Thomas M. Laz

GABA B receptors function as a heteromeric assembly of the subunits GABA B R1 and GABA B R2

The principal inhibitory neurotransmitter GABA (γ-aminobutyric acid) exerts its effects through two ligand-gated channels, GABAA and GABAC receptors, and a third receptor, GABAB (ref. 1), which acts through G proteins to regulate potassium and calcium channels. Cells heterologously expressing the cloned DNA encoding the GABABR1 protein exhibit high-affinity antagonist-binding sites, but they produce little of the functional activity expected from studies of endogenous GABAB receptors in the brain. Here we describe a new member of the GABAB polypeptide family, GABABR2, that shows sequence homology to GABABR1. Neither GABABR1 nor GABABR2, when expressed individually, activates GIRK-type potassium channels; however, the combination of GABABR1 and GABABR2 confers robust stimulation of channel activity. Both genes are co-expressed in individual neurons, and both proteins co-localize in transfected cells. Moreover, immunoprecipitation experiments indicate that the two polypeptides associate with each other, probably as heterodimers. Several G-protein-coupled receptors (GPCRs) exist as high-molecular-weight species, consistent with the formation of dimers by these receptors, but the relevance of these species for the functioning of GPCRs has not been established. We have now shown that co-expression of two GPCR structures, GABABR1 and GABABR2, belonging to the samesubfamily is essential for signal transduction by GABAB receptors.

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.

Human members of the eukaryotic protein kinase family

BackgroundEukaryotic protein kinases (EPKs) constitute one of the largest recognized protein families represented in the human genome. EPKs, which are similar to each other in sequence, structure and biochemical properties, are important players in virtually every signaling pathway involved in normal development and disease. Near completion of projects to sequence the human genome and transcriptome provide an opportunity to identify and perform sequence analysis on a nearly complete set of human EPKs.ResultsPublicly available genetic sequence data were searched for human sequences that potentially represent EPK family members. After removal of duplicates, splice variants and pseudogenes, this search yielded 510 sequences with recognizable similarity to the EPK family. Protein sequences of putative EPK catalytic domains identified in the search were aligned, and a phonogram was constructed based on the alignment. Representative sequence records in GenBank were identified, and derived information about gene mapping and nomenclature was summarized.ConclusionsThis work represents a nearly comprehensive census and early bioinformatics overview of the EPKs encoded in the human genome. Evaluation of the sequence relationships between these proteins contributes contextual information that enhances understanding of individual family members. This curation of human EPK sequences provides tools and a framework for the further characterization of this important class of enzymes.

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.

Molecular biology and pharmacology of multiple NPY Y5 receptor species homologs

NPY is a 36-amino acid peptide which exerts its physiological effects through the activation of a family of G-protein coupled receptors. In vivo and in vitro characterization of the recently cloned rat Y5 receptor suggests that it is a primary mediator of NPY-induced feeding (Gerald et al., Nature 1996;382:168-171). We now report the molecular cloning and pharmacological characterization of the human, dog and mouse homologs of the Y5 receptor. With the exception of a 21 amino acid repeat in the amino terminus of the mouse Y5 receptor, the sequence of the four species homologs appear to be highly conserved, with 88% to 97% amino acid identities between any two species. Similarly, the pharmacological profiles of the four species homologs as determined in porcine 125I-PYY binding assays show a great deal of conservation, with the following rank order of affinity: human or porcine NPY, PYY, [Leu31,Pro34]NPY, NPY(2-36), human PP > human [D-Trp32]NPY > rat PP, C2-NPY. Northern blot analysis reveals that the Y5 receptor is widely distributed in the human brain, with the strongest signals detected in the cortex, putamen and caudate nucleus. The chromosomal localization of the human Y5 receptor, previously shown to be overlapping and in the opposite orientation to the Y1 receptor, is determined to be 4q31, the same locus as previously demonstrated for the human Y1 receptor (Herzog et al., J Biol Chem 1993;268:6703-6707), suggesting that these receptors may be coregulated. These Y5 species homologs along with corresponding animal models may be useful in the search for novel therapeutics in the treatment of obesity and related feeding disorders.