David Shire

Molecular cloning, expression and function of the murine CB2 peripheral cannabinoid receptor.

We have cloned the peripheral cannabinoid receptor, mCB2, from a mouse splenocyte cDNA library. The 3.7 kb sequence contains an open reading frame encoding a protein of 347 residues sharing 82% overall identity with the only other known peripheral receptor, human CB2 (hCB2) and shorter than hCB2 by 13 amino acids at the carboxyl terminus. Binding experiments with membranes from COS-3 cells transiently expressing mCB2 showed that the synthetic cannabinoid WIN 55212-2 had a 6-fold lower affinity for mCB2 than for hCB2, whereas both receptors showed similar affinities for the agonists CP 55,940, delta(9)-THC and anandamide and almost no affinity for the central receptor- (CB1) specific antagonist SR 141716A. Both hCB2 and mCB2 mediate agonist-stimulated inhibition of forskolin-induced cAMP production in CHO cell lines permanently expressing the receptors. SR 141716A failed to antagonize this activity in either cell line, confirming its specificity for CB1.

Distribution profile and properties of peripheral-type benzodiazepine receptors on human hemopoietic cells

The cellular localization of peripheral-type benzodiazepine receptors (PBRs) was characterized in several human blood cell subpopulations including erythrocytes, platelets, monocytes and polymorphonuclear neutrophils (PMN), B, NK, T8 and T4-cells. Pharmacological properties of the PBR were established by binding studies and PBR mRNA expression was measured by quantitative polymerase chain reaction based method. These data clearly indicate 1) the PBR is pharmacologically homogeneous in the various types of blood cells, 2) the rank order of PBR cell density is monocytes = PMN > lymphocytes >> platelets > erythrocytes, 3) the PBR appears to be transcriptionally regulated since mRNA levels are roughly correlated with PBR density.

Targeted inactivation of the neurotensin type 1 receptor reveals its role in body temperature control and feeding behavior but not in analgesia.

Three subtypes of neurotensin receptor have been described, two members of the heptahelical transmembrane domain G protein-coupled receptor superfamily NT-1R and NT-2R, and NT-3R unrelated to this family. We have generated NT-1R deficient (NT-1R(-/-)) mice. NT-1R(-/-) mice were born at the expected Mendelian frequency without obvious abnormalities and they were fertile. The NT-induced analgesia on the writhing induced by phenyl-p-benzoquinone administration remained at wild-type levels in the NT-1R(-/-) mice demonstrating that the NT-1R is not implicated in the analgesic effect of NT in this test. The NT-1R(-/-) mice were hyperthermic; their body temperature was not affected by intracerebroventricular (i.c.v.) administration of NT, contrasting with the hypothermia induced in NT-1R(+/+) mice. NT-1R(-/-) mice showed a small significant increase in body weight compared to the NT-1R(+/+) congeners as early as 10 weeks after birth, correlated with a higher food intake. NT-1R(-/-) mice showed similar spontaneous locomotion to the control littermates, but did not respond to i.c.v. NT-induced hypolocomotion. I.c.v. injection of NT inhibited feeding in fasted wild-type mice, but had no effect on feeding of the NT-1R(-/-) mice. I.c.v. administration of the orexigenic neuropeptide Y (NPY) stimulated feeding to the same extent in both wild-type and NT-1R(-/-) mice. This analysis of NT-1R-deficient mice shows that the NT-1R does not play a role in NT-induced analgesia, but that it is clearly implicated in thermal and feeding regulation, weight control, and NT-induced hypolocomotion.

Emopamil-binding Protein, a Mammalian Protein That Binds a Series of Structurally Diverse Neuroprotective Agents, Exhibits Δ8-Δ7 Sterol Isomerase Activity in Yeast

Δ8-Δ7 sterol isomerase is an essential enzyme on the sterol biosynthesis pathway in eukaryotes. This endoplasmic reticulum-resident membrane protein catalyzes the conversion of Δ8-sterols to their corresponding Δ7-isomers. No sequence data for high eukaryote sterol isomerase being available so far, we have cloned a murine sterol isomerase-encoding cDNA by functional complementation of the corresponding deficiency in the yeast Saccharomyces cerevisiae. The amino acid sequence deduced from the cDNA open reading frame is highly similar to human emopamil-binding protein (EBP), a protein of unknown function that constitutes a molecular target for neuroprotective drugs. A yeast strain in which the sterol isomerase coding sequence has been replaced by that of human EBP or its murine homologue recovers the ability to convert Δ8-sterol into Δ7-sterol, both in vivo and in vitro. In these recombinant strains, both cell proliferation and the sterol isomerization reaction are inhibited by the high affinity EBP ligand trifluoperazine, as is the case in mammalian cells but not in wild type yeast cell. In contrast, the recombinant strains are much less susceptible to the sterol inhibition effect of haloperidol and fenpropimorph, as compared with wild type yeast strains. Our results strongly suggest that EBP and Δ8-Δ7 sterol isomerase are identical proteins in mammals.

Topological Analysis of the Peripheral Benzodiazepine Receptor in Yeast Mitochondrial Membranes Supports a Five-transmembrane Structure

The peripheral benzodiazepine receptor, implicated in the transport of cholesterol from the outer to the inner mitochondrial membrane, is predicted by hydropathy analysis to feature five membrane-spanning domains, with the amino terminus within the mitochondrial periplasm and the carboxyl terminus in the external cytoplasm. We have tested these structural predictions directly by immunodetection of c-Myc-tagged peripheral benzodiazepine receptor on intact yeast mitochondria and by specific labeling in yeast membranes of cysteine residues introduced by site-directed mutagenesis. The combined results support the model originally proposed with some minor but important modifications. The theoretical model predicted relatively short α-helical domains, only long enough to span a phospholipid monolayer, whereas the results presented here would support a model with extended α-helices sufficiently long to span an entire membrane bilayer, with concomitant shorter loop and tail regions.

Cannabinoid receptor interactions with the antagonists SR 141716A and SR 144528.

The G protein-coupled cannabinoid receptor subtypes CB1 and CB2 have been cloned from several species. The CB1 receptor is highly conserved across species, whereas the CB2 receptor shows considerable cross-species variations. The two human receptors share only 44% overall identity, ranging from 35% to 82% in the transmembrane regions. Despite this structural disparity, the most potent cannabinoid agonists currently available are largely undiscriminating and are therefore unsatisfactory tools for investigating the architecture of ligand binding sites. However, the availability of two highly specific antagonists, SR 141716A for the CB1 receptor and SR 144528 for the CB2 receptor, has allowed us to adopt a systematic approach to defining their respective binding sites through the use of chimeric CB1 receptor/CB2 receptor constructs, coupled with site-directed mutagenesis. We identified the region encompassed by the fourth and fifth transmembrane helices as being critical for antagonist specificity. Both the wild type human receptors overexpressed in heterologous systems are autoactivated; SR 141716A and SR 144528 exhibit classical inverse agonist properties with their respective target receptors. In addition, through its interaction with the CB1 receptor SR 141716A blocks the Gi protein-mediated activation of mitogen-activated protein kinase stimulated by insulin or insulin-like growth factor I. An in-depth analysis of this discovery has led to a modified three-state model for the CB1 receptor, one of which implicates the SR 141716A-mediated sequestration of Gi proteins, with the result that the growth factor-stimulated intracellular pathways are effectively impeded.

Dual intracellular signaling pathways mediated by the human cannabinoid CB1 receptor

It has long been established that the cannabinoid CB1 receptor transduces signals through a pertussis toxin-sensitive Gi/Go inhibitory pathway. Although there have been reports that the cannabinoid CB1 receptor can also mediate an increase in cyclic AMP levels, in most cases the presence of an adenylyl cyclase costimulant or the use of very high amounts of agonist was necessary. Here, we present evidence for dual coupling of the cannabinoid CB receptor to the classical pathway and to a pertussis toxin-insensitive adenylyl cyclase stimulatory pathway initiated with low quantities of agonist in the absence of any costimulant. Treatment of Chinese hamster ovary (CHO) cells expressing the cannabinoid CB1 receptor with the cannabinoid CP 55,940, {(-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hyd roxypropyl) cyclohexan-1-ol} resulted in cyclic AMP accumulation in a dose-response manner, an accumulation blocked by the cannabinoid CB1 receptor-specific antagonist SR 141716A, {N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-me thyl-1H-pyrazole-3-carboxamide hydrochloride}. In CHO cells coexpressing the cannabinoid CB1 receptor and a cyclic AMP response element (CRE)-luciferase reporter gene system, CP 55,940 induced luciferase expression by a pathway blocked by the protein kinase A inhibitor N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide hydrochloride (H-89). Under the same conditions the peripheral cannabinoid CB2 receptor proved to be incapable of inducing cAMP accumulation or luciferase activity. This incapacity allowed us to study the luciferase activation mediated by CB /CB2 chimeric constructs, from which we determined that the first and second internal loop regions of the cannabinoid CB1 receptor were involved in transducing the pathway leading to luciferase gene expression.

Mutational analysis and molecular modelling of the antagonist SR 144528 binding site on the human cannabinoid CB2 receptor

We have investigated the binding site of the subtype specific antagonist SR 144528, (N-[(1S)-endo-1,3,3-trimethyl bicyclo [2.2. 1]heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methoxybenzyl)- pyrazo le-3-carboxamide) on the human cannabinoid CB(2) receptor based on functional studies with mutated receptors. Two serine residues in the fourth transmembrane region, Ser(161) and Ser(165), were singly mutated to the cognate cannabinoid CB(1) receptor residue, alanine, and each gave receptors with wild-type properties for the cannabinoid agonists CP 55,940 (1R,3R,4R)-3-[2-hydroxy-4-(1, 1-dimethylheptyl)phenyl]-4-(3-hydroxypropyl)cyclohexan-1-ol) and WIN 55212-2 (R)-(+)[2, 3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]pyrrolo[1,2,3-de]-1, 4-benzoxazin-6-yl](1-naphthalenyl) methanone, which SR 144528 completely failed to antagonise. Molecular modelling studies show that SR 144528 interacts with residues in transmembrane domains 3, 4, and 5 of the cannabinoid CB(2) receptor through a combination of hydrogen bonds and aromatic and hydrophobic interactions. In addition, the replacement by serine of a nearby cannabinoid CB(2) receptor-specific residue, Cys(175) resulted in wild-type receptor properties with CP 55,940, loss of SR 144528 binding and eight-fold reduced binding and activity of WIN 55212-2, a result compatible with a recently-proposed binding site model for WIN 55212-2.

Evidence for the presence of β3-adrenergic receptor mRNA in the human brain

Abstract The β3-adrenergic receptor (AR) is widely distributed in peripheral tissues, but up to now it has not been detected in the central nervous system. By using the polymerase chain reaction (PCR) technique, we found the β3-AR mRNA to be present in all the regions of the human brain we investigated. The quantities found were very low compared to those of the β1-AR and β2-AR mRNAs, being hardly detectable in adult brain. In contrast, the brain of very young infants contained about 100 times more β3-AR mRNA than the adult brain, whereas the amounts of β1-AR and β2-AR transcripts were essentially the same. In addition, using PCR we have cloned a central β-AR coding region from a human frontal cortex cDNA library and have found it to be identical to the corresponding peripheral sequence.

Molecular basis for the different binding properties of benzodiazepines to human and bovine peripheral-type benzodiazepine receptors

The 18 kDa peripheral benzodiazepine receptor (PBR) can be labelled by benzodiazepines, such as Ro5‐4864, and isoquinoline carboxamides such as PK11195. These two compounds are reversible competitive inhibitors of each other. However, while the binding affinity of Ro5‐4864 varies enormously across species, PK11195 always displays high affinity, suggesting that their binding domains are overlapping but not identical. We report here that recombinant human and bovine PBR produced in yeast, a microorganism devoid of endogenous PBR, can be labelled with [3H]PK11195, but only the human receptor can be labelled with [3H]Ro5‐4864. Furthermore, we identified, through the binding analysis of human‐bovine chimaeric receptors, a region near the C‐terminal end of the PBR, with only five non‐conserved amino acids between human and bovine sequences, as responsible for the difference in high affinity binding of Ro5‐4864 to the two receptors.