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C M Fraser

Publications and source records attributed to C M Fraser.

At least 109 records · Page 6Linked to original sources

Site-directed mutagenesis of m1 muscarinic acetylcholine receptors: conserved aspartic acids play important roles in receptor function.

Muscarinic acetylcholine receptors contain a region encompassing the second and third transmembrane domains that is rich in conserved aspartic acid residues. To investigate the role of four conserved aspartic acids at positions 71, 99, 105, and 122 in muscarinic receptor function, point mutations in the rat m1 muscarinic receptor gene were made that converted each Asp to Asn, and wild type or mutant genes were stably expressed in Chinese hamster ovary cells that normally lack muscarinic receptors. Substitution of Asp71 or Asp122 with Asn produced mutant receptors that displayed high affinity for carbachol but decreased efficacy and potency, respectively, in agonist-induced activation of phosphoinositide hydrolysis, suggesting that these residues may mediate receptor-GTP binding protein interactions. Substitution of Asp99 or Asp105 with Asn produced marked decreases in ligand binding affinities and/or covalent incorporation of [3H] propylbenzilylcholine mustard, suggesting that these residues may be involved in receptor-ligand interactions.

Animals↗

Site-directed mutagenesis and continuous expression of human beta-adrenergic receptors. Identification of a conserved aspartate residue involved in agonist binding and receptor activation.

Using a new expression vector that allows stable and steroid inducible expression of the human beta 2-adrenergic receptor in mouse L cells, we have examined the functional significance of the highly conserved aspartate residue in the putative second transmembrane region of the receptor. Substitution of aspartate 79 with asparagine produced a mutant receptor that displays the expected affinity and stereoselectivity for antagonists but a 40-, 140-, and 240-fold reduction in its affinity for isoproterenol, epinephrine, and norepinephrine, respectively. This receptor mutant does not display guanine nucleotide-sensitive high affinity binding of agonists. Addition of saturating concentrations of isoproterenol to cell cultures expressing the mutant receptor produces a slight, albeit significant, increase in intracellular levels of cyclic AMP as compared to cells expressing wild type receptor. These observations demonstrate that substitution of aspartate with asparagine at residue 79 in the human beta-adrenergic receptor differentially affects the binding of catecholamines and produces a functional uncoupling of receptors and stimulatory guanine nucleotide regulatory proteins (Gs). These data are consistent with a role for aspartate 79 as a counterion to the amine in catecholamines and in agonist-induced activation of the beta-adrenergic receptor associated with high affinity ligand binding, Gs coupling, and adenylate cyclase stimulation.

Aspartic Acid↗

Evolution of neurotransmitter receptor systems.

The presence of hormones, neurotransmitters, their receptors and biosynthetic and degradative enzymes is clearly not only associated with the present and the recent past but with the past several hundred million years. Evidence is mounting which indicates substantial conservation of protein structure and function of these receptors and enzymes over these tremendous periods of time. These findings indicate that the evolution and development of the nervous system was not dependent upon the formation of new or better transmitter substances, receptor proteins, transducers and effector proteins but involved better utilization of these highly developed elements in creating advanced and refined circuitry. This is not a new concept; it is one that is now substantiated by increasingly sophisticated studies. In a 1953 article discussing chemical aspects of evolution (Danielli, 1953) Danielli quotes Medawar, "... endocrine evolution is not an evolution of hormones but an evolution of the uses to which they are put; an evolution not, to put it crudely, of chemical formulae but of reactivities, reaction patterns and tissue competences." To also quote Danielli, "In terms of comparative biochemistry, one must ask to what extent the evolution of these reactivities, reaction patterns and competences is conditional upon the evolution of methods of synthesis of new proteins, etc., and to what extent the proteins, etc., are always within the synthetic competence of an organism. In the latter case evolution is the history of changing uses of molecules, and not of changing synthetic abilities." (Danielli, 1953). Figure 4 outlines a phylogenetic tree together with an indication of where evidence exists for both the enzymes that determine the biosynthesis and metabolism of the cholinergic and adrenergic transmitters and their specific cholinergic and adrenergic receptors. This figure illustrates a number of important points. For example, the evidence appears to show that the transmitters and their associated enzymes existed for a substantial period before their respective receptor proteins. While the transmitters and enzymes appear to exist in single cellular organisms, there is no solid evidence for the presence of adrenergic or cholinergic receptors until multicellular organisms where the receptors appear to be clearly associated with specific cellular and neuronal communication (Fig. 4). One can only speculate as to the possible role for acetylcholine and the catecholamine in single cell organisms.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

Site-directed mutagenesis of human beta-adrenergic receptors: substitution of aspartic acid-130 by asparagine produces a receptor with high-affinity agonist binding that is uncoupled from adenylate cyclase.

By using oligonucleotide-directed mutagenesis, we have produced a point mutation (guanine to adenine) at nucleotide 388 of the gene for human beta-adrenergic receptor (beta AR) that results in a substitution of asparagine for the highly conserved aspartic acid at position 130 in the putative third transmembrane domain of the human beta AR ([Asn130]beta AR). We have examined the functional significance of this mutation in B-82 cells continuously expressing the mutant [Asn130]beta AR. The mutant [Asn130]beta AR displayed normal antagonist binding but unusually high-affinity agonist binding (5- to 10-fold higher than wild-type beta AR), consistent with a single class of high-affinity binding sites. The mutant beta AR displayed guanine nucleotide-sensitive changes in agonist affinity (3- to 5-fold shift) implying an interaction between the beta AR and the stimulatory guanine nucleotide-binding regulatory protein; however, the ability of guanine nucleotides to alter agonist affinity was attenuated. Addition of saturating concentrations of isoproterenol to cell cultures expressing mutant [Asn130]-beta ARs had no effect on intracellular levels of cAMP, indicating that the mutant beta AR is unable to affect stimulation of adenylate cyclase. These results indicate that substitution of the aspartic acid with asparagine at residue 130 of the human beta AR dissociates the well-characterized guanine nucleotide effects on agonist affinity from those on activation of the stimulatory guanine nucleotide-binding regulatory protein and adenylate cyclase and suggests the existence of two distinct counterions for the amine portion of catecholamines that are associated with high- and low-affinity agonist binding states of beta AR.

Adenylyl Cyclases↗

Biochemical characterization of brown adipose tissue beta-adrenergic receptor.

The beta-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 beta-receptor of rat interscapular brown adipose tissue plasma membranes was found to bind the beta-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 dodecylsulfate polyacrylamide gel electrophoresis and analyzed by autoradiography. Autoradiograms revealed a peptide of 62 kDa whose labeling was stereoselectively displaced by alprenolol and isoproterenol. The beta 1-selective antagonist betaxolol was about 100 times more potent in displacing the labeling of this 62 kDa peptide than the beta 2-selective antagonist ICI 118,551. Based upon these data, it appears that the beta-receptor in brown adipose tissue is a beta 1 subtype with molecular weight of 62 kDa.

Adipose Tissue, Brown↗

Continuous high density expression of human beta 2-adrenergic receptors in a mouse cell line previously lacking beta-receptors.

The PvuII fragment of human genomic clone LCV-517 which contains the entire coding region of a beta-adrenergic receptor gene was cloned into the SmaI site of the expression vector pMSG. The recombinant DNA was cotransfected with pRSVneo into mouse B-82 cells using the CaPO4 precipitation method. B-82 cells do not possess beta-adrenergic receptors but do contain prostaglandin E1 receptors that stimulate adenylate cyclase. Following transfection, several colonies expressing beta-adrenergic receptors were isolated. Analysis of ligand binding to expressed beta-receptors indicated that the protein encoded by the gene in clone LCV-517 was a beta 2-adrenergic subtype. Human beta 2-adrenergic receptors photoaffinity labeled with [125I]iodocyanopindolol diazirine migrated on sodium dodecyl sulfate-polyacrylamide gels consistent with a molecular mass of 68,000, demonstrating that the receptor is glycosylated to an extent of 25-30% by weight. Addition of isoproterenol to cultures of transfected cells resulted in a 3-4-fold stimulation of adenylate cyclase, an effect similar to that seen in control B-82 cells with prostaglandin E1. These data describe the production of stable murine clonal cell lines expressing human beta 2-adrenergic receptors and illustrate the utility of such lines in the biochemical and pharmacological characterization of receptor proteins.

Adenylyl Cyclases↗

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

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.

Amino Acid Sequence↗

Structural analysis of purified beta-adrenergic receptors.

We have characterized the structure of purified beta-adrenergic receptors by a combination of photoaffinity labeling, high performance liquid chromatography (HPLC)-tryptic mapping, CNBr fragmentation, target size analysis, and electron microscopy of purified receptor molecules. Guinea pig lung beta-adrenergic receptors purified by affinity chromatography, ion exchange chromatography, and HPLC size exclusion chromatography or photoaffinity labeled with [125]-iodocyanopindolol diazirine displayed mobilities on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) that corresponded to Mr = 68,000. Purified, radioiodinated guinea pig lung beta-receptors were subjected to complete trypsin digestion and subsequent reverse-phase HPLC analysis, which revealed nine peptides. Active site labeling and tryptic digestion of partially purified hamster lung beta-receptors produced one peptide, whereas CNBr digestion of the same material produced two labeled fragments, yielding information about the location of the active site within the primary sequence. Purified guinea pig lung receptors were examined with transmission electron microscopy. Electron micrographs revealed slightly asymmetric, rod-shaped structures with an average length of 13 nm and width of 3.4 nm. Many receptors were arranged as apparent dimeric structures. These findings confirm data obtained from target size analysis of guinea pig lung beta-receptors in situ which suggest that receptors may exist as oligomeric arrays in the native membrane. Taken together, these data provide information about putative functional domains of the beta-adrenergic receptor and its quaternary structure.

Amino Acid Sequence↗

Primary structure of rat cardiac beta-adrenergic and muscarinic cholinergic receptors obtained by automated DNA sequence analysis: further evidence for a multigene family.

Two cDNA clones, lambda RHM-MF and lambda RHB-DAR, encoding the muscarinic cholinergic receptor and the beta-adrenergic receptor, respectively, have been isolated from a rat heart cDNA library. The cDNA clones were characterized by restriction mapping and automated DNA sequence analysis utilizing fluorescent dye primers. The rat heart muscarinic receptor consists of 466 amino acids and has a calculated molecular weight of 51,543. The rat heart beta-adrenergic receptor consists of 418 amino acids and has a calculated molecular weight of 46,890. The two cardiac receptors have substantial amino acid homology (27.2% identity, 50.6% with favored substitutions). The rat cardiac beta receptor has 88.0% homology (92.5% with favored substitutions) with the human brain beta receptor and the rat cardiac muscarinic receptor has 94.6% homology (97.6% with favored substitutions) with the porcine cardiac muscarinic receptor. The muscarinic cholinergic and beta-adrenergic receptors appear to be as conserved as hemoglobin and cytochrome c but less conserved than histones and are clearly members of a multigene family. These data support our hypothesis, based upon biochemical and immunological evidence, that suggests considerable structural homology and evolutionary conservation between adrenergic and muscarinic cholinergic receptors. To our knowledge, this is the first report utilizing automated DNA sequence analysis to determine the structure of a gene.

Amino Acid Sequence↗

A test battery to measure the recovery of voluntary movement control following stroke.

Tests of upper limb function and an activities of daily living (ADL) index were selected to measure recovery following stroke. Thirty stroke patients were assessed at intervals for up to 6 months to 1 year post-stroke using the battery. The results showed the ADL index is insensitive to upper limb recovery. All the tests measured recovery in some of the patients after 24 weeks post-stroke. Since the presentation and recovery of patients was variable, it is argued that it is necessary to offer a selection of assessment tests to measure recovery and to aid treatment planning.

Activities of Daily Living↗

Alpha and beta adrenergic and muscarinic cholinergic receptor structure.

Purification and characterization of the neurotransmitter receptors of the autonomic nervous system have revealed considerable structural and functional homology between these pharmacologically distinct classes of information transduction molecules. Alpha 1- and alpha 2-adrenergic receptors are single polypeptides with molecular mass 85,000 Da and pI 4.6. Beta 1- and beta 2-adrenergic receptors are single polypeptides with molecular mass 68,000 Da and pI 5.0. Muscarinic cholinergic receptors from a variety of tissues and species are single polypeptides with molecular mass 80,000 Da and pI 4.2. Proteolytic digestion and analysis of affinity-labelled adrenergic and cholinergic receptors indicates a striking similarity in the number and sizes of peptides produced. Topographical analysis of the receptors has shown that they have a similar membrane orientation with more than half of the protein exposed to the extracellular environment. Peptide-mapping studies of soluble and membrane-bound receptors suggest that the ligand-binding domain of adrenergic and cholinergic receptors is localized near the end of the protein that is exposed to the extracellular environment. The marked similarity between alpha- and beta-adrenergic and muscarinic cholinergic receptor structure is perhaps not unexpected in light of the fact that these receptors interact with the same transmitters (in the case of alpha- and beta-adrenergic receptors) and/or with the same effector proteins in the membrane (stimulatory and inhibitory guanine nucleotide regulatory proteins, ion channels). Yet, depending on the tissue distribution of receptors and their effectors, this limited number of proteins can modulate dramatically different physiological effects. It may be that the differences in pharmacological specificity of ligand binding and the differences in receptor-effector interactions observed among adrenergic receptor subtypes and muscarinic cholinergic receptors are due to minor structural differences within the active sites of these proteins. Obviously, the real answers as to the extent of structural homology between the receptor classes will be derived from the amino acid sequencing of the purified proteins or from the cloning of the receptor genes and recently the genes coding for the beta-adrenergic receptor have been cloned and sequenced from human brain, hamster lung, and turkey erythrocytes. Comparison of the derived protein sequences reveals a high degree of structural homology between the avian and mammalian receptors with approximately 50% primary sequence identity and highly conserved secondary structure.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

Molecular comparison of alpha 1- and alpha 2-adrenergic receptors suggests that these proteins are structurally related "isoreceptors".

The structures of human platelet alpha 2-adrenergic receptors and rat liver alpha 1-adrenergic receptors were compared by utilizing isoelectric focusing, NaDodSO4/PAGE, and monoclonal antibody crossreactivity. Digitonin-solubilized alpha 1- and alpha 2-adrenergic receptors have an identical isoelectric point of 4.6. Under reducing conditions in NaDodSO4/polyacrylamide gels, the alpha 1-adrenergic receptor has an apparent molecular mass of 85 kDa. Similarly, the alpha 2-adrenergic receptor, which had been affinity-labeled with [3H]phenoxybenzamine and partially purified by isoelectric focusing or photoaffinity-labeled with p-[3,5-3H]azidoclonidine, was also found to have an apparent molecular mass of 85 kDa. One hybridoma, developed from a fusion between SP2/O myeloma cells and splenic lymphocytes from BALB/c mice immunized with human platelet alpha 2-adrenergic receptors, secreted a monoclonal antibody (alpha 2-116p) against the ligand binding site of alpha 2-adrenergic but not alpha 1-adrenergic receptors. In contrast, three monoclonal antibodies raised against the alpha 1-receptor polypeptide backbone but not the ligand binding site were found to specifically immunoprecipitate human platelet alpha 2-adrenergic receptors. These data suggest that the alpha 1- and alpha 2-adrenergic receptors are "isoreceptors," sharing immunogenic and, by implication, structural determinants that most likely evolved as a result of gene duplication.

Adrenergic alpha-Agonists↗