activation of heterotrimeric G proteins. The GPCRs expressed in neutrophils recognize a number of structurally diverse ligands (activating agonists, allosteric modulators, and inhibiting antagonists) and share significant sequence homologies. Studies of receptor structure and function have during the last 40 years generated important information on GPCR biology in general; this knowledge aids in the overall understanding of general pharmacological principles, governing regulation of neutrophil function and inflammatory processes, including novel leukocyte receptor activities related to ligand recognition, biased/functional selective signaling, allosteric modulation, desensitization, and reactivation mechanisms as well as communication (receptor transactivation/cross-talk) between GPCRs. This review summarizes the recent discoveries and pharmacological hallmarks with focus on some of the neutrophil expressed pattern recognition GPCRs. In addition, unmet challenges, including recognition by the receptors of diverse ligands and how biased signaling mediate different biological effects are described/discussed.     

Neuropeptides and neuropeptide receptors in the Drosophila melanogaster genome

Recent genetic analyses in worms, flies, and mammals illustrate the importance of bioactive peptides in controlling numerous complex behaviors, such as feeding and circadian locomotion. To pursue a comprehensive genetic analysis of bioactive peptide signaling, we have scanned the recently completed Drosophila genome sequence for G protein-coupled receptors sensitive to bioactive peptides (peptide GPCRs). Here we describe 44 genes that represent the vast majority, and perhaps all, of the peptide GPCRs encoded in the fly genome. We also scanned for genes encoding potential ligands and describe 22 bioactive peptide precursors. At least 32 Drosophila peptide receptors appear to have evolved from common ancestors of 15 monophyletic vertebrate GPCR subgroups (e.g., the ancestral gastrin/cholecystokinin receptor). Six pairs of receptors are paralogs, representing recent gene duplications. Together, these findings shed light on the evolutionary history of peptide GPCRs, and they provide a template for physiological and genetic analyses of peptide signaling in Drosophila.     

A new key neurohormone controlling reproduction,gonadotropin-inhibitory hormone (GnIH): Biosynthesis,mode of action and functional significance

Identification of novel neurohormones that play important roles in the regulation of pituitary function is essential for the progress of neurobiology. The decapeptide gonadotropin-releasing hormone (GnRH) is the primary factor responsible for the hypothalamic control of gonadotropin secretion. Gonadal sex steroids and inhibin inhibit gonadotropin secretion via feedback from the gonads, but a neuropeptide inhibitor of gonadotropin secretion was, until recently, unknown in vertebrates. In 2000, a novel hypothalamic dodecapeptide that inhibits gonadotropin release was identified in quail and termed gonadotropin-inhibitory hormone (GnIH). This was the first demonstration of a hypothalamic neuropeptide inhibiting gonadotropin release in any vertebrate. GnIH acts on the pituitary and GnRH neurons in the hypothalamus via a novel G protein-coupled receptor for GnIH to inhibit gonadal development and maintenance by decreasing gonadotropin release and synthesis. GnIH neurons express the melatonin receptor and melatonin stimulates the expression of GnIH. Because GnIH exists and functions in several avian species, GnIH is considered to be a new key neurohormone controlling avian reproduction. From a broader perspective, subsequently the presence of GnIH homologous peptides has been demonstrated in other vertebrates. Mammalian GnIH homologous peptides also act to inhibit reproduction by decreasing gonadotropin release in several mammalian species. Thus, the discovery of GnIH has opened the door to a new research field in reproductive neurobiology. This review summarizes the advances made in our understanding of the biosynthesis, mode of action and functional significance of GnIH, a newly discovered key neurohormone, and its homologous peptides.     

Tango knock-ins visualize endogenous activity of G protein-coupled receptors in Drosophila

G protein-coupled receptors (GPCRs) represent a family of seven-pass transmembrane protein receptors whose ligands include neuropeptides and small-molecule neuromodulators such as dopamine and serotonin. These neurotransmitters act at long distances and are proposed to define the ground state of the nervous system. The Drosophila genome encodes approximately 50 neuropeptides and their functions in physiology and behavior are now under intensive studies. Key information currently lacking in the field is the spatiotemporal activation patterns of endogenous GPCRs. Here we report application of the Tango system, a reporter assay to detect GPCR activity, to endogenous GPCRs in the fly genome. We developed a method to integrate the sensor component of the Tango system to the C-terminus of endogenous genes by using genome editing techniques. We demonstrate that Tango sensors in the Sex-peptide receptor (SPR) locus allow sensitive detection of mating-dependent SPR activity in the female reproductive organ. The method is easily applicable to any GPCR and will provide a way to systematically characterize GPCRs in the fly brain.     

Genomics, transcriptomics, and peptidomics of neuropeptides and protein hormones in the red flour beetle Tribolium castaneum

Neuropeptides and protein hormones are ancient molecules that mediate cell-to-cell communication. The whole genome sequence from the red flour beetle Tribolium castaneum, along with those from other insect species, provides an opportunity to study the evolution of the genes encoding neuropeptide and protein hormones. We identified 41 of these genes in the Tribolium genome by using a combination of bioinformatic and peptidomic approaches. These genes encode >80 mature neuropeptides and protein hormones, 49 peptides of which were experimentally identified by peptidomics of the central nervous system and other neuroendocrine organs. Twenty-three genes have orthologs in Drosophila melanogaster: Sixteen genes in five different groups are likely the result of recent gene expansions during beetle evolution. These five groups contain peptides related to antidiuretic factor-b (ADF-b), CRF-like diuretic hormone (DH37 and DH47 of Tribolium), adipokinetic hormone (AKH), eclosion hormone, and insulin-like peptide. In addition, we found a gene encoding an arginine-vasopressin-like (AVPL) peptide and one for its receptor. Both genes occur only in Tribolium and not in other holometabolous insects with a sequenced genome. The presence of many additional osmoregulatory peptides in Tribolium agrees well with its ability to live in very dry surroundings. In contrast to these extra genes, there are at least nine neuropeptide genes missing in Tribolium, including the genes encoding the prepropeptides for corazonin, kinin, and allatostatin-A. The cognate receptor genes for these three peptides also appear to be absent in the Tribolium genome. Our analysis of Tribolium indicates that, during insect evolution, genes for neuropeptides and protein hormones are often duplicated or lost.     

Ligand stabilization of CXCR4/delta-opioid receptor heterodimers reveals a mechanism for immune response regulation

The CXCR4 chemokine receptor and the delta opioid receptor (DOR) are pertussis toxin-sensitive G protein-coupled receptors (GPCR). Both are widely distributed in brain tissues and immune cells, and have key roles in inflammation processes and in pain sensation on proximal nerve endings. We show that in immune cells expressing CXCR4 and DOR, simultaneous addition of their ligands CXCL12 and [D-Pen2, D-Pen5]enkephalin does not trigger receptor function. This treatment does not affect ligand binding or receptor expression, nor does it promote heterologous desensitization. Our data indicate that CXCR4 and DOR form heterodimeric complexes that are dynamically regulated by the ligands. This is compatible with a model in which GPCR oligomerization leads to suppression of signaling, promoting a dominant negative effect. Knockdown of CXCR4 and DOR signaling by heterodimerization might have repercussions on physiological and pathological processes such as inflammation, pain sensation and HIV-1 infection.     

Glycosylation of G-protein-coupled receptors for hormones central to normal reproductive functioning: its occurrence and role.

Many hormones that are central to normal reproductive functioning mediate their physiological effects by activating a receptor which belongs to the large family of G-protein-coupled receptors (GPCR). Members of this family of receptor proteins are usually glycosylated on extracellular domains. In recent years the role of this glycosylation in cell surface expression/protein folding, ligand recognition and receptor-effector coupling has been investigated. This review summarises current knowledge of the role of glycosylation in the functioning of the receptors for gonadotrophin-releasing hormone (GnRH), luteinizing hormone/human chorionic gonadotrophin (LH/HCG), follicle stimulating hormone (FSH), oxytocin (OT) and vasopressin (AVP).     

A novel Schistosoma mansoni G protein-coupled receptor is responsive to histamine.

A new cDNA was cloned from the bloodfluke, Schistosoma mansoni and shown to encode a protein with structural characteristics of a biogenic amine G protein-coupled receptor (GPCR). At the amino acid level, the parasite receptor (SmGPCR) shared about the same level of sequence homology (approximately 30%) with all major types of amine GPCRs and could not be identified on the basis of sequence. SmGPCR exhibited several nonconservative substitutions at key GPCR positions, including an unusual asparagine substitution (Asn(111)) for the highly conserved aspartate of transmembrane (TM) 3. The full-length SmGPCR cDNA was double-tagged with N-terminal FLAG and C-terminal hexahistidine epitopes, and was codon-optimized for expression in cultured HEK293 and COS7 cells. In situ immunofluorescence analyses targeting the two N- and C-terminal epitopes demonstrated that the modified SmGPCR was expressed at high level in mammalian cells and assumed a typical GPCR topology, the N-terminus being extracellular and the C-terminus intracellular. Functional activity assays revealed that SmGPCR was responsive to histamine, which caused a dose-dependent elevation in intracellular Ca2+ (EC50=0.54+/-0.05 microM). An Asn(111)-->Asp mutation had no effect on the responsiveness to histamine, suggesting that SmGPCR does not require the TM3 aspartate for agonist activation, in contrast to most amine GPCRs. None of the other monoamines tested had any significant effect on receptor activity, using assays that measured both Ca2+- and cAMP-mediated signaling. The results suggest that SmGPCR is a novel structural class of histamine receptor that may be unique to flatworms.     

Cytomegalovirus-encoded homologs of G protein-coupled receptors and chemokines.

BACKGROUND: Cytomegaloviruses (CMVs) have developed various sophisticated strategies to manipulate and evade the defense mechanisms of their hosts. Among the CMV genes that are predicted to be involved in these strategies are genes that encode mimics of cellular proteins, such as G protein-coupled receptors (GPCRs) and chemokines (CKs). These genes may have been pirated from the host genome during the long co-evolution of virus and host. OBJECTIVES: In this report, the putative functions of the CMV-encoded homologs of GPCRs and CKs in the pathogenesis of infection will be discussed. STUDY DESIGN: In order to present an overview of the current state of knowledge, the literature on the CMV-encoded homologs of GPCRs and CKs was reviewed. RESULTS: The GPCR and CK homologs that are encoded by the CMVs represent immunomodulatory proteins with crucial roles in the pathogenesis of infection. CONCLUSIONS: In light of their function as well as accessibility on the cell surface, the CMV-encoded GPCR homologs are attractive targets for the development of new anti-viral therapies.     

The nicotinic acetylcholine receptor gene family of the honey bee, Apis mellifera

Nicotinic acetylcholine receptors (nAChRs) mediate fast cholinergic synaptic transmission and play roles in many cognitive processes. They are under intense research as potential targets of drugs used to treat neurodegenerative diseases and neurological disorders such as Alzheimer's disease and schizophrenia. Invertebrate nAChRs are targets of anthelmintics as well as a major group of insecticides, the neonicotinoids. The honey bee, Apis mellifera, is one of the most beneficial insects worldwide, playing an important role in crop pollination, and is also a valuable model system for studies on social interaction, sensory processing, learning, and memory. We have used the A. mellifera genome information to characterize the complete honey bee nAChR gene family. Comparison with the fruit fly Drosophila melanogaster and the malaria mosquito Anopheles gambiae shows that the honey bee possesses the largest family of insect nAChR subunits to date (11 members). As with Drosophila and Anopheles, alternative splicing of conserved exons increases receptor diversity. Also, we show that in one honey bee nAChR subunit, six adenosine residues are targeted for RNA A-to-I editing, two of which are evolutionarily conserved in Drosophila melanogaster and Heliothis virescens orthologs, and that the extent of editing increases as the honey bee lifecycle progresses, serving to maximize receptor diversity at the adult stage. These findings on Apis mellifera enhance our understanding of nAChR functional genomics and provide a useful basis for the development of improved insecticides that spare a major beneficial insect species.     

Relaxin-3 systems in the brain--the first 10 years

The relaxin-3 gene was identified in 2001 by searching the human genome database for homologues of the relaxin hormone, and was subsequently discovered to encode a highly conserved neuropeptide in mammals and lower species. In the decade since its discovery there have been significant advances in our knowledge of the peptide, including the identification of its cognate receptor (a type 1 G-protein coupled receptor, GPCR135 or RXFP3), an understanding of its structure–activity and associated cellular signalling, and the elucidation of key neuroanatomical aspects of relaxin-3/RXFP3 networks in mammalian brain. The latter studies revealed that relaxin-3 is expressed within GABA neurons of the brainstem including an area known as the nucleus incertus, and that ascending relaxin-3 projections innervate a broad range of RXFP3-rich forebrain areas. These maps provided a foundation for pharmacological and physiological studies to elucidate the neurobiological nature of relaxin-3/RXFP3 signalling in vivo. Recent findings from our laboratory and others suggest the relaxin-3 neural network represents a newly identified ascending arousal system, able to modulate a range of interrelated functions including responses to stress, spatial and emotional memory, feeding and metabolism, motivation and reward, and circadian rhythm and sleep/wake states. More research is now required to discover further important facts about relaxin-3 neurons, such as their various regulatory inputs, and to characterise populations of RXFP3-positive neurons and determine their influence on particular neural circuits, physiology and complex behaviour.     

Immunofluorescent identification of neuropeptide B-containing nerve fibers and terminals in the rat hypothalamus

Neuropeptide B (NPB) and the structurally related neuropeptide W (NPW) have recently been identified as the endogenous ligands of the orphan G protein-coupled receptors GPR7 and GPR8. Whereas NPW is a high-affinity ligand for both GPR7 and GPR8, NPB activates only GPR7 in sub-nanomolar concentrations. GPR7 is highly conserved in both human and rodent orthologs while GPR8 has not been found in rodents. GPR7 mRNA is expressed in discrete regions of the hypothalamus suggesting a role in the regulation of energy homeostasis and neuroendocrine axes. In the present study, we have generated and extensively characterized antibodies that exert selective specificity for NPB. In dot-blot assays, these antibodies detected NPB but not NPW. Immunofluorescent staining of rat brain sections revealed moderately dense plexus of NPB-immunoreactive fibers and terminals in discrete areas of the hypothalamus. Neuronal somata were only seen in colchicine-treated rats. This immunostaining was completely abolished by preincubation of the antibodies with NPB but not with NPW. NPB-immunoreactivity was enriched in many regions within the hypothalamus which also contained high levels of GPR7 mRNA including the ventromedial hypothalamic nucleus, dorsomedial hypothalamic nucleus, arcuate nucleus, supraoptic retrochiasmatic nucleus, and in the area ventral to the zona incerta. Together, NPB and its receptor GPR7 exist in close proximity in the rat hypothalamus and are, hence, ideally positioned to modulate neuroendocrine functions.     

Functional significance of serotonin receptor dimerization

The original model of G-protein activation by a single G-protein-coupled receptor (GPCR) is giving way to a new model, wherein two protomers of a GPCR dimer interact with a single G-protein. This article will review the evidence suggesting that 5-HT receptors form dimers/oligomers and will compare the findings with the results obtained from the studies with other biogenic amine receptors. Topics to be covered include the origin or biogenesis of dimer formation, potential dimer interface(s), and oligomer size (dimer vs. tetramer or higher order). The functional significance will be discussed in terms of G-protein activation following ligand binding to one or two protomers in a dimeric structure, the formation of heterodimers, and the development of bivalent ligands.     

Evolution of the Yellow/Major Royal Jelly Protein family and the emergence of social behavior in honey bees

The genomic architecture underlying the evolution of insect social behavior is largely a mystery. Eusociality, defined by overlapping generations, parental brood care, and reproductive division of labor, has most commonly evolved in the Hymenopteran insects, including the honey bee Apis mellifera. In this species, the Major Royal Jelly Protein (MRJP) family is required for all major aspects of eusocial behavior. Here, using data obtained from the A. mellifera genome sequencing project, we demonstrate that the MRJP family is encoded by nine genes arranged in an approximately 60-kb tandem array. Furthermore, the MRJP protein family appears to have evolved from a single progenitor gene that encodes a member of the ancient Yellow protein family. Five genes encoding Yellow-family proteins flank the genomic region containing the genes encoding MRJPs. We describe the molecular evolution of these protein families. We then characterize developmental-stage-specific, sex-specific, and caste-specific expression patterns of the mrjp and yellow genes in the honey bee. We review empirical evidence concerning the functions of Yellow proteins in fruit flies and social ants, in order to shed light on the roles of both Yellow and MRJP proteins in A. mellifera. In total, the available evidence suggests that Yellows and MRJPs are multifunctional proteins with diverse, context-dependent physiological and developmental roles. However, many members of the Yellow/MRJP family act as facilitators of reproductive maturation. Finally, it appears that MRJP protein subfamily evolution from the Yellow protein family may have coincided with the evolution of honey bee eusociality.     

ProtoBee: hierarchical classification and annotation of the honey bee proteome

The recently sequenced genome of the honey bee (Apis mellifera) has produced 10,157 predicted protein sequences, calling for a computational effort to extract biological insights from them. We have applied an unsupervised hierarchical protein-clustering method, which was previously used in the ProtoNet system, to nearly 200,000 proteins consisting of the predicted honey bee proteins, the SWISS-PROT protein database, and the complete set of proteins of the mouse (Mus musculus) and the fruit fly (Drosophila melanogaster). The hierarchy produced by this method has been entitled ProtoBee. In ProtoBee, the proteins are hierarchically organized into 18,936 separate tree hierarchies, each representing a protein functional family. By using the mouse and Drosophila complete proteomes as reference, we are able to highlight functional groups of putative gene-loss events, putative novel proteins of unique functionality, and bee-specific paralogs. We have studied some of the ProtoBee findings and suggest their biological relevance. Examples include novel opsin genes and intriguing nuclear matches of mitochondrial genes. The organization of bee sequences into functional clusters suggests a natural way of automatically inferring functional annotation. Following this notion, we were able to assign functional annotation to about 70% of the sequences. ProtoBee is available at http://www.protobee.cs.huji.ac.il.     

The Brugia malayi neuropeptide receptor-4 is activated by FMRFamide-like peptides and signals via Gαi

Genetic studies undertaken in the model organism Caenorhabditis elegans have demonstrated the importance of neuropeptidergic signalling in nematode physiology. Disruption of this signalling may have deleterious phenotypic consequences, including altered locomotion, feeding behaviour, and reproduction. Neuropeptide G protein-coupled receptors (GPCRs) that transduce many of these signals therefore represent cogent drug targets. Recently published genomic sequencing data for a number of parasitic helminths of medical and veterinary importance has revealed the apparent conservation of a number of neuropeptides, and neuropeptide receptors between parasitic and free-living species, raising the intriguing possibility of developing broad-spectrum anthelmintic therapeutics. Here, we identify and clone a neuropeptide receptor, NPR-4, from the human filarial nematode Brugia malayi and demonstrate its activation in vitro, by FMRFamide-like peptides of the FLP-18 family, and intracellular signalling via Gα_i mediated pathways. These data represent the first example of deorphanisation of a neuropeptide GPCR in any parasitic helminth species.