Cloning of a cDNA encoding a non-isopeptide-selective subtype of the endothelin receptor

Endothelin-1 was initially identified as a 21-residue potent vasoconstrictor peptide produced by vascular endothelial cells, but was subsequently found to have many effects on both vascular and non-vascular tissues. The discovery of three isopeptides of the endothelin family, ET-1, ET-2 and ET-3, each possessing a diverse set of pharmacological activities of different potency, suggested the existence of several different endothelin receptor subtypes. Endothelins may elicit biological responses by various signal-transduction mechanisms, including the G protein-coupled activation of phospholipase C and the activation of voltage-dependent Ca2+ channels. Thus, different subtypes of the endothelin receptor may use different signal-transduction mechanisms. Here we report the cloning of a complementary DNA encoding one subtype belonging to the superfamily of G protein-coupled receptors. COS-7 cells transfected with the cDNA express specific and high-affinity binding sites for endothelins, responding to binding by the production of inositol phosphates and a transient increase in the concentration of intracellular free Ca2+. The three endothelin isopeptides are roughly equipotent in displacing 125I-labelled ET-1 binding and causing Ca2+ mobilization. A messenger RNA corresponding to the cDNA is detected in many rat tissues including the brain, kidney and lung but not in vascular smooth muscle cells. These results indicate that this cDNA encodes a 'nonselective' subtype of the receptor which is different from the vascular smooth muscle receptor.     

Differential activation by atrial and brain natriuretic peptides of two different receptor guanylate cyclases

Alpha atrial natriuretic peptide (alpha-ANP) and brain natriuretic peptide are homologous polypeptide hormones involved in the regulation of fluid and electrolyte homeostasis. These two natriuretic peptides apparently share common receptors and stimulate the intracellular production of cyclic GMP as a second messenger. Molecular cloning has defined two types of natriuretic peptide receptors: the ANP-C receptor of relative molecular mass (Mr) 60-70,000 (60-70 K), which is not coupled to cGMP production and may function in the clearance of ANP and the ANP-A receptor of Mr 120-140 K, which is a membrane form of guanylate cyclase in which ligand binding to the extracellular domain activates the cytoplasmic domain of the enzyme. Here we report the cloning and expression of a second human natriuretic peptide-receptor guanylate cyclase, the ANP-B receptor. The ANP-B receptor is preferentially activated by porcine brain natriuretic peptide rather than human alpha-ANP, whereas the ANP-A receptor responds similarly to both natriuretic peptides. These observations may have important implications for our understanding of the central and peripheral control of cardiovascular homeostasis.     

Molecular cloning and expression of a rat V1a arginine vasopressin receptor.

The neurohypophyseal hormone arginine vasopressin has diverse actions, including the inhibition of diuresis, contraction of smooth muscle, stimulation of liver glycogenolysis and modulation of adrenocorticotropic hormone release from the pituitary. Arginine vasopressin receptors are G protein-coupled and have been divided into at least three types; the V1a (vascular/hepatic) and V1b (anterior pituitary) receptors which act through phosphatidylinositol hydrolysis to mobilize intracellular Ca2+, and the V2 (kidney) receptor which is coupled to adenylate cyclase. We report here the cloning of a complementary DNA encoding the hepatic V1a arginine vasopressin receptor. The liver cDNA encodes a protein with seven putative transmembrane domains, which binds arginine vasopressin and related compounds with affinities similar to the native rat V1a receptor. The messenger RNA corresponding to the cDNA is distributed in rat tissues known to contain V1a receptors.     

Molecular cloning and expression of the gene for a human D1 dopamine receptor

The diverse physiological actions of dopamine are mediated by its interaction with two basic types of G protein-coupled receptor, D1 and D2, which stimulate and inhibit, respectively, the enzyme adenylyl cyclase. Alterations in the number or activity of these receptors may be a contributory factor in diseases such as Parkinson's disease and schizophrenia. Here we describe the isolation and characterization of the gene encoding a human D1 dopamine receptor. The coding region of this gene is intronless, unlike the gene encoding the D2 dopamine receptor. The D1 receptor gene encodes a protein of 446 amino acids having a predicted relative molecular mass of 49,300 and a transmembrane topology similar to that of other G protein-coupled receptors. Transient or stable expression of the cloned gene in host cells established specific ligand binding and functional activity characteristic of a D1 dopamine receptor coupled to stimulation of adenylyl cyclase. Northern blot analysis and in situ hybridization revealed that the messenger RNA for this receptor is most abundant in caudate, nucleus accumbens and olfactory tubercle, with little or no mRNA detectable in substantia nigra, liver, kidney, or heart. Several observations from this work in conjunction with results from other studies are consistent with the idea that other D1 dopamine receptor subtypes may exist.     

Cloning and expression of a rat D2 dopamine receptor cDNA

Dopamine receptors are classified into D1 and D2 subtypes on the basis of their pharmacological and biochemical characteristics. The D2 dopamine receptor has been implicated in the pathophysiology and treatment of movement disorders, schizophrenia and drug addiction. The D2 dopamine receptor interacts with guanine nucleotide-binding proteins to induce second messenger systems. Other members of the family of receptors that are coupled to G proteins share a significant similarity in primary amino-acid sequence and exhibit an archetypical topology predicted to consist of seven putative transmembrane domains. We have taken advantage of the expected nucleotide sequence similarities among members of this gene family to isolate genes coding for new receptors. Using the hamster beta 2-adrenergic receptor gene as a hybridization probe we have isolated related genes including a cDNA encoding the rat D2 dopamine receptor. This receptor has been characterized on the basis of three criteria: the deduced amino-acid sequence which reveals that it is a member of the family of G-protein-coupled receptors; the tissue distribution of the mRNA which parallels that of the D2 dopamine receptor; and the pharmacological profile of mouse fibroblast cells transfected with the cDNA.     

Cloning and expression of human and rat D1 dopamine receptors

The importance of the dopaminergic system in brain function has been emphasized by its association with neurological and psychiatric disorders such as Parkinson's disease and schizophrenia. On the basis of their biochemical and pharmacological characteristics, dopamine receptors are classified into D1 and D2 subtypes. As the most abundant dopamine receptor in the central nervous system, D1 receptors seem to mediate some behavioural responses, modulate activity of D2 dopamine receptors, and regulate neuron growth and differentiation. The D dopamine receptor has been cloned by low-stringency screening. We report here the cloning of human and rat D1 dopamine receptors by applying an approach based on the polymerase chain reaction. The cloned human D1 dopamine receptor has been characterized on the basis of four criteria: the deduced amino-acid sequence, which reveals that it is a G protein-coupled receptor; the tissue distribution of its messenger RNA, which is compatible with that of the D1 dopamine receptor; its pharmacological profile when transfected into COS-7 cells; and its ability to stimulate the accumulation of cyclic AMP in human 293 cells.     

Cloning of the gene for a human dopamine D5 receptor with higher affinity for dopamine than D1.

Dopamine receptors belong to a superfamily of receptors that exert their biological effects through guanine nucleotide-binding (G) proteins. Two main dopamine receptor subtypes have been identified, D1 and D2, which differ in their pharmacological and biochemical characteristics. D1 stimulates adenylyl cyclase activity, whereas D2 inhibits it. Both receptors are primary targets for drugs used to treat many psychomotor diseases, including Parkinson's disease and schizophrenia. Whereas the dopamine D1 receptor has been cloned, biochemical and behavioural data indicate that dopamine D1-like receptors exist which either are not linked to adenylyl cyclase or display different pharmacological activities. We report here the cloning of a gene encoding a 477-amino-acid protein with strong homology to the cloned D1 receptor. The receptor, called D5, binds drugs with a pharmacological profile similar to that of the cloned D1 receptor, but displays a 10-fold higher affinity for the endogenous agonist, dopamine. As with D1, the dopamine D5 receptor stimulates adenylyl cyclase activity. Northern blot and in situ hybridization analyses reveal that the receptor is neuron-specific, localized primarily within limbic regions of the brain; no messenger RNA was detected in kidney, liver, heart or parathyroid gland. The existence of a dopamine D1-like receptor with these characteristics had not been predicted and may represent an alternative pathway for dopamine-mediated events and regulation of D2 receptor activity.     

Mast cells promote homeostasis by limiting endothelin-1-induced toxicity

Endothelin-1 (ET-1) is a 21-amino-acid peptide, derived from vascular endothelial cells, with potent vasoconstrictor activity. ET-1 has been implicated in diverse physiological or pathological processes, including the vascular changes associated with sepsis. However, the factors that regulate ET-1-associated toxicity during bacterial infections, or in other settings, are not fully understood. Both the pathology associated with certain allergic and autoimmune disorders, and optimal host defence against bacterial and parasitic infections are mediated by mast cells. In vitro, mast cells can produce ET-1 (ref. 11), undergo ET-1-dependent and endothelin-A receptor (ET(A))-dependent activation, and release proteases that degrade ET-1 (ref. 14). Although the potential relationships between mast cells and the ET-1 system thus may be complex, the importance of interactions between ET-1 and mast cells in vivo is obscure. Here we show that ET(A)-dependent mast-cell activation can diminish both ET-1 levels and ET-1-induced pathology in vivo, and also can contribute to optimal survival during acute bacterial peritonitis. These findings identify a new biological function for mast cells: promotion of homeostasis by limiting the toxicity associated with an endogenous mediator.     

Cloning and expression of a complementary DNA encoding a bovine adrenal angiotensin II type-1 receptor

Angiotensin II elicits different responses which affect cardiovascular, neuronal and electrolyte transport regulation. To understand the mechanisms responsible for its various actions, the receptor for angiotensin II has long been sought, but numerous attempts to purify the receptor have been unsuccessful owing to its instability and low concentration. We report here the expression cloning of a complementary DNA encoding a bovine angiotensin II receptor to overcome these difficulties. The receptor cDNA encodes a protein of 359 amino-acid residues with a transmembrane topology similar to that of other G protein-coupled receptors. COS-7 cells transfected with the cDNA expressed specific and high-affinity binding sites for angiotensin II, angiotensin II antagonist and a non-peptide specific antagonist for type-1 receptor. Dithiothreitol inhibited ligand binding. The concentration of intracellular Ca2+ and of inositol-1,4,5-trisphosphate increased in the transfected COS-7 cells in response to angiotensin II or angiotensin III, indicating that this receptor is the type-1 receptor for angiotensin II. Northern blot analysis revealed that the messenger RNA for this receptor is expressed in bovine adrenal medulla, cortex and kidney.     

Importance of a novel GABAA receptor subunit for benzodiazepine pharmacology

Neurotransmission effected by GABA (gamma-aminobutyric acid) is predominantly mediated by a gated chloride channel intrinsic to the GABAA receptor. This heterooligomeric receptor exists in most inhibitory synapses in the vertebrate central nervous system (CNS) and can be regulated by clinically important compounds such as benzodiazepines and barbiturates. The primary structures of GABAA receptor alpha- and beta-subunits have been deduced from cloned complementary DNAs. Co-expression of these subunits in heterologous systems generates receptors which display much of the pharmacology of their neural counterparts, including potentiation by barbiturates. Conspicuously, however, they lack binding sites for, and consistent electrophysiological responses to, benzodiazepines. We now report the isolation of a cloned cDNA encoding a new GABAA receptor subunit, termed gamma 2, which shares approximately 40% sequence identity with alpha- and beta-subunits and whose messenger RNA is prominently localized in neuronal subpopulations throughout the CNS. Importantly, coexpression of the gamma 2 subunit with alpha 1 and beta 1 subunits produces GABAA receptors displaying high-affinity binding for central benzodiazepine receptor ligands.     

A synthetic peptide that is a bombesin receptor antagonist

The tetradecapeptide bombesin was originally isolated from frog skin. Bombesin-like peptides have since been detected in mammalian gastrointestinal tract, brain and lung. These peptides have potent pharmacological effects on the central nervous system; they cause contraction of intestinal, uterine and urinary tract smooth muscle; and stimulate the release of other peptides including gastrin, cholecystokinin, motilin, pancreatic polypeptide, neurotensin, insulin, enteroglucagon, prolactin and growth hormone. Specific plasma membrane receptors for bombesin have been demonstrated on pancreatic acinar cells, brain membranes and pituitary cells. Studies defining the physiological importance of bombesin have been impeded by the lack of a bombesin receptor antagonist. Here we describe experiments which demonstrate that a peptide originally described as a substance P receptor antagonist, [D-Arg, D-Pro, D-Trp, Leu ]substance P, is also a bombesin receptor antagonist. This peptide competitively inhibits the ability of bombesin to stimulate enzyme secretion from dispersed pancreatic acini, and also inhibits the action of other peptides that interact with the bombesin receptor.     

Association of CD2 and CD45 on human T lymphocytes

At least two membrane receptors have been defined through which human T lymphocytes can be induced to proliferate and differentiate, namely the CD3-Ti antigen receptor complex and the CD2 molecule. Monoclonal antibodies directed at either CD2 or CD3 induce intracellular second messenger production and subsequent protein phosphorylation. On most human non-B lymphocytes, CD3-Ti and CD2 are coexpressed and seem to be functionally interrelated. But there are minor subpopulations in which these receptor systems can transduce signals despite a mutually exclusive expression, indicating that CD3-Ti and CD2 can act independently of each other. This view is supported by the finding that most monoclonal antibodies directed at the CD45 molecules are strongly co-mitogenic with CD2 but not CD3 monoclonal antibodies. As the intracytoplasmic domains of CD45 have tyrosine phosphatase activity these functional effects could be explained by a physical association between CD2 and CD45. Using chemical crosslinking techniques, we now show that CD45 is linked to CD2 on the surface of human T lymphocytes.     

Distinct molecular mechanism for initiating TRAF6 signalling

Tumour-necrosis factor (TNF) receptor-associated factor 6 (TRAF6) is the only TRAF family member that participates in signal transduction of both the TNF receptor (TNFR) superfamily and the interleukin-1 receptor (IL-1R)/Toll-like receptor (TLR) superfamily; it is important for adaptive immunity, innate immunity and bone homeostasis. Here we report crystal structures of TRAF6, alone and in complex with TRAF6-binding peptides from CD40 and TRANCE-R (also known as RANK), members of the TNFR superfamily, to gain insight into the mechanism by which TRAF6 mediates several signalling cascades. A 40 degrees difference in the directions of the bound peptides in TRAF6 and TRAF2 shows that there are marked structural differences between receptor recognition by TRAF6 and other TRAFs. The structural determinant of the petide TRAF6 interaction reveals a Pro-X-Glu-X-X-(aromatic/acidic residue) TRAF6-binding motif, which is present not only in CD40 and TRANCE-R but also in the three IRAK adapter kinases for IL-1R/TLR signalling. Cell-permeable peptides with the TRAF6-binding motif inhibit TRAF6 signalling, which indicates their potential as therapeutic modulators. Our studies identify a universal mechanism by which TRAF6 regulates several signalling cascades in adaptive immunity, innate immunity and bone homeostasis.     

Cloning of a putative high-affinity kainate receptor expressed predominantly in hippocampal CA3 cells.

Kainic acid is a potent neurotoxin for certain neurons. Its neurotoxicity is thought to be mediated by an excitatory amino-acid-gated ion channel (ionotropic receptor) possessing nanomolar affinity for kainate. Here we describe a new member of the rat excitatory amino-acid receptor gene family, KA-1, that has a 30% sequence similarity with the previously characterized alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor subunits GluR-A to -D. The pharmacological profile of expressed recombinant KA-1 determined in binding experiments with [3H]kainate is different from that of the cloned AMPA receptors and similar to the mammalian high-affinity kainate receptor (kainate greater than quisqualate greater than glutamate much greater than AMPA) with a dissociation constant of about 5 nM for kainate. The selectively high expression of KA-1 messenger RNA in the CA3 region of the hippocampus closely corresponds to autoradiographically located high-affinity kainate binding sites. This correlation, as well as the particular in vivo pattern of neurodegeneration observed on kainate-induced neurotoxicity, suggests that KA-1 participates in receptors mediating the kainate sensitivity of neurons in the central nervous system.     

A histamine-activated chloride channel involved in neurotransmission at a photoreceptor synapse

Compared with the variety of neuromodulatory agents acting through second messenger systems, the number of fast neurotransmitters which directly activate ion channels is limited. Thus, synaptic receptors that act as ligand-gated ion channels have been firmly established only for acetylcholine, glycine, GABA and glutamate, with the first three of these belonging to the same molecular superfamily. Recently, however, a possible addition to this list has been suggested as a result of evidence implicating histamine as the neurotransmitter released by a variety of arthropod photoreceptors. Neurotransmission at this synapse has been studied extensively, particularly in the fly. The postsynaptic elements, large monopolar cells, respond to light with a rapid, chloride-mediated hyperpolarization that can be mimicked by the application of histamine. In this report I document some basic properties of the histamine receptors present on large monopolar cells isolated from blowfly optic lobes. The receptor is a ligand-gated chloride channel showing properties consistent with its presumed role of mediating neurotransmission at the photoreceptor synapse.     

Cloning and characterization of a vasopressin V2 receptor and possible link to nephrogenic diabetes insipidus.

The antidiuretic effect of arginine vasopressin (AVP) is mediated by renal-type (V2) receptors linked to adenylyl cyclase. We report here the cloning of the rat kidney V2 AVP receptor complementary DNA that encodes a 370-amino-acid protein with a transmembrane topography characteristic of G protein-coupled receptors, and with similarity to the V1a (hepatic) AVP receptor in its seven membrane-spanning domains. Expression of the cloned cDNA in mammalian cells showed specific ligand binding and activity characteristic of the native V2 AVP receptor. The receptor messenger RNA is detected only in the kidney. The human V2 receptor gene has been localized to the long arm of the X chromosome close to the locus for nephrogenic diabetes insipidus, an X-linked recessive disorder characterized by renal resistance to the antidiuretic action of AVP.     

Drosophila odorant receptors are both ligand-gated and cyclic-nucleotide-activated cation channels

From worm to man, many odorant signals are perceived by the binding of volatile ligands to odorant receptors that belong to the G-protein-coupled receptor (GPCR) family. They couple to heterotrimeric G-proteins, most of which induce cAMP production. This second messenger then activates cyclic-nucleotide-gated ion channels to depolarize the olfactory receptor neuron, thus providing a signal for further neuronal processing. Recent findings, however, have challenged this concept of odorant signal transduction in insects, because their odorant receptors, which lack any sequence similarity to other GPCRs, are composed of conventional odorant receptors (for example, Or22a), dimerized with a ubiquitously expressed chaperone protein, such as Or83b in Drosophila. Or83b has a structure akin to GPCRs, but has an inverted orientation in the plasma membrane. However, G proteins are expressed in insect olfactory receptor neurons, and olfactory perception is modified by mutations affecting the cAMP transduction pathway. Here we show that application of odorants to mammalian cells co-expressing Or22a and Or83b results in non-selective cation currents activated by means of an ionotropic and a metabotropic pathway, and a subsequent increase in the intracellular Ca(2+) concentration. Expression of Or83b alone leads to functional ion channels not directly responding to odorants, but being directly activated by intracellular cAMP or cGMP. Insect odorant receptors thus form ligand-gated channels as well as complexes of odorant-sensing units and cyclic-nucleotide-activated non-selective cation channels. Thereby, they provide rapid and transient as well as sensitive and prolonged odorant signalling.     

Structure of a cannabinoid receptor and functional expression of the cloned cDNA

Marijuana and many of its constituent cannabinoids influence the central nervous system (CNS) in a complex and dose-dependent manner. Although CNS depression and analgesia are well documented effects of the cannabinoids, the mechanisms responsible for these and other cannabinoid-induced effects are not so far known. The hydrophobic nature of these substances has suggested that cannabinoids resemble anaesthetic agents in their action, that is, they nonspecifically disrupt cellular membranes. Recent evidence, however, has supported a mechanism involving a G protein-coupled receptor found in brain and neural cell lines, and which inhibits adenylate cyclase activity in a dose-dependent, stereoselective and pertussis toxin-sensitive manner. Also, the receptor is more responsive to psychoactive cannabinoids than to non-psychoactive cannabinoids. Here we report the cloning and expression of a complementary DNA that encodes a G protein-coupled receptor with all of these properties. Its messenger RNA is found in cell lines and regions of the brain that have cannabinoid receptors. These findings suggest that this protein is involved in cannabinoid-induced CNS effects (including alterations in mood and cognition) experienced by users of marijuana.