Claire M. Fraser

Cloning, localization, and permanent expression of a Drosophila octopamine receptor

A cDNA for a member of the G protein-coupled receptor family was isolated from Drosophila using a probe derived from a human beta 2-adrenergic receptor cDNA. This Drosophila receptor gene is localized at 99A10-B1 on the right arm of chromosome 3 and is preferentially expressed in Drosophila heads. The insect octopamine receptor has been permanently expressed in mammalian cells, where it mediates the attenuation of adenylate cyclase activity and exhibits a pharmacological profile consistent with an octopamine type 1 receptor. Sequence and pharmacological comparisons indicate that the octopamine receptor is unique but closely related to mammalian adrenergic receptors, perhaps as an evolutionary precursor.

Discrete activation of transduction pathways associated with acetylcholine m1 receptor by several muscarinic ligands.

Activation of transfected muscarinic m1 acetylcholine receptors (m1AChR) has been linked to several signal transduction pathways which include phosphoinositide hydrolysis, arachidonic acid release and cAMP accumulation. In Chinese hamster ovary cells stably transfected with the rat m1AChR gene, carbachol elicited all three responses with EC50 values of 2.6, 3.8 and 76 microM, respectively. However, pilocarpine and the selective muscarinic agonist AF102B activated phosphoinositide hydrolysis (by 94 and 27% vs. carbachol, respectively), while antagonizing carbachol-mediated cAMP accumulation. Carbachol also activated (by 4-fold) adenylyl cyclase in membranes prepared from these cells, indicating independence of this signal from intracellular mediators. Moreover, carbachol and AF102B similarly elevated cytosolic Ca2+ in intact m1AChR-transfected cells. The ligand-selective cAMP accumulation, its independence from Ca2+ and the carbachol-activated adenylyl cyclase in membranes suggest that it represents an independent m1AChR-mediated signal, unrelated to phosphoinositide hydrolysis. Selective muscarinic ligands such as AF102B may independently activate distinct signalling pathways, which may be important for designing cholinergic replacement therapy for treating Alzheimers disease.

Muscarinic cholinergic receptor structure: molecular biological support for subtypes

Abstract Pharmacological evidence has indicated the existence of multiple subtypes of muscarinic receptors, while biochemical and immunological data have uncovered the highly conserved nature of muscarinic receptor structure. Molecular biological data now appear to have confirmed the notion of subtypes. Tony Kerlavage, Claire Fraser and Craig Venter discuss recent cloning and sequence analyses of the genes encoding muscarinic receptors in brain and heart which have revealed the existence of four distinct but highly homologous gene products.

Cloning and sequence analysis of the human brain β-adrenergic receptor: Evolutionary relationship to rodent and avian β-receptors and porcine muscarinic receptors

Two cDNA clones, λ‐CLFV‐108 and λ‐CLFV‐119, encoding for the β‐adrenergic receptor, have been isolated from a human brain stem cDNA library. One human genomic clone, LCV‐517 (20 kb), was characterized by restriction mapping and partial sequencing. The human brain β‐receptor consists of 413 amino acids with a calculated M r of 46 480. The gene contains three potential glucocorticoid receptor‐binding sites. The β‐receptor expressed in human brain was homology with rodent (88%) and avian (52%) β‐receptors and with porcine muscarinic cholinergic receptors (31%), supporting our proposal [(1984) Proc. Natl. Acad. Sci USA 81, 272‐276] that adrenergic and muscarinic cholinergic receptors are structurally related. This represents the first cloning of a neurotransmitter receptor gene from human brain.Two cDNA clones, lambda-CLFV-108 and lambda-CLFV-119, encoding for the beta-adrenergic receptor, have been isolated from a human brain stem cDNA library. One human genomic clone, LCV-517 (20 kb), was characterized by restriction mapping and partial sequencing. The human brain beta-receptor consists of 413 amino acids with a calculated Mr of 46480. The gene contains three potential glucocorticoid receptor-binding sites. The beta-receptor expressed in human brain was homology with rodent (88%) and avian (52%) beta-receptors and with porcine muscarinic cholinergic receptors (31%), supporting our proposal [(1984) Proc. Natl. Acad. Sci. USA 81, 272 276] that adrenergic and muscarinic cholinergic receptors are structurally related. This represents the first cloning of a neurotransmitter receptor gene from human brain.

Cloning, sequence analysis and chromosome localization of a Drosophila muscarinic acetylcholine receptor

Two cDNA clones (3.7 kb and 4.8 kb) encoding a Drosophila muscarinic acetylcholine receptor were isolated from a Drosophila head cDNA library and characterized by automated DNA sequence analysis. The Drosophila muscarinic receptor contains 788 amino acids with a calculated M r of 84 807 and displays greater than 60% homology with mammalian muscarinic receptors. The muscarinic receptor maps to the tip of the right arm of the second chromosome of the Drosophila genome.

Radioligand binding studies of the atypical β3‐adrenergic receptor in rat brown adipose tissue using [3H]CGP 12177

Two populations of [3H]CGP 12177 binding sites exist in rat interscapular brown adipose tissue (IBAT) plasma membrane. The majority of binding sites are of low affinity with a K d of 31 nM, a value in close agreement with that for the K d or [3H]CGP 12177 binding to a cloned rat β3‐adrenergic receptor (AR) expressed in CHO cells (44 nM). Competition binding studies demonstrate that the K i values of the cloned rat β3‐AR and of the low affinity sites in IBAT are 45 and 29 nM, respectively, for BRL 37344 and 1.4 and 1.0 μM, for (−)‐propranolol. These findings strongly suggest that the low affinity [3H]CGP 12177 binding site measured in IBAT plasma membranes represents the atypical β3‐AR in this tissue.

Muscarinic acetylcholine receptor subtypes which selectively couple to phospholipase C: Pharmacological and biochemical properties

The pharmacological and biochemical properties of rat m1 and m3 muscarinic acetylcholine receptors (mAChR) stably transfected into Chinese hamster ovary-K1 (CHO) cells were characterized with ligand binding, affinity labeling and biochemical assays. Both mAChR subtypes display saturable, high affinity binding of [3H]-quinuclidinyl benzilate (QNB) and a rank order of antagonist potency of QNB greater than atropine greater than pirenzepine greater than AF-DX 116. Carbachol displacement of [3H]-QNB binding to the m3 mAChR revealed an approximate 17-fold higher affinity than observed with the m1 mAChR. [3H]-propylbenzilylcholine mustard (PrBCM) labeling of mAChR revealed that m1 and m3 mAChR migrated on SDS-polyacrylamide gels with apparent molecular masses of 80,000 and 94,000 daltons, respectively, consistent with the known differences in their molecular sizes. Both m1 and m3 mAChR elicited dose-dependent increases in the hydrolysis of phosphoinositides; however, the maximal increase in total inositol phosphates elicited with the m1 mAChR was approximately 2-fold greater than that observed in cells expressing similar densities of m3 mAChR. Agonist activation of the m1 mAChR also elicited increases in basal and forskolin-stimulated cAMP, whereas the m3 mAChR had no effect on intracellular cAMP levels. These data suggest that although m1 and m3 mAChR display a considerable degree of structural homology, they exhibit distinct pharmacological and biochemical properties.

Discrepancies between the affinities of binding and action of the novel β-adrenergic agonist BRL 37344 in rat brown adipose tissue

The novel brown adipose tissue (BAT) selective beta-adrenergic agonist, BRL 37344, is 31-fold more potent than (-)-isoproterenol in stimulating the respiratory rate of interscapular BAT fragments. BRL 37344 is also more potent (9-fold) than (-)-isoproterenol in stimulating adenylate cyclase activity of IBAT purified plasma membranes whereas, in the same preparation, it is 81-fold less potent than (-)-isoproterenol in competition displacement studies with the beta-adrenergic ligand, [125I]cyanopindolol. We have previously demonstrated that the photoaffinity reagent [125I]cyanopindolol-diazirine selectively labels a 62 kDa protein in IBAT plasma membranes that displays pharmacological properties of a beta 1-adrenergic subtype. Relatively high concentrations of BRL 37344 (10 microM) are required to displace [125I]cyanopindolol-diazirine binding to the 62 kDa protein. Taken together, the results suggest that two different populations of beta-adrenergic receptors may co-exist in BAT plasma membranes: a small population (about 15%) of atypical beta-receptors and a large population of beta 1-receptors that exhibit high and low affinities for BRL 37344, respectively.

Biochemical Characterization of Brown Adipose Tissue β-Adrenergic Receptor

AbstractThe β-adrenergic receptor of rodent brown fat plays a key role in the control of energy dissipation by this tissue. The aim of the present study was to further characterize the biochemical properties of this receptor. The β-receptor of rat interscapular brown adipose tissue plasma membranes was found to bind the β-adrenergic antagonist [125I] cyanopindolol with a high affinity (KD 67 pM). The [125I] cyanopindolol receptor complex could be solubilized by digitonin and the isoelectric point of the solubilized receptor was found to be 5.8. Brown adipose tissue plasma membranes were labeled with the photoaffinity ligand [125I] cyanopindolol diazirine and labeled membrane proteins were separated by sodium dodecylsufate polyacrylamide gelelectrophoresis and analyzed by autoradiography. Autoradiograms revealed a peptide of 62 kDa whose labeling was stereoselectively displaced by alprenolol and isoproterenol. The β1-selective antagonist betaxolol was about 100 times more potent in displacing the labeling ...

Cloning and Expression of Adrenergic and Muscarinic Cholinergic Receptor Genes

We have extensively characterized the structure and evolution of the adrenergic and muscarinic cholinergic receptors. This analysis has included studies with monoclonal antibodies, protein purification, target size analysis, gene cloning and sequencing and gene expression (1–7). Based upon the results of biochemical and immunological studies, we proposed that adrenergic and muscarinic cholinergic receptors are highly conserved proteins. This hypothesis has been confirmed by gene cloning experiments which have provided the primary structures of a number of adrenergic and muscarinic cholinergic receptors.