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M G Caron

Publications and source records attributed to M G Caron.

At least 235 records · Page 13Linked to original sources
PubMed1990

Multiple second messenger pathways of alpha-adrenergic receptor subtypes expressed in eukaryotic cells.

The alpha-adrenergic receptors mediate the effects of epinephrine and norepinephrine on cellular signaling systems via guanine nucleotide binding regulatory proteins (G-proteins). Three alpha-adrenergic receptor subtypes have been cloned: the alpha 1, the alpha 2-C10, and the alpha 2-C4 adrenergic receptors. To investigate functional differences between the different subtypes, we assessed the ability of each to interact with adenylyl cyclase and polyphosphoinositide metabolism by permanently and transiently expressing the DNAs encoding the alpha 1, the alpha 2-C10, and the alpha 2-C4 adrenergic receptors in cells lacking endogenous alpha-adrenergic receptors. Both alpha 2-C10 and alpha 2-C4 couple primarily to inhibition of adenylyl cyclase and to a lesser extent to stimulation of polyphosphoinositide hydrolysis. alpha 2-C10 inhibits adenylyl cyclase more efficiently than alpha 2-C4. Effects of the alpha 2-adrenergic receptors on adenylyl cyclase inhibition and on polyphosphoinositide hydrolysis are both mediated by pertussis toxin-sensitive G-proteins. The major coupling system of the alpha 1-adrenergic receptor is activation of phospholipase C via a pertussis toxin-insensitive G-protein. alpha 1-Adrenergic receptor stimulation can also increase intracellular cAMP by a mechanism that does not involve direct activation of adenylyl cyclase. As with the muscarinic cholinergic receptor family our results show that each of the alpha-adrenergic receptor subtypes can couple to multiple signal transduction pathways and suggest several generalities about the effector coupling mechanisms of G-protein-coupled receptors.

Adenylate Cyclase Toxin↗
PubMed1990

Role of phosphorylation in desensitization of the beta-adrenoceptor.

Regulation of receptors allows their responses to be modified rapidly and appropriately according to the needs of the environment. Multiple mechanisms are involved in the loss of sensitivity that follows exposure to agonists. Receptor sequestration, a rapid and transient event, and receptor downregulation, which requires more prolonged agonist exposure, contribute to this effect. However, in this article Bob Lefkowitz and colleagues focus primarily on the recent developments in understanding mechanisms of rapid desensitization involving receptor phosphorylation. Various molecular biological techniques have been used to demonstrate the important roles of two particular kinases--beta ARK and protein kinase A--in this regard.

Animals↗
PubMed1990

Dopamine receptor subtypes: beyond the D1/D2 classification.

The D1/D2 dopamine receptor classification is widely accepted. However, intense investigative efforts over the last several years using pharmacological, biochemical and behavioral approaches have produced results that are increasingly difficult to reconcile with the existence of only two dopamine receptor subtypes. Recent developments, including cloning of the cDNAs and/or genes for several members of the large family of G-protein-coupled receptors, have revealed that heterogeneity in the pharmacological or biochemical characteristics of individual receptors often indicates the presence of previously unsuspected molecular subtypes. In this article, Marc Caron and colleagues have assembled the main lines of evidence that suggest the presence of several novel subtypes for both D1 and D2 dopamine receptors and predict that molecular cloning will, in the near future, confirm their existence.

Animals↗
PubMed1990

Involvement of tyrosine residues located in the carboxyl tail of the human beta 2-adrenergic receptor in agonist-induced down-regulation of the receptor.

Chronic exposure of various cell types to adrenergic agonists leads to a decrease in cell surface beta 2-adrenergic receptor (beta 2AR) number. Sequestration of the receptor away from the cell surface as well as a down-regulation of the total number of cellular receptors are believed to contribute to this agonist-mediated regulation of receptor number. However, the molecular mechanisms underlying these phenomena are not well characterized. Recently, tyrosine residues located in the cytoplasmic tails of several membrane receptors, such as the low density lipoprotein and mannose-6-phosphate receptors, have been suggested as playing an important role in the agonist-induced internalization of these receptors. Accordingly, we assessed the potential role of two tyrosine residues in the carboxyl tail of the human beta 2AR in agonist-induced sequestration and down-regulation of the receptor. Tyr-350 and Tyr-354 of the human beta 2AR were replaced with alanine residues by site-directed mutagenesis and both wild-type and mutant beta 2AR were stably expressed in transformed Chinese hamster fibroblasts. The mutation dramatically decreased the ability of the beta 2AR to undergo isoproterenol-induced down-regulation. However, the substitution of Tyr-350 and Tyr-354 did not affect agonist-induced sequestration of the receptor. These results suggest that tyrosine residues in the cytoplasmic tail of human beta 2AR are crucial determinants involved in its down-regulation.

Adenylyl Cyclases↗
PubMed1990

Expansion of the alpha 2-adrenergic receptor family: cloning and characterization of a human alpha 2-adrenergic receptor subtype, the gene for which is located on chromosome 2.

Pharmacologic, biochemical, and genetic analyses have demonstrated the existence of multiple alpha 2-adrenergic receptor (alpha 2AR) subtypes. We have cloned a human alpha 2AR by using the polymerase chain reaction with oligonucleotide primers homologous to conserved regions of the previously cloned alpha 2ARs, the genes for which are located on human chromosomes 4 (C4) and 10 (C10). The deduced amino acid sequence encodes a protein of 450 amino acids whose putative topology is similar to that of the family of guanine nucleotide-binding protein-coupled receptors, but whose structure most closely resembles that of the alpha 2ARs. Competition curve analysis of the binding properties of the receptor expressed in COS-7 cells with a variety of adrenergic ligands demonstrates a unique alpha 2AR pharmacology. Hybridization with somatic cell hybrids shows that the gene for this receptor is located on chromosome 2. Northern blot analysis of various rat tissues shows expression in liver and kidney. The unique pharmacology and tissue localization of this receptor suggest that this is an alpha 2AR subtype not previously identified by classical pharmacological or ligand binding approaches.

Amino Acid Sequence↗
PubMed1990

Chromosomal organization of adrenergic receptor genes.

The adrenergic receptors (ARs) (subtypes alpha 1, alpha 2, beta 1, and beta 2) are a prototypic family of guanine nucleotide binding regulatory protein-coupled receptors that mediate the physiological effects of the hormone epinephrine and the neurotransmitter norepinephrine. We have previously assigned the genes for beta 2- and alpha 2-AR to human chromosomes 5 and 10, respectively. By Southern analysis of somatic cell hybrids and in situ chromosomal hybridization, we have now mapped the alpha 1-AR gene to chromosome 5q32----q34, the same position as beta 2-AR, and the beta 1-AR gene to chromosome 10q24----q26, the region where alpha 2-AR is located. In mouse, both alpha 2- and beta 1-AR genes were assigned to chromosome 19, and the alpha 1-AR locus was localized to chromosome 11. Pulsed field gel electrophoresis has shown that the alpha 1- and beta 2-AR genes in humans are within 300 kilobases (kb) and the distance between the alpha 2- and beta 1-AR genes is less than 225 kb. The proximity of these two pairs of AR genes and the sequence similarity that exists among all the ARs strongly suggest that they are evolutionarily related. Moreover, they likely arose from a common ancestral receptor gene and subsequently diverged through gene duplication and chromosomal duplication to perform their distinctive roles in mediating the physiological effects of catecholamines. The AR genes thus provide a paradigm for understanding the evolution of such structurally conserved yet functionally divergent families of receptor molecules.

Animals↗
PubMed1990

Regions of the alpha 1-adrenergic receptor involved in coupling to phosphatidylinositol hydrolysis and enhanced sensitivity of biological function.

Regions of the hamster alpha 1-adrenergic receptor (alpha 1 AR) that are important in GTP-binding protein (G protein)-mediated activation of phospholipase C were determined by studying the biological functions of mutant receptors constructed by recombinant DNA techniques. A chimeric receptor consisting of the beta 2-adrenergic receptor (beta 2AR) into which the putative third cytoplasmic loop of the alpha 1AR had been placed activated phosphatidylinositol metabolism as effectively as the native alpha 1AR, as did a truncated alpha 1AR lacking the last 47 residues in its cytoplasmic tail. Substitutions of beta 2AR amino acid sequence in the intermediate portions of the third cytoplasmic loop of the alpha 1AR or at the N-terminal portion of the cytoplasmic tail caused marked decreases in receptor coupling to phospholipase C. Conservative substitutions of two residues in the C terminus of the third cytoplasmic loop (Ala293----Leu, Lys290----His) increased the potency of agonists for stimulating phosphatidylinositol metabolism by up to 2 orders of magnitude. These data indicate (i) that the regions of the alpha 1AR that determine coupling to phosphatidylinositol metabolism are similar to those previously shown to be involved in coupling of beta 2AR to adenylate cyclase stimulation and (ii) that point mutations of a G-protein-coupled receptor can cause remarkable increases in sensitivity of biological response.

Amino Acid Sequence↗
PubMed1990

Alpha 2-adrenergic agonists stimulate DNA synthesis in Chinese hamster lung fibroblasts transfected with a human alpha 2-adrenergic receptor gene.

To test the hypothesis that agents activating receptors negatively coupled to adenylyl cyclase (AC) can stimulate cell proliferation, we have expressed a human alpha 2-adrenergic receptor (alpha 2-C10) in CCL39 cells and studied the effects of alpha 2-agonists on reinitiation of DNA synthesis in quiescent cells. We report that the alpha 2-agonists epinephrine and clonidine stimulate [3H]-thymidine incorporation in synergy with fibroblast growth factor and that the alpha 2-antagonist yohimbine efficiently inhibits this response. Epinephrine- and clonidine-stimulated DNA synthesis is completely blocked by pertussis toxin and correlates well with the inhibition of prostaglandin E1-stimulated AC. Thus, their action closely resembles the action of serotonin in the same cell system, which is mediated through 5-HT1b receptors. In fact, serotonin- and epinephrine-stimulated DNA synthesis reinitiation is not additive, suggesting that both agents act through a common pathway. Interestingly, alpha 2-agonists also induced a moderate release of inositol phosphates, indicating that alpha 2-adrenergic receptors can interact both with the AC and phospholipase C messenger system. Activation of phosphoinositide (PI) turnover by epinephrine leads to a significant stimulation of Na+/H+ exchange but is insufficient to trigger a mitogenic response in CCL39 cells, as will be discussed. We found no evidence for epinephrine-induced activation of Na+/H+ exchange by a mechanism independent of PI breakdown.Our data show that alpha 2-adrenergic receptors can play a role in the regulation of cell proliferation in an appropriate context; also, the data support the hypothesis that receptors negatively coupled to AC must be taken into account as mediators of growth factor action in fibroblasts, in particular when activated in parallel with receptor tyrosine kinases.

Adrenergic alpha-Agonists↗
PubMed1990

Molecular characterization of dopamine receptors.

The D1 and D2 dopamine receptors have been biochemically characterized using specific probes based on the subtype selective antagonists SCH 23390 and spiperone, respectively. The D2 dopamine receptor was identified from several tissues by photoaffinity labeling and was purified from bovine anterior pituitary to homogeneity using a combination of affinity, lectin and hydroxylapatite chromatography. A complementary DNA (cDNA) encoding a rat brain D2 dopamine receptor has been cloned via low stringency hybridization using a portion of the beta 2-adrenergic receptor gene as a probe. Photoaffinity crosslinking and affinity chromatography have also been used to identify and purify the rat brain D1 dopamine receptor.

Affinity Labels↗
PubMed1990

Synthetic peptides of the hamster beta 2-adrenoceptor as substrates and inhibitors of the beta-adrenoceptor kinase.

1. The beta-adrenoceptor is one of a number of G protein-coupled receptors which have been proposed to contain seven transmembrane alpha-helices. The function of this receptor appears to be regulated by phosphorylation by a specific enzyme, the beta-adrenoceptor kinase. Synthetic peptides which comprise each of the proposed intra- and extracellular domains of the beta 2-adrenoceptor have been tested as potential substrates and inhibitors of the beta-adrenoceptor kinase. 2. Two peptides which encompass the middle and terminal portions of the carboxyl tail of the receptor served as substrates by beta-adrenoceptor kinase. The kinetics of the phosphorylation reaction, however, suggest that these peptides are 10(6)-fold poorer substrate than the agonist occupied receptor. 3. A number of synthetic peptides also served as inhibitors of beta 2-adrenoceptor phosphorylation by beta-adrenoceptor kinase. In particular, a peptide which comprised the first intracellular loop of the beta 2-adrenoceptor (amino acids 56-74) inhibited most effectively with an IC50 of 40 microM. 4. These results suggest that multiple intracellular regions of the beta-receptor may serve as potential sites of interaction with beta-adrenoceptor kinase. Moreover, these regions may serve as potential targets for the development of specific inhibitors of beta-adrenoceptor kinase which could be used to block homologous desensitization.

Amino Acid Sequence↗
PubMed1990

Adrenergic receptors. Models for regulation of signal transduction processes.

Adrenergic receptors are prototypic models for the study of the relations between structure and function of G protein-coupled receptors. Each receptor is encoded by a distinct gene. These receptors are integral membrane proteins with several striking structural features. They consist of a single subunit containing seven stretches of 20-28 hydrophobic amino acids that represent potential membrane-spanning alpha-helixes. Many of these receptors share considerable amino acid sequence homology, particularly in the transmembrane domains. All of these macromolecules share other similarities that include one or more potential sites of extracellular N-linked glycosylation near the amino terminus and several potential sites of regulatory phosphorylation that are located intracellularly. By using a variety of techniques, it has been demonstrated that various regions of the receptor molecules are critical for different receptor functions. The seven transmembrane regions of the receptors appear to form a ligand-binding pocket. Cysteine residues in the extracellular domains may stabilize the ligand-binding pocket by participating in disulfide bonds. The cytoplasmic domains contain regions capable of interacting with G proteins and various kinases and are therefore important in such processes as signal transduction, receptor-G protein coupling, receptor sequestration, and down-regulation. Finally, regions of these macromolecules may undergo posttranslational modifications important in the regulation of receptor function. Our understanding of these complex relations is constantly evolving and much work remains to be done. Greater understanding of the basic mechanisms involved in G protein-coupled, receptor-mediated signal transduction may provide leads into the nature of certain pathophysiological states.

Amino Acid Sequence↗
PubMed1990

Two kinases mediate agonist-dependent phosphorylation and desensitization of the beta 2-adrenergic receptor.

Binding of the catecholamine agonists epinephrine and norepinephrine to the beta-adrenergic receptor (BAR) rapidly activates adenylate cyclase via the stimulatory guanine nucleotide regulatory protein Gs, and results in rises in cellular levels of cAMP. However, continuous exposure to these agonists leads within minutes to a dampening of the enzymatic response. Both in vivo and in vitro studies have implicated agonist-induced phosphorylation of BAR in this process. These results include the isolation of a novel beta-adrenergic receptor kinase (BARK), which has been shown to preferentially phosphorylate receptors that are occupied by agonist when assessed in vitro. Recent studies in our laboratory have examined the desensitization process in intact cells to determine where on the receptor molecule functionally relevant phosphorylation occurs, and to identify the kinase(s) involved. In one set of studies, site-specific mutagenic techniques with the cloned gene for the human beta 2-adrenergic receptor were utilized to delete putative sites of phosphorylation by BARK and/or the cAMP-dependent protein kinase (PKA). Following expression of the mutated receptors in mammalian cells, the cells were challenged with different concentrations of agonist for 10-15 min and the functional and phosphorylation properties of the mutant receptors were then assessed. In another set of studies human A431 cells were permeabilized with low concentrations of digitonin and treated with selective inhibitors of both BARK and PKA. The cells were then exposed to desensitizing concentrations of agonist, and similar measurements performed. Taken together, the results from both sets of studies suggest that exposure of cells to low (nanomolar) concentrations of agonist leads to phosphorylation of the receptor on one or both consensus sites for PKA, and that the predominant effect of this phosphorylation on the adenylyl cyclase response is a loss in sensitivity of the receptor to further stimulation by the agonist. In contrast, exposure of cells to higher (micromolar) concentrations of agonist leads to BAR phosphorylation by both PKA and BARK, the latter on the carboxyl terminal region of the receptor. Phosphorylation of the receptor by both kinases appears to be required for the full desensitization effect seen with the high concentration of agonist, which includes both losses in sensitivity and in the maximal responsiveness of the adenylyl cyclase response upon subsequent challenge with the agonist. Such a dual kinase control of BAR phosphorylation may have important implications for understanding the regulation of desensitization under different physiological circumstances.

Adenylyl Cyclases↗
PubMed1990

Turning off the signal: desensitization of beta-adrenergic receptor function.

Cellular responses to many hormones and neurotransmitters wane rapidly despite continuous exposure of cells to these stimuli. This phenomenon, termed desensitization, has been particularly well studied for the stimulation of cAMP levels by plasma membrane beta-adrenergic receptors (beta AR). The molecular mechanisms underlying rapid beta AR desensitization do not appear to require internalization of the receptors, but rather an alteration in the functioning of beta AR themselves that uncouples the receptors from the stimulatory G protein Gs. This uncoupling phenomenon involves phosphorylation of beta AR by at least two kinases, PKA and the beta AR kinase (beta ARK), which are activated under different desensitizing conditions. Receptor phosphorylation by the two kinases leads to desensitization of the receptor response via distinct biochemical mechanisms, and additional cytosolic factors appear to be involved in the case of beta ARK. Numerous experimental approaches have been used recently to elucidate the molecular details of this ubiquitous biological process.

Amino Acid Sequence↗
PubMed1990

Expression of three alpha 2-adrenergic receptor subtypes in rat tissues: implications for alpha 2 receptor classification.

Based on biochemical and ligand binding studies in various tissues and species, evidence for several alpha 2-adrenergic receptor subtypes has accumulated. The current alpha 2-adrenergic receptor classification (alpha 2A, alpha 2B, alpha 2C) is based exclusively on pharmacological criteria. The molecular cloning of three distinct genes for human alpha 2-adrenergic receptors has confirmed the existence of multiple alpha 2-adrenergic receptor subtypes. According to their localization on different human chromosomes, the receptor genes were termed alpha 2-C10, alpha 2-C4, and alpha 2-C2. The relationship, however, between the pharmacologically characterized alpha 2-adrenergic receptors and the isolated genes has yet to be clarified. Using Northern blot hybridization, we analyzed the expression of the three cloned alpha 2-adrenergic receptor genes in 13 rat tissues, as well as in cell lines previously described as model systems for the pharmacologically defined alpha 2-adrenergic receptor subtypes. The alpha 2-C10 receptor corresponds to the alpha 2A subtype and is expressed in rat brainstem, cerebral cortex, hippocampus, pituitary gland, cerebellum, kidney, aorta, skeletal muscle, spleen, and lung. Messenger RNA coding for the alpha 2-C4 receptor was detected only in brain regions, not in peripheral tissues, whereas the alpha 2-C2 message was found only in liver and kidney. Hybridization experiments with RNA derived from tissues and cells from which the pharmacological alpha 2-receptor classification has been developed lead to the conclusion that the alpha 2B subtype represents two distinct receptor molecules, the alpha 2-C4 and a subtype previously undetected by classical ligand binding approaches. Furthermore, our results suggest that the alpha 2C subtype characterized in opossum kidney cells is an interspecies variation of alpha 2-C4 rather than a separate subtype. Finally, the cloned alpha 2-C2 receptor was found to be "alpha 2B-like" and not covered by the current pharmacological classification.

Animals↗