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.     

Primary structure of human T-cell receptor alpha-chain

The T-cell receptor has been studied intensely over the past 10 years in an effort to understand the molecular basis for major histocompatibility complex (MHC) restricted antigen recognition. The use of anti-receptor monoclonal antibodies to isolate and characterize the receptor from human and murine T-cell clones has shown that the protein consists of two disulphide-linked glycopeptides, alpha and beta, distinct from known immunoglobulin light and heavy chains. Like immunoglobulin light and heavy chains, however, both the alpha- and beta-chains are composed of variable and constant regions. Molecular cloning has revealed that the beta-chain is evolutionarily related to immunoglobulins, and is encoded in separate V (variable), D (diversity), J (joining) and C (constant) segments that are rearranged in T cells to produce a functional gene. We report here cDNA clones encoding the alpha-chain of the receptor of the human T-cell leukaemia line HPB-MLT. Using these cDNA probes, we find that expression of alpha-chain mRNA and rearrangement of an alpha-chain V-gene segment occur only in T cells. The protein sequence predicted by these cDNAs is homologous to T-cell receptor beta-chains and to immunoglobulin heavy and light chains, particularly in the V and J segments.     

Cloning and expression of cDNA for a human thromboxane A2 receptor.

Thromboxane A2 is a very unstable arachidonate metabolite, yet a potent stimulator of platelet aggregation and a constrictor of vascular and respiratory smooth muscles. It has been implicated as a mediator in diseases such as myocardial infarction, stroke and bronchial asthma. Using a stable analogue of this compound we recently purified the human platelet thromboxane A2 receptor to apparent homogeneity. Using an oligonucleotide probe corresponding to its partial amino-acid sequence, we have obtained a complementary DNA clone encoding this receptor from human placenta and a partial clone from cultured human megakaryocytic leukaemia cells. The placenta cDNA encodes a protein of 343 amino acids with seven putative transmembrane domains. The protein expressed in COS-7 cells binds drugs with affinities identical to those of the platelet receptor, and that in Xenopus oocytes opens Ca2(+)-activated Cl- channel on agonist stimulation. Northern blot analysis and nucleotide sequences of the two clones suggest that an identical species of the thromboxane A2 receptor is present in platelets and vascular tissues. This first report on the molecular structure of an eicosanoid receptor will promote the molecular pharmacology and pathophysiology of these bioactive compounds.     

Primary structure and functional expression of a developmentally regulated skeletal muscle chloride channel.

Skeletal muscle is unusual in that 70-85% of resting membrane conductance is carried by chloride ions. This conductance is essential for membrane-potential stability, as its block by 9-anthracene-carboxylic acid and other drugs causes myotonia. Fish electric organs are developmentally derived from skeletal muscle, suggesting that mammalian muscle may express a homologue of the Torpedo mamorata electroplax chloride channel. We have now cloned the complementary DNA encoding a rat skeletal muscle chloride channel by homology screening to the Cl- channel from Torpedo. It encodes a 994-amino-acid protein which is about 54% identical to the Torpedo channel and is predominantly expressed in skeletal muscle. Messenger RNA amounts in that tissue increase steeply in the first 3-4 weeks after birth, in parallel with the increase in muscle Cl- conductance. Expression from cRNA in Xenopus oocytes leads to 9-anthracene-carboxylic acid-sensitive currents with time and voltage dependence typical for macroscopic muscle Cl- conductance. This and the functional destruction of this channel in mouse myotonia suggests that we have cloned the major skeletal muscle chloride channel.     

Primary structure and functional expression from complementary DNA of a brain calcium channel.

The primary structure of a voltage-dependent calcium channel from rabbit brain has been deduced by cloning and sequencing the complementary DNA. Calcium channel activity expressed from the cDNA is dramatically increased by coexpression of the alpha 2 and beta subunits, known to be associated with the dihydropyridine receptor. This channel is a high voltage-activated calcium channel that is insensitive both to nifedipine and to omega-conotoxin. We suggest that it is expressed predominantly in cerebellar Purkinje cells and granule cells.     

Primary structure and functional expression of a cyclic nucleotide-activated channel from olfactory neurons.

Odorant signal transduction occurs in the specialized cilia of the olfactory sensory neurons. Considerable biochemical evidence now indicates that this process could be mediated by a G protein-coupled cascade using cyclic AMP as an intracellular second messenger. A stimulatory G protein alpha subunit is expressed at high levels in olfactory neurons and is specifically enriched in the cilia, as is a novel form of adenylyl cyclase. This implies that the olfactory transduction cascade might involve unique molecular components. Electrophysiological studies have identified a cyclic nucleotide-activated ion channel in olfactory cilia. These observations provide evidence for a model in which odorants increase intracellular cAMP concentration, which in turn activates this channel and depolarizes the sensory neuron. An analogous cascade regulating a cGMP-gated channel mediates visual transduction in photoreceptor cells. The formal similarities between olfactory and visual transduction suggest that the two systems might use homologous channels. Here we report the molecular cloning, functional expression and characterization of a channel that is likely to mediate olfactory transduction.     

Primary structure and functional expression of the cardiac dihydropyridine-sensitive calcium channel

In cardiac muscle, where Ca2+ influx across the sarcolemma is essential for contraction, the dihydropyridine (DHP)-sensitive L-type calcium channel represents the major entry pathway of extracellular Ca2+. We have previously elucidated the primary structure of the rabbit skeletal muscle DHP receptor by cloning and sequencing the complementary DNA. An expression plasmid carrying this cDNA, microinjected into cultured skeletal muscle cells from mice with muscular dysgenesis, has been shown to restore both excitation-contraction coupling and slow calcium current missing from these cells, so that a dual role for the DHP receptor in skeletal muscle transverse tubules is suggested. We report here the complete amino-acid sequence of the rabbit cardiac DHP receptor, deduced from the cDNA sequence. We also show that messenger RNA derived from the cardiac DHP receptor cDNA is sufficient to direct the formation of a functional DHP-sensitive calcium channel in Xenopus oocytes. Furthermore, higher calcium-channel activity is observed when mRNA specific for the polypeptide of relative molecular mass approximately 140,000 (alpha 2-subunit) associated with skeletal muscle DHP receptor is co-injected.     

Cloning and expression of a cDNA encoding an endothelin receptor

Endothelins are a newly described peptide family consisting of three peptides (ET-1, ET-2 and ET-3) which are the most potent vasoconstrictive peptides known. They are crucial in the regulation of vascular smooth muscle tone. The diverse functions of endothelins are thought to be mediated by interaction with many different receptors coupled to the inositol phosphate/calcium ion messenger pathway. However, because of the structural resemblance of the three peptides, the presence and nature of multiple endothelin receptors remain to be elucidated. We report here the cloning of a complementary DNA encoding a bovine endothelin receptor, which has a transmembrane topology similar to that of other G protein-coupled receptors and shows specific binding, with the highest selectivity to ET-1 in animal cells transfected with the cloned cDNA. This receptor messenger RNA is widely distributed in the central nervous system and peripheral tissues, particularly in the heart and lung. Our results support the view that there are other receptor subtypes.     

Domain structure of human glucocorticoid receptor and its relationship to the v-erb-A oncogene product

The interaction of steroids with their nuclear receptors induces a cascade of regulatory events that results from the activation of specific sets of genes by the hormone/receptor complex. Steroids, either acting alone or possibly synergistically with other growth factors, can influence the DNA synthesis and proliferation of specific target cells, initiate developmental pathways and activate expression of the differentiated phenotype. Moreover, steroid hormones have been implicated in abnormal growth regulation both in tumours and tumour-derived cell lines. The identification of complementary DNAs encoding the human glucocorticoid receptor (hGR) predicts two protein forms (alpha and beta; 777 and 742 amino acids long, respectively) which differ at their carboxy termini. We report here that both forms of the receptor are related, with respect to their domain structure, to the v-erb-A oncogene product of avian erythroblastosis virus (AEV), which suggests that steroid receptor genes and the c-erb-A proto-oncogene are derived from a common primordial regulatory gene. Therefore, oncogenicity by AEV may result, in part, from the inappropriate activity of a truncated steroid receptor or a related regulatory molecule encoded by v-erb-A. This suggests a mechanism by which transacting factors may facilitate transformation. We also identify a short region of hGR that is homologous with the Drosophila homoeotic proteins encoded by Antennapedia and fushi tarazu.     

Primary structure of alpha-subunit precursor of Torpedo californica acetylcholine receptor deduced from cDNA sequence

DNA sequences complementary to the Torpedo californica electroplax mRNA coding for the alpha-subunit precursor of the acetylcholine receptor were cloned. The nucleotide sequence of the cloned cDNA indicates that the precursor consists of 461 amino acids including a prepeptide of 24 amino acids. Possible sites for acetylcholine binding and antigenic determinants on the alpha-subunit molecule are discussed.     

Primary structure and expression from complementary DNA of skeletal muscle ryanodine receptor

The sequence of 5,037 amino acids composing the ryanodine receptor from rabbit skeletal muscle sarcoplasmic reticulum has been deduced by cloning and sequencing the complementary DNA. The predicted structure suggests that the calcium release channel activity resides in the C-terminal region of the receptor molecule, whereas the remaining portion constitutes the 'foot' structure spanning the junctional gap between the sarcoplasmic reticulum and the transverse tubule.     

Primary structure and functional expression of the inositol 1,4,5-trisphosphate-binding protein P400

Cloning and expression of functional P400 protein from cerebellar Purkinje neurons shows that this protein is a receptor for inositol 1,4,5-trisphosphate, a second messenger that mediates the release of intracellular calcium.     

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.     

Expression of functional human interleukin-2 receptor in mouse T cells by cDNA transfection

Interleukin-2 (IL-2) in combination with the IL-2 receptor has an essential role in antigen-stimulated proliferation of T lymphocytes. It has been proposed that the constitutive expression of the IL-2 receptor on adult T-cell leukaemia (ATL) cells may be associated with transformation of T cells. Although we and others have isolated complementary DNA clones encoding a protein that binds IL-2, formal proof that this protein is the IL-2 receptor requires demonstration of IL-2-dependent growth stimulation of cells expressing the protein. In addition, a functional assay system other than binding of IL-2 is required to investigate the molecular mechanism of signal transmission through the IL-2 receptor using artificially mutated cDNA. The IL-2 receptor expressed in non-lymphoid cells by cDNA transfection did not mediate a growth signal, implying that lymphoid cells expressing the functional receptor might have specific accessory molecule(s) for signal transmission by the receptor. Therefore, we established a line of IL-2-dependent mouse cells (CT/hR) expressing both murine (endogenous) and human IL-2 receptors. Here, by blocking the endogenous mouse IL-2 receptors with monoclonal antibodies, we show that the human IL-2 receptor of CT/hR cells is functionally active. Although CT/hR expressed the human IL-2 receptor constitutively, growth of these cells was strictly dependent on IL-2, indicating that uncontrolled over-expression of the IL-2 receptor was not by itself sufficient for T-cell transformation.     

Structure and expression of a cloned cDNA for human interleukin-2

A cDNA coding for human interleukin-2 (IL-2) has been cloned from a cDNA library prepared from partially purified IL-2 mRNA. The DNA sequence codes for a polypeptide which consists of 153 amino acids including a putative signal sequence. A biologically active polypeptide, characteristic of human IL-2, was produced when the cDNA was fused to a simian virus 40 promoter sequence and used to transfect cultured monkey COS cells.     

Primary structure and functional expression from complementary DNA of the rod photoreceptor cyclic GMP-gated channel

The complete amino-acid sequence of the cyclic GMP-gated channel from bovine retinal rod photoreceptors, deduced by cloning and sequencing its complementary DNA, shows that the protein contains several putative transmembrane segments, followed by a region that is similar to the cyclic GMP-binding domains of cyclic GMP-dependent protein kinase. Expression of the complementary DNA produces cyclic GMP-gated channel activity in Xenopus oocytes.     

Complete primary structure of a heterodimeric T-cell receptor deduced from cDNA sequences

Two related, but distinct, cDNA clones have been isolated and sequenced from a functional murine cytotoxic T-lymphocyte clone. The genes corresponding to these cDNA are expressed and rearranged specifically in T cells and both have similarities to immunoglobulin variable and constant region genes. It is concluded that these genes code for the two subunits of the heterodimeric antigen receptor on the surface of the T cell; its complete deduced primary structure is presented.     

Sequence and functional expression of the GABA A receptor shows a ligand-gated receptor super-family

Amino-acid sequences derived from complementary DNAs encoding the alpha- and beta-subunits of the GABA/benzodiazepine receptor from bovine brain show homology with other ligand-gated receptor subunits, suggesting that there is a super-family of ion-channel-containing receptors. Co-expression of the in vitro-generated alpha-subunit and beta-subunit RNAs in Xenopus oocytes produces a functional receptor and ion channel with the pharmacological properties characteristic of the GABAA receptor.