Marvin Bayne

ADP Is the Cognate Ligand for the Orphan G Protein-coupled Receptor SP1999

P2Y receptors are a class of G protein-coupled receptors activated primarily by ATP, UTP, and UDP. Five mammalian P2Y receptors have been cloned so far including P2Y1, P2Y2, P2Y4, P2Y6, and P2Y11. P2Y1, P2Y2, and P2Y6 couple to the activation of phospholipase C, whereas P2Y4 and P2Y11 couple to the activation of both phospholipase C and the adenylyl cyclase pathways. Additional ADP receptors linked to Gαi have been described but have not yet been cloned. SP1999 is an orphan G protein-coupled receptor, which is highly expressed in brain, spinal cord, and blood platelets. In the present study, we demonstrate that SP1999 is a Gαi-coupled receptor that is potently activated by ADP. In an effort to identify ligands for SP1999, fractionated rat spinal cord extracts were assayed for Ca2+ mobilization activity against Chinese hamster ovary cells transiently transfected with SP1999 and chimeric Gα subunits (Gαq/i). A substance that selectively activated SP1999-transfected cells was identified and purified through a series of chromatographic steps. Mass spectral analysis of the purified material definitively identified it as ADP. ADP was subsequently shown to inhibit forskolin-stimulated adenylyl cyclase activity through selective activation of SP1999 with an EC50 of 60 nm. Other nucleotides were able to activate SP1999 with a rank order of potency 2-MeS-ATP = 2-MeS-ADP > ADP = adenosine 5′-O-2-(thio)diphosphate > 2-Cl-ATP > adenosine 5′-O-(thiotriphosphate). Thus, SP1999 is a novel, Gαi-linked receptor for ADP.

Cloning and Expressional Characterization of a Novel Galanin Receptor IDENTIFICATION OF DIFFERENT PHARMACOPHORES WITHIN GALANIN FOR THE THREE GALANIN RECEPTOR SUBTYPES

Galanin, a 29–30 amino acid neuropeptide, is found in the central and peripheral nervous systems and displays several important physiological activities. The actions are believed to be mediated through distinct G protein-coupled receptors. To date, two galanin receptor subtypes have been cloned. In this report, we describe the cloning and expression of a cDNA encoding a novel galanin receptor (GalR3). The receptor has 370 amino acids and shares 36 and 54% homology with the rat GalR1 and GalR2 receptors.125I-Porcine galanin binds the rat GalR3 receptor expressed in COS-7 cells with high affinity (K d = 0.6 nm) and could be displaced by galanin and galanin fragments and galanin-chimeric peptides. The pharmacological profile of this novel receptor is distinct from those of GalR1 and GalR2, revealing different pharmacophores within galanin for the three galanin receptor subtypes. Northern blot analysis showed expression in heart, spleen, and testis. Unlike GalR1 and GalR2, no expression of GalR3 was detectable in the brain, suggesting that GalR3 may mediate some of the peripheral functions of galanin.

Presenilins Interact with Armadillo Proteins Including Neural‐Specific Plakophilin‐Related Protein and β‐Catenin

Abstract : Missense substitutions in the presenilin 1 (PS1) and presenilin 2 (PS2) proteins are associated with early‐onset familial Alzheimers disease. We have used yeast‐two‐hybrid and coimmunoprecipitation methods to show that the large cytoplasmic loop domains of PS1 and PS2 interact specifically with three members of the armadillo protein family, including β‐catenin, p0071, and a novel neuronal‐specific armadillo protein—neural plakophilin‐related armadillo protein (NPRAP). The PS1 : NPRAP interaction occurs between the arm repeats of NPRAP and residues 372‐399 at the C‐terminal end of the large cytoplasmic loop of PS1. The latter residues contain a single arm‐like domain and are highly conserved in the presenilins, suggesting that they form a functional armadillo protein binding site for the presenilins.

Identification of a Novel Neuromedin U Receptor Subtype Expressed in the Central Nervous System

Neuromedin U is a neuropeptide prominently expressed in the upper gastrointestinal tract and central nervous system. Recently, GPR66/FM-3 (NmU-R1) was identified as a specific receptor for neuromedin U. A BLAST search of the GenBankTM genomic database using the NmU-R1 cDNA sequence revealed a human genomic fragment encoding a G protein-coupled receptor that we designated NmU-R2 based on its homology to NmU-R1. The full-length NmU-R2 cDNA was subsequently cloned, stably expressed in 293 cells, and shown to mobilize intracellular calcium in response to neuromedin U. This response was dose-dependent (EC50 = 5 nm) and specific in that other neuromedins did not induce a calcium flux in receptor-transfected cells. Expression analysis of human NmU-R2 demonstrated its mRNA to be most highly expressed in central nervous system tissues. Based on these data, we conclude that NmU-R2 is a novel neuromedin U receptor subtype that is likely to mediate central nervous system-specific neuromedin U effects.

A novel hepatointestinal leukotriene B4 receptor. Cloning and functional characterization.

Leukotriene B4 (LTB4) is a product of eicosanoid metabolism and acts as an extremely potent chemotactic mediator for inflammation. LTB4 exerts positive effects on the immigration and activation of leukocytes. These effects suggest an involvement of LTB4 in several diseases: inflammatory bowel disease, psoriasis, arthritis, and asthma. LTB4 elicits actions through interaction with one or more cell surface receptors that lead to chemotaxis and inflammation. One leukotriene B4 receptor has been recently identified (LTB4-R1). In this report we describe cloning of a cDNA encoding a novel 358-amino acid receptor (LTB4-R2) that possesses seven membrane-spanning domains and is homologous (42%) and genetically linked to LTB4-R1. Expression of LTB4-R2 is broad but highest in liver, intestine, spleen, and kidney. In radioligand binding assays, membranes prepared from COS-7 cells transfected with LTB4-R2 cDNA displayed high affinity (K d = 0.17 nm) for [3H]LTB4. Radioligand competition assays revealed high affinities of the receptor for LTB4 and LTB5, and 20-hydroxy-LTB4, and intermediate affinities for 15(S)-HETE and 12-oxo-ETE. Three LTB4 receptor antagonists, 14,15-dehydro-LTB4, LTB4-3-aminopropylamide, and U-75302, had high affinity for LTB4-R1 but not for LTB4-R2. No apparent affinity binding for the receptors was detected for the CysLT1-selective antagonists montelukast and zafirlukast. LTB4 functionally mobilized intracellular calcium and inhibited forskolin-stimulated cAMP production in 293 cells. The discovery of this new receptor should aid in further understanding the roles of LTB4 in pathologies in these tissues and may provide a tool in identification of specific antagonists/agonists for potential therapeutic treatments.

Genomic organization and functional characterization of the mouse GalR1 galanin receptor.

Galanin mediates diverse physiological functions in digestive, endocrine, and central nervous systems through G‐protein‐coupled receptors. Two galanin receptors have been cloned but the gene structures are unknown. We report genomic and cDNA cloning of the mouse GalR1 galanin receptor and demonstrate that the coding sequence is uniquely divided into three exons encoding the N‐terminal portion through the fifth transmebrane domain, the third intracellular loop, and the sixth transmembrane domain through the C‐terminus. Functional analysis of the encoded cDNA revealed active ligand binding and intracellular signaling. The expression is detected in brain, spinal cord, heart and skeletal muscle.

The GalR2 galanin receptor mediates galanin‐induced jejunal contraction, but not feeding behavior, in the rat: differentiation of central and peripheral effects of receptor subtype activation

The neuropeptide galanin mediates a diverse array of physiological functions through activation of specific receptors. Roles of the three recently cloned galanin receptors (GalRs) in rat intestinal contraction and food intake were examined using GalR‐selective ligands and the results were compared with the pharmacological profiles of defined GalRs. The action profile of these ligands in jejunal contraction resembled only that of GalR2 and only a high level of GalR2 mRNA was detected in the tissue, supporting GalR2 as the receptor mediating jejunal contraction. The action profile for food intake in rats excluded GalR2, GalR3 and the putative pituitary galanin receptor as the ‘feeding receptor’, suggesting that either GalR1 or an unidentified GalR is responsible for mediating this function.

The Mouse GalR2 Galanin Receptor: Genomic Organization, cDNA Cloning, and Functional Characterization

Abstract: The diverse physiological actions of galanin are thought to be mediated through activation of galanin receptors (GalRs). We report the genomic and cDNA cloning of a mouse GalR that possesses a genomic structure distinct from that of GalR1 and encodes a functional galanin receptor. The mouse GalR gene consists of two exons separated by a single intron within the protein‐coding region. The splicing site for the intron is located at the junction between the third transmembrane domain and the second intracellular loop. The cDNA encodes a 370‐amino acid putative G protein‐coupled receptor that is markedly different from human GalR1 and rat GalR3 (38 and 57%) but shares high homology with rat GalR2 (94%). In binding studies utilizing membranes from COS‐7 cells transfected with mouse GalR2 cDNA, the receptor displayed high affinity (KD = 0.47 nM) and saturable binding with 125I‐galanin (Bmax = 670 fmol/mg). The radioligand binding can be displaced by galanin and its analogues in a rank order: galanin ⋍ M40 ⋍ M15 ⋍ M35 ⋍ C7 ⋍ galanin (2–29) ⋍ galanin (1–16) ≫ galanin (10–29) ⋍ galanin (3–29), which resembles the pharmacological profile of the rat GalR2. Receptor activation by galanin in COS‐7 cells stimulated phosphoinositide metabolism, which was not reversed by pertussis toxin. Thus, the galanin receptor encoded in the cloned mouse GalR gene is the type 2 galanin receptor and is active in both ligand binding and signaling assays.

Cloning and characterization of murine neuromedin U receptors

Neuromedin U (NmU) is a neuropeptide involved in various physiological functions such as feeding behavior, muscle contractile activity, and regulation of intestinal ion transport. Recently, two human G protein-coupled receptors have been identified as NmU-specific receptors, NmU-R1 and NmU-R2, which share 55% amino acid identity. It is unclear however, which of the two receptors mediates responses to NmU observed in rodent models. Attempts to define the pharmacological profile of the two receptors are confounded by overlapping expression of the two receptors and a lack of subtype-selective compounds. In order to establish a basis to further our understanding of the function of these receptors, we cloned and characterized the mouse homologues of the two human NmU receptors. Mouse NmU-R1 and mouse NmU-R2 are 79 and 81% identical to their respective human homologues. Expression of NmU-R1 was mainly observed in testis, gastrointestinal (GI) tract, and immune system, while NmU-R2 was primarily expressed in brain tissues. Each mouse receptor was independently expressed in HEK293 cells and demonstrated a dose-dependent calcium flux in response to NmU-8, NmU-23 and NmU-25. In an attempt to identify a synthetic NmU peptide that would exhibit selectivity at one of the two receptors, we examined the functional activity of eight alanine-substituted NmU-8 peptides. These experiments demonstrated that alanine substitution at positions 5 and 7 affects the functional activity of the peptide at both receptors. The arginine residue at position 7 is required for NmU-8 activity at either receptor while alanine substitution at position 5 selectively affects the potency and the efficacy at mNmU-R1. These experiments validate the use of rodent models to characterize NmU function relative to humans and suggest that substitution at Arginine-5 of NmU-8 may provide a receptor selective peptide.

Peppat, a pattern-based oligopeptide homology search method and the identification of a novel tachykinin-like peptide

PepPat, a hybrid method that combines pattern matching with similarity scoring, is described. We also report PepPats application in the identification of a novel tachykinin-like peptide. PepPat takes as input a query peptide and a user-specified regular expression pattern within the peptide. It first performs a database pattern match and then ranks candidates on the basis of their similarity to the query peptide. PepPat calculates similarity over the pattern spanning region, enhancing PepPats sensitivity for short query peptides. PepPat can also search for a user-specified number of occurrences of a repeated pattern within the target sequence. We illustrate PepPats application in short peptide ligand mining. As a validation example, we report the identification of a novel tachykinin-like peptide, C14TKL-1, and show it is an NK1 (neuokinin receptor 1) agonist whose message is widely expressed in human periphery. Availability: PepPat is offered online at: http://peppat.cbi.pku.edu.cn