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C M Fraser

Publications and source records attributed to C M Fraser.

At least 73 records · Page 4Linked to original sources
PubMed1997

The involvement of adenosine receptors in the effect of dizocilpine on mice in the elevated plus-maze.

It has been claimed that blockade of receptors for N-methyl-D-aspartate (NMDA) can enhance adenosine receptor function on single neurones. Previous work has also indicated that the NMDA channel blocker dizocilpine, and the A1 selective agonist N6-cyclopentyladenosine (CPA) both had anxiolytic profiles in the elevated plus-maze. The anxiolytic effect of dizocilpine was accompanied by an increase in locomotor activity. In the present study, the elevated plus-maze has been used to determine whether dizocilpine's effects on behaviour are mediated through activation of adenosine receptors. When co-administered with dizocilpine (0.05 mg/kg), CPA (0.05 mg/kg) reduced the anxiolytic and locomotor effects of dizocilpine. The A1 selective antagonist 1,3-dipropyl-8-cyclopentylxanthine (CPX, 0.05 mg/kg) had no effect when administered alone. When co-administered with dizocilpine, CPX reversed the anxiolytic and increased locomotor effects induced by dizocilpine. The A2 receptor selective agonist N6-[2-(3,5-dimethoxyphenyl)-2(2-methylphenyl)ethyladenosine (DPMA) (1 mg/kg) reversed both the anxiolytic effect and the increased locomotion induced by dizocilpine, while the A2 selective antagonist 3,7-dimethyl-1-propargylxanthine (DMPX) (1 mg/kg) did not. It is concluded that at least part of the anxiolytic and locomotor stimulant properties of dizocilpine may be explained by the release of endogenous adenosine acting at A1, but not A2 receptors.

Adenosine↗
PubMed1996

Sequence analysis of the 5'-untranslated region of the human H1 histamine receptor-encoding gene.

A clone containing the H1 histamine receptor (H1HR)-encoding gene was isolated from a human genomic DNA library. The 5'-UTR of the H1HR gene reported here differs upstream from bp -142 from that reported previously [Fukui et al., Biochem. Biophys. Res. Comm. 201 (1994) 894-901]. PCR amplification utilizing primer pairs derived from the 5'-UTR reported herein amplified a DNA fragment of the expected size from human genomic DNA whereas 5'-UTR primers derived from the Fukui et al. sequence did not yield a PCR product. The 5'-UTR of H1HR contains potential TATA and CCAAT boxes, a CACCC sequence, potential GREs and other DNA-binding motifs.

Base Sequence↗
PubMed1996

Interactions between ifenprodil and dizocilpine on mouse behaviour in models of anxiety and working memory.

The N-methyl-D-aspartate (NMDA) receptor polyamine site antagonist, ifenprodil, had no effect on spontaneous alteration or locomotor activity in the Y-maze when given alone. The NMDA receptor/ion channel blocker, dizocilpine, induced a deficit in spontaneous alteration, but when ifenprodil was co-administered with dizocilpine, it showed a strong tendency to attenuate the dizocilpine-induced deficit. In the plus-maze, ifenprodil had an anxiolytic profile which was accompanied by an increase in locomotion. Dizocilpine had an anxiolytic profile in this model and increased locomotor activity. When co-administered with dizocilpine, ifenprodil reduced both the anxiolytic and locomotor effects of dizocilpine. When co-administered with ifenprodil, cyclopentyladenosine (CPA) and 1,3-dipropyl-8-cyclopentylxanthine (CPX) reduced the anxiolytic effect of ifenprodil. CPA and CPX in combination did not reverse the anxiolytic effect of ifenpropil. It is concluded that NMDA antagonists with different sites of action can show distinct behavioural profiles, with dizocilpine but not ifenprodil inducing a deficit in working memory, while both are anxiolytic. Blockade of NMDA receptors by ifenprodil, however, can preclude any response to dizocilpine. The anxiolytic activity of ifenprodil may involve the release of purines acting at adenosine receptors.

Animals↗
PubMed1996

Alanine scanning mutagenesis of conserved arginine/lysine-arginine/lysine-X-X-arginine/lysine G protein-activating motifs on m1 muscarinic acetylcholine receptors.

Alanine scanning mutagenesis of B-B-X-X-B motifis (where B is a basic residue and X is any nonbasic residue) in m1 muscarinic acetylcholine receptors was performed to determine the relative roles of basic amino acids in receptor coupling. This conserved motif is found in many G protein-coupled receptors and has been implicated in G protein activation. The KKAAR365 motif, located at the carboxyl-terminal third intracellular loop of m1 receptors, was mutated to AAAAA365, thereby generating a triple-substitution mutant devoid of ability to stimulate either phosphoinositide (PI) hydrolysis or cAMP accumulation. In contrast, a triple-alanine substitution of the KRTPR140 motif in the carboxyl-terminal second intracellular loop, yielding mutant AATPA140, had no effect on receptor coupling to the two independent second messenger pathways. Analysis of a series of single- and double-substitution mutants demonstrate that all three basic residues of the KKAAR365 motif participate in efficient m1 receptor coupling. The presence of second and third basic residues in this motif was absolutely critical for full agonist recognition of a high and low affinity state of the receptor. Mutation of either Lys362 or Lys365, but not-Lys361, abolished guanine nucleotide-dependent conversion of agonist affinity states and correlated with an inability of full agonists to fully activate PI hydrolysis. The different combinatorial double-substitution mutants also revealed that Lys365 was necessary but not sufficient, in the context of the KKAAR365 motif, for efficient receptor coupling. This residue cannot facilitate full agonist-stimulated Pl hydrolysis in the absence of both Lys361 and Lys362. In comparison, the critical residue Lys362 was both necessary and sufficient. Substitution of nearby basic residues Lys361 and Lys365 with alanine yielded mutant AKAAA365, which exhibited partial ability to couple PI hydrolysis after full agonist stimulation. Therefore, Lys365 seems to function in a hierarchal (interdependent) manner with nearby basic residues, whereas Lys361 and Lys362 can act independent of surrounding basic residues to facilitate partial m1 receptor coupling after full agonist stimulation. In contrast, all three residues must be present for stimulation of PI hydrolysis by a partial agonist.

Alanine↗
PubMed1995

The minimal gene complement of Mycoplasma genitalium.

The complete nucleotide sequence (580,070 base pairs) of the Mycoplasma genitalium genome, the smallest known genome of any free-living organism, has been determined by whole-genome random sequencing and assembly. A total of only 470 predicted coding regions were identified that include genes required for DNA replication, transcription and translation, DNA repair, cellular transport, and energy metabolism. Comparison of this genome to that of Haemophilus influenzae suggests that differences in genome content are reflected as profound differences in physiology and metabolic capacity between these two organisms.

Antigenic Variation↗
PubMed1995

Regulation of muscarinic receptor expression by changes in mRNA stability.

Regulation of muscarinic acetylcholine receptor (mAChR) subtype mRNAs was investigated in the human neuroblastoma cell line IMR-32 and in transfected CHO cells. IMR-32 cells express both m1 and m3 subtypes of mAChR. Exposure of IMR-32 cells to the muscarinic agonist, carbamylcholine (CBC) leads to a time dependent down-regulation of mAChRs which was maximal by 9 hours. mAChR activation resulted in a differential regulation of mAChR subtype mRNAs. m1 mAChR mRNA was down-regulated following 12 hours of agonist treatment and was associated with a decreased stability of the receptor transcript. In contrast, the m3 mAChR mRNA was resistant to agonist treatment for up to 24 hours. Using transfected CHO cells, we identified sequence elements within the 3'-untranslated region (3'-UTR) of the m1 mAChR gene which dictate agonist-induced destabilization of the m1 mAChR mRNA. Removal of these sequences abolished the ability of chronic agonist exposure to destabilize m1 mAChR mRNA. These findings suggest that sequence specific differences between m1 and m3 mAChR subtypes, which both preferentially couple to hydrolysis of phosphoinositides, may be responsible for differences in the regulation of mAChR gene expression.

3' Untranslated Regions↗
PubMed1995

Structure and functional analysis of G protein-coupled receptors and potential diagnostic ligands.

G protein-coupled receptors are a diverse class of proteins that mediate signal transduction across the plasma membrane. More than 200 receptors in this extended gene family have been cloned, and comparison of the deduced amino-acid sequences indicates that these proteins have marked homology and share a common membrane topology consisting of seven transmembrane helices. Although there is considerable variability in the physiologic ligands responsible for receptor activation, all receptors in this group interact with trimeric, guanine nucleotide-binding proteins to initiate signaling cascades in the cell cytosol. To investigate the structural motifs responsible for ligand binding, we have established a model system to express heterologously human G protein-coupled receptors in a mammalian cell line. This experimental system allows each receptor subtype to be studied in isolation and provides a direct means to link receptor activation to a particular second messenger cascade. Furthermore, the efficacy and specificity of new pharmaceuticals can now be evaluated readily with cloned human receptors, eliminating the need for animal tissues. We have used this expression system in conjunction with an experimental strategy of site-directed mutagenesis to identify amino-acid residues that have a functional role in ligand binding. Because of the strong homology that exists within this family of receptor proteins, the results of this work are applicable to other systems and, therefore, can help to establish a more complete understanding of ligand-receptor interactions. This combined molecular and biochemical approach to the study of G protein-coupled receptors can pave the way for the development of isoform-specific ligands that may be used for radionuclide imaging and therapy.

Amino Acid Sequence↗
PubMed1994

Mutations of two PMS homologues in hereditary nonpolyposis colon cancer.

Hereditary nonpolyposis colorectal cancer (HNPCC) is one of man's commonest hereditary diseases. Several studies have implicated a defect in DNA mismatch repair in the pathogenesis of this disease. In particular, hMSH2 and hMLH1 homologues of the bacterial DNA mismatch repair genes mutS and mutL, respectively, were shown to be mutated in a subset of HNPCC cases. Here we report the nucleotide sequence, chromosome localization and mutational analysis of hPMS1 and hPMS2, two additional homologues of the prokaryotic mutL gene. Both hPMS1 and hPMS2 were found to be mutated in the germline of HNPCC patients. This doubles the number of genes implicated in HNPCC and may help explain the relatively high incidence of this disease.

Adenosine Triphosphatases↗
PubMed1994

Mutation of a mutL homolog in hereditary colon cancer.

Some cases of hereditary nonpolyposis colorectal cancer (HNPCC) are due to alterations in a mutS-related mismatch repair gene. A search of a large database of expressed sequence tags derived from random complementary DNA clones revealed three additional human mismatch repair genes, all related to the bacterial mutL gene. One of these genes (hMLH1) resides on chromosome 3p21, within 1 centimorgan of markers previously linked to cancer susceptibility in HNPCC kindreds. Mutations of hMLH1 that would disrupt the gene product were identified in such kindreds, demonstrating that this gene is responsible for the disease. These results suggest that defects in any of several mismatch repair genes can cause HNPCC.

Adaptor Proteins, Signal Transducing↗
PubMed1994

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 Alzheimer's disease.

Adenylyl Cyclases↗
PubMed1994

Agonist-mediated destabilization of m1 muscarinic acetylcholine receptor mRNA. Elements involved in mRNA stability are localized in the 3'-untranslated region.

The effects of chronic agonist exposure on receptor number (down-regulation) have been shown, in part, to be due to effects on mRNA levels. Agonist-mediated effects on muscarinic acetylcholine receptor (mAChR) mRNA were investigated in Chinese hamster ovary (CHO) cells stably transfected with m1 mAChR gene constructs containing the open reading frame and a series of deletions of the flanking 3'-untranslated region (3'-UTR). Carbachol (CBC) down-regulated m1 mAChRs encoded by the construct m1C1, an m1 mAChR transcript containing the entire flanking 3'UTR (nucleotides 1526-2622), in a time-dependent fashion with maximal decreases occurring by 12 h. Steady-state levels of m1C1 mRNA declined in a parallel fashion beginning 6 h after CBC pretreatment. Similar findings were obtained with m1C2, a construct which is missing all but 261 bases of flanking 3'-UTR (nucleotides (nt) 1526-1786). Since the rate of mRNA degradation represents an important potential regulatory mechanism to control the level of gene expression, we investigated the effects of CBC treatment on m1C1 and m1C2 mRNA stability. The half-life of either transcript in untreated cells was approximately 14 h, whereas m1C1 and m1C2 transcript half-lives decreased to approximately 3 h in cells treated with CBC. Agonist-induced destabilization of m1C2 mRNA could be mimicked by phorbol esters in a concentration-dependent manner and blocked by the protein kinase inhibitor, H-7. In contrast, m1 mAChR mRNA constructs missing nt 1526-1786 of the 3'-UTR (m1C3 and m1C4) did not undergo agonist- or phorbol ester-induced destabilization. In the neuroblastoma cell line IMR-32, endogenous m1 mAChR mRNA was down-regulated and destabilized following CBC treatment. These results demonstrate that agonist-induced mRNA destabilization is a potential mechanism for regulating m1 mAChR levels. Furthermore, deletion studies identify a 261 base region of the 3'-UTR having the potential to form stable stem-loop structures which likely harbors element(s) responsible for message destabilization.

Animals↗
PubMed1994

Beta-3 adrenoceptor selectivity of the dioxolane dicarboxylate phenethanolamines.

The beta-1, beta-2 and beta-3 adrenergic properties of several benzodioxole-containing phenethanolamines were determined in vitro in both functional and binding assays. In addition, two of the compounds were evaluated for their effects on radioligand binding and cyclic AMP (cAMP) production in stably transfected Chinese Hamster Ovary (CHO) cells expressing the cloned rat or human beta-3 adrenoceptor or the human beta-2 or beta-1 adrenoceptor. The (+/-)-R*,R*-racemate, CL 314,514, and the pure (-)-R,R enantiomer, CL 316,243, stimulated rat adipocyte lipolysis (beta-3 effect) with EC50 values in the low nanomolar range, while having no effect on the rate of contraction of guinea pig atria (beta-1 effect) and little or no ability to prevent the insulin-stimulated incorporation of [14C]glucose into rat soleus muscle glycogen (beta-2 effect) with concentrations as great as 100 microM. The lack of beta-1 and beta-2 adrenergic activity was confirmed by the low affinity of the compounds for beta-1 or beta-2 adrenoceptors in plasma membranes from rat heart or rat soleus muscle, respectively. In CHO cells expressing each human beta adrenoceptor subtype, CL 314,514 bound to beta-3-CHO cells with a Ki of 2 microM and stimulated cAMP production with an activation constant (Kact) of 1 microM, whereas it did not bind to either beta-1- or beta-2-CHO cells at 100 microM. CL 316,243 bound to membranes from rat beta-3-CHO cells with a Ki of 1 microM and stimulated cAMP production in beta-3-CHO cells with a Kact of 0.7 nM.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenergic beta-Agonists↗
PubMed1994

Site-directed mutagenesis of N-linked glycosylation sites on the gamma-aminobutyric acid type A receptor alpha 1 subunit.

Oligonucleotide-directed mutagenesis was used to mutate the two potential sites for N-linked glycosylation on the rat gamma-aminobutyric acid (GABA)A receptor alpha 1 subunit. Wild-type (WT) or mutant alpha 1 subunits [asparagine to glutamine substitutions at position 10 (alpha 1Q10), 110 (alpha 1Q110), or both 10 and 110 (alpha 1Q10/110)] were coexpressed with beta 1 and gamma 2 subunits in Xenopus oocytes. Removal of either one or both potential sites for N-linked glycosylation resulted in expression, in Xenopus oocytes, of functional GABAA receptors with pharmacological properties similar to those observed for the WT receptor. WT and mutant alpha 1 subunits were co-transfected with beta 1 and gamma 2 subunits in human embryonic kidney 293 cells. WT and mutant alpha 1 subunits expressed in 293 cells were photoaffinity labeled with [3H]flunitrazepam. Co-transfection of alpha 1WT, alpha 1Q10, or alpha 1Q110 subunits in combination with beta 1 and gamma 2 GABAA receptor subunits resulted in the labeling of single bands, with approximate molecular masses of 54, 49, and 50 kDa, respectively. The decrease in molecular mass for both the alpha 1Q10 and alpha 1Q110 mutants suggests that both consensus sequences for N-linked glycosylation are used in 293 cells. Low levels of [3H]flunitrazepam binding prevented visualization of the alpha 1Q10/110 double mutant. The 293 cells transfected with either the alpha 1Q10 or alpha 1Q110 mutant in combination with beta 1 and gamma 2 subunits expressed significantly lower levels of [3H]Ro15-1788 binding, relative to WT levels. In addition, [3H]Ro15-1788 binding was undetectable in 293 cells expressing the alpha 1Q10/110 double mutant. When transfected 293 cells were grown at 30 zero, [3H]Ro15-1788 binding to alpha 1Q10 and alpha 1Q110 GABAA receptors was restored to levels comparable to that for WT receptors. [3H]Ro15-1788 binding to alpha 1Q10/110 was not reliably detected at 30 zero. Similar results were observed using [3H]muscimol. These data suggest that intracellular processing and transport of the glycosylation-deficient GABAA receptor alpha 1 subunit is temperature sensitive. Furthermore, the observed differences between the two expression systems may be accounted for by the typically lower temperature used for maintaining microinjected Xenopus oocytes. Thus, although glycosylation is not an absolute requirement for GABAA receptor expression, it has a profound effect on the processing of at least the alpha 1 receptor and its subsequent assembly into a mature receptor.

Affinity Labels↗
PubMed1993

Cross-talk between m1 muscarinic acetylcholine and beta 2-adrenergic receptors. cAMP and the third intracellular loop of m1 muscarinic receptors confer heterologous regulation.

Genes encoding the m1 muscarinic (m1 mAChR) and beta 2-adrenergic receptors (beta 2AR) were stably co-expressed into Chinese hamster ovary (CHO) cells to study receptor regulation and cross-talk. Persistent activation of the beta 2AR/adenylate cyclase pathway by isoproterenol leads to heterologous desensitization, internalization, and down-regulation of the m1 mAChR which is comparable, but smaller in magnitude, with that seen with persistent activation of the m1 mAChR by carbachol. This heterologous effect was mimicked by dibutyryl cAMP and forskolin and antagonized by the protein kinase A (PKA) inhibitor H-8. A potential consensus sequence for phosphorylation by PKA (Lys351-Arg-Lys-Thr354) exists on the third intracellular loop of the m1 mAChR, suggesting that receptor phosphorylation by PKA may be involved in heterologous regulation. The loss of m1 mAChRs induced by carbachol was not reversed by H-8, indicating that homologous regulation is not dependent on PKA. Recent evidence suggests that muscarinic agonist-mediated internalization of the m1 mAChR involves the third intracellular loop (i3) (Maeda, S., Lameh, J., Mallet, W. G., Philip, M., Ramachandran, J., and Sadee, W. (1990) FEBS Lett. 269, 386-388). Three deletion mutant receptors were constructed in which the majority, or small regions, of i3 were eliminated but the membrane proximal portions of the loop were left intact. Each of the mutants was co-expressed with the beta 2AR in CHO cells. A small region in i3 was identified which is crucial for carbachol- and isoproterenol-promoted internalization and down-regulation. This region contains a series of 6 serine residues within an 8-amino acid stretch. A similar domain has been identified in the carboxyl tail of the beta 2AR and has been proposed to participate in receptor internalization (Hausdorff, W. P., Campbell, P. T., Ostrowski, J., Yu, S. S., Caron, M. G., and Lefkowitz, R. J. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 2979-2983).

Adenylyl Cyclases↗
PubMed1993

A strong promoter element is located between alternative exons of a gene encoding the human gamma-aminobutyric acid-type A receptor beta 3 subunit (GABRB3).

The gene that encodes the beta 3 subunit of the gamma-aminobutyrate-Type A (GABAA) receptor is widely expressed in brain tissue and has been associated with imprinted genetic disorders. Here, the 5' regions of the human and rat genes were characterized and found to be highly conserved in both coding and non-coding sequences. A novel transcript of the human gene revealed the existence of an alternative exon 1 (exon 1a) that encodes a variant signal sequence. Relative levels of the alternative transcripts were found to vary between fetal and adult brain, and between different brain regions. Endogenous beta 3 subunit transcripts were also detected in several immortalized cell lines, including human kidney 293 cells. Transcription of exon 1 is initiated from multiple sites within a pyrimidine-rich region of the gene. This region of the human gene also exhibits strong promoter activity and binds nuclear factors at a site which overlaps the transcriptional start sites. The promoter element was shown to bind Sp1 and at least one other unidentified nuclear factor.

Amino Acid Sequence↗
PubMed1992

Site-directed mutagenesis of the rat m1 muscarinic acetylcholine receptor. Role of conserved cysteines in receptor function.

There are 9 cysteine residues in the rat m1 muscarinic acetylcholine receptor (mAChR) that are conserved among all five mammalian mAChR subtypes sequenced to date. To study the role of these cysteines in rat m1 mAChR function, site-directed mutagenesis was used to convert each Cys residue to Ser, and the mutant receptor genes were transfected into mAChR-deficient Chinese hamster ovary (CHO) cells. Substitution of Cys391 (extracellular loop III) or Cys421 and Cys435 (carboxyl terminus) produces receptors with wild type phenotype. Cells transfected with Ser98 or Ser178 (extracellular loops I and II, respectively) receptor genes display no carbachol-mediated hydrolysis of phosphoinositides (PI), and membranes prepared from these cells do not bind the muscarinic antagonist [3H] quinuclidinyl benzilate, even though the cells express transcripts of the m1 mAChR as determined by RNA hybridization analysis. Since biochemical evidence suggests that these cysteines form a disulfide bridge (Curtis, C. A. M., Wheatley, M., Bansal, S., Birdsall, N. J. M., Eveleigh, P., Pedder, E. K., Poyner, D., and Hulme, E. C. (1989) 264, 489-495), our findings imply that this disulfide linkage may be critical for formation of the ligand binding domain or for proper protein folding. The Ser394 mAChR (extracellular loop III) exhibits a 44% decrease in efficacy for carbachol-mediated stimulation of PI hydrolysis relative to the wild type receptor, but displays normal ligand binding affinities. The Ser407 m1 mAChR (transmembrane helix VII) displays a decreased efficacy for eliciting carbachol-mediated PI hydrolysis (39% that of CHO cells transfected with the wild type receptor) and a 4-fold shift to the right in the carbachol dose-response curve, which is consistent with the 4-fold decrease in carbachol affinity at the Ser407 m1 mAChR. In contrast, the Ser417 m1 mAChR (transmembrane helix VII) displays an increase in carbachol affinity and a shift to the left in the carbachol dose-response curve for PI hydrolysis. These findings suggest that cysteine residues in the seventh transmembrane helix of the m1 mAChR may influence agonist binding and the efficiency of receptor activation.

Amino Acid Sequence↗