Biomedical subjects

R K Sunahara

Publications and source records attributed to R K Sunahara.

At least 19 recordsLinked to original sources

PubMed2001

Cloning and mitochondrial localization of full-length D-AKAP2, a protein kinase A anchoring protein.

Differential compartmentalization of signaling molecules in cells and tissues is being recognized as an important mechanism for regulating the specificity of signal transduction pathways. A kinase anchoring proteins (AKAPs) direct the subcellular localization of protein kinase A (PKA) by binding to its regulatory (R) subunits. Dual specific AKAPs (D-AKAPs) interact with both RI and RII. A 372-residue fragment of mouse D-AKAP2 with a 40-residue C-terminal PKA binding region and a putative regulator of G protein signaling (RGS) domain was previously identified by means of a yeast two-hybrid screen. Here, we report the cloning of full-length human D-AKAP2 (662 residues) with an additional putative RGS domain, and the corresponding mouse protein less the first two exons (617 residues). Expression of D-AKAP2 was characterized by using mouse tissue extracts. Full-length D-AKAP2 from various tissues shows different molecular weights, possibly because of alternative splicing or posttranslational modifications. The cloned human gene product has a molecular weight similar to one of the prominent mouse proteins. In vivo association of D-AKAP2 with PKA in mouse brain was demonstrated by using cAMP agarose pull-down assay. Subcellular localization for endogenous mouse, rat, and human D-AKAP2 was determined by immunocytochemistry, immunohistochemistry, and tissue fractionation. D-AKAP2 from all three species is highly enriched in mitochondria. The mitochondrial localization and the presence of RGS domains in D-AKAP2 may have important implications for its function in PKA and G protein signal transduction.

A Kinase Anchor Proteins↗

PubMed2000

Isolation and characterization of constitutively active mutants of mammalian adenylyl cyclase.

A genetic screen in Saccharomyces cerevisiae identified mutations in mammalian adenylyl cyclase that activate the enzyme in the absence of G(s)alpha. Thirteen of these mutant proteins were characterized biochemically in an assay system that depends on a mixture of the two cytosolic domains (C(1) and C(2)) of mammalian adenylyl cyclases. Three mutations, I1010M, K1014N, and P1015Q located in the beta4-beta5 loop of the C(2) domain of type II adenylyl cyclase, increase enzymatic activity in the absence of activators. The K1014N mutation displays both increased maximal activity and apparent affinity for the C(1) domain of type V adenylyl cyclase in the absence of activators of the enzyme. The increased affinity of the mutant C(2) domain of adenylyl cyclase for the wild type C(1) domain was exploited to isolate a complex containing VC(1), IIC(2), and G(s)alpha-guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) in the absence of forskolin and a complex of VC(1), IIC(2), forskolin, and P-site inhibitor in the absence of G(s)alpha-GTPgammaS. The isolation of these complexes should facilitate solution of crystal structures of low activity states of adenylyl cyclase and thus determination of the mechanism of activation of the enzyme by forskolin and G(s)alpha.

Adenylyl Cyclases↗

PubMed2000

Molecular basis for P-site inhibition of adenylyl cyclase.

P-site inhibitors are adenosine and adenine nucleotide analogues that inhibit adenylyl cyclase, the effector enzyme that catalyzes the synthesis of cyclic AMP from ATP. Some of these inhibitors may represent physiological regulators of adenylyl cyclase, and the most potent may ultimately serve as useful therapeutic agents. Described here are crystal structures of the catalytic core of adenylyl cyclase complexed with two such P-site inhibitors, 2'-deoxyadenosine 3'-monophosphate (2'-d-3'-AMP) and 2',5'-dideoxyadenosine 3'-triphosphate (2',5'-dd-3'-ATP). Both inhibitors bind in the active site yet exhibit non- or uncompetitive patterns of inhibition. While most P-site inhibitors require pyrophosphate (PP(i)) as a coinhibitor, 2',5'-dd-3'-ATP is a potent inhibitor by itself. The crystal structure reveals that this inhibitor exhibits two binding modes: one with the nucleoside moiety bound to the nucleoside binding pocket of the enzyme and the other with the beta and gamma phosphates bound to the pyrophosphate site of the 2'-d-3'-AMP.PP(i) complex. A single metal binding site is observed in the complex with 2'-d-3'-AMP, whereas two are observed in the complex with 2', 5'-dd-3'-ATP. Even though P-site inhibitors are typically 10 times more potent in the presence of Mn(2+), the electron density maps reveal no inherent preference of either metal site for Mn(2+) over Mg(2+). 2',5'-dd-3'-ATP binds to the catalytic core of adenylyl cyclase with a K(d) of 2.4 microM in the presence of Mg(2+) and 0.2 microM in the presence of Mn(2+). Pyrophosphate does not compete with 2',5'-dd-3'-ATP and enhances inhibition.

Adenylyl Cyclase Inhibitors↗

PubMed1999

Two-metal-Ion catalysis in adenylyl cyclase.

Adenylyl cyclase (AC) converts adenosine triphosphate (ATP) to cyclic adenosine monophosphate, a ubiquitous second messenger that regulates many cellular functions. Recent structural studies have revealed much about the structure and function of mammalian AC but have not fully defined its active site or catalytic mechanism. Four crystal structures were determined of the catalytic domains of AC in complex with two different ATP analogs and various divalent metal ions. These structures provide a model for the enzyme-substrate complex and conclusively demonstrate that two metal ions bind in the active site. The similarity of the active site of AC to those of DNA polymerases suggests that the enzymes catalyze phosphoryl transfer by the same two-metal-ion mechanism and likely have evolved from a common ancestor.

Adenosine Triphosphate↗

PubMed1998

Exchange of substrate and inhibitor specificities between adenylyl and guanylyl cyclases.

The active sites of guanylyl and adenylyl cyclases are closely related. The crystal structure of adenylyl cyclase and modeling studies suggest that specificity for ATP or GTP is dictated in part by a few amino acid residues, invariant in each family, that interact with the purine ring of the substrate. By exchanging these residues between guanylyl cyclase and adenylyl cyclase, we can completely change the nucleotide specificity of guanylyl cyclase and convert adenylyl cyclase into a nonselective purine nucleotide cyclase. The activities of these mutant enzymes remain fully responsive to their respective stimulators, sodium nitroprusside and Gsalpha. The specificity of nucleotide inhibitors of guanylyl and adenylyl cyclases that do not act competitively with respect to substrate are similarly altered, indicative of their action at the active sites of these enzymes.

Adenylyl Cyclases↗

PubMed1997

Crystal structure of the catalytic domains of adenylyl cyclase in a complex with Gsalpha.GTPgammaS.

The crystal structure of a soluble, catalytically active form of adenylyl cyclase in a complex with its stimulatory heterotrimeric G protein alpha subunit (Gsalpha) and forskolin was determined to a resolution of 2.3 angstroms. When P-site inhibitors were soaked into native crystals of the complex, the active site of adenylyl cyclase was located and structural elements important for substrate recognition and catalysis were identified. On the basis of these and other structures, a molecular mechanism is proposed for the activation of adenylyl cyclase by Gsalpha.

Adenosine Triphosphate↗

PubMed1997

Crystal structure of the adenylyl cyclase activator Gsalpha.

The crystal structure of Gsalpha, the heterotrimeric G protein alpha subunit that stimulates adenylyl cyclase, was determined at 2.5 A in a complex with guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS). Gsalpha is the prototypic member of a family of GTP-binding proteins that regulate the activities of effectors in a hormone-dependent manner. Comparison of the structure of Gsalpha.GTPgammaS with that of Gialpha.GTPgammaS suggests that their effector specificity is primarily dictated by the shape of the binding surface formed by the switch II helix and the alpha3-beta5 loop, despite the high sequence homology of these elements. In contrast, sequence divergence explains the inability of regulators of G protein signaling to stimulate the GTPase activity of Gsalpha. The betagamma binding surface of Gsalpha is largely conserved in sequence and structure to that of Gialpha, whereas differences in the surface formed by the carboxyl-terminal helix and the alpha4-beta6 loop may mediate receptor specificity.

Adenylyl Cyclases↗

PubMed1997

Interaction of Gsalpha with the cytosolic domains of mammalian adenylyl cyclase.

Forskolin- and Gsalpha-stimulated adenylyl cyclase activity is observed after mixture of two independently-synthesized approximately 25-kDa cytosolic fragments derived from mammalian adenylyl cyclases (native Mr approximately 120,000). The C1a domain from type V adenylyl cyclase (VC1) and the C2 domain from type II adenylyl cyclase (IIC2) can both be expressed in large quantities and purified to homogeneity. When mixed, their maximally stimulated specific activity, 150 micromol/min/mg protein, substantially exceeds values observed previously with the intact enzyme. A soluble, high-affinity complex containing one molecule each of VC1, IIC2, and guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS)-Gsalpha is responsible for the observed enzymatic activity and can be isolated. In addition, GTPgammaS-Gsalpha interacts with homodimers of IIC2 to form a heterodimeric complex (one molecule each of Gsalpha and IIC2) but not detectably with homodimers of VC1. Nevertheless, Gsalpha can be cross-linked to VC1 in the activated heterotrimeric complex of VC1, IIC2, and Gsalpha, indicating its proximity to both components of the enzyme that are required for efficient catalysis. These results and those in the accompanying report (Dessauer, C. W., Scully, T. T., and Gilman, A. G. (1997) J. Biol. Chem. 272, 22272-22277) suggest that activators of adenylyl cyclase facilitate formation of a single, high-activity catalytic site at the interface between C1 and C2.

Adenylyl Cyclases↗

PubMed1997

cAMP induces heme oxygenase-1 gene expression and carbon monoxide production in vascular smooth muscle.

Recent studies indicate that vascular smooth muscle cells generate carbon monoxide (CO) via the action of heme oxygenase (HO). Because adenosine 3',5'-cyclic monophosphate (cAMP) is an important intracellular signaling molecule in the regulation of vascular cell function, we examined whether this second messenger modulates the expression of HO and the production of CO by rat aortic smooth muscle cells. Treatment of smooth muscle cells with the membrane-permeable cAMP derivative dibutyryl cAMP or with compounds that increase intracellular cAMP levels (isoproterenol and forskolin) resulted in a concentration- and time-dependent increase in the levels of HO-1 mRNA and protein, whereas the expression of HO-2 remained unchanged. Both actinomycin D and cycloheximide blocked the basal expression of HO-1 mRNA and protein and prevented the cAMP-mediated induction of HO-1. Incubation of platelets with cAMP-treated smooth muscle cells resulted in a significant increase in platelet cGMP concentration that was partially reversed by treatment of smooth muscle cells with the nitric oxide synthase inhibitor NG-monomethyl-L-arginine or the HO blocker zinc protoporphyrin-IX. However, the combined addition of these two inhibitors to cAMP-treated smooth muscle cells or the addition of the CO and NO scavenger hemoglobin to platelets completely blocked the stimulatory effect on platelet cGMP levels. These results demonstrate that cAMP induces the expression of the HO-1 gene and stimulates the formation of CO and NO in vascular smooth muscle cells. The capacity of cAMP to induce the synthesis of guanylate cyclase-stimulatory CO from smooth muscle cells may represent a novel mechanism by which this nucleotide regulates vascular tone.

Animals↗

PubMed1996

Complexity and diversity of mammalian adenylyl cyclases.

Molecular cloning has permitted identification of several novel isoforms of mammalian adenylyl cyclase; these proteins now comprise a family of at least 10. All of the membrane-bound enzymes are activated by the alpha subunit of G alpha, a receptor-regulated, heterotrimeric guanine nucleotide-binding protein, and by the diterpene forskolin. Certain cyclases are also activated by Ca(2+)-calmodulin, while some are inhibited by the alpha subunits of the three Gi proteins. The discovery of new isoforms has also revealed unanticipated mechanisms of regulation, including activation or inhibition by the G-protein beta gamma subunit complex, inhibition by G(o) alpha, inhibition by Ca2+, and phosphorylation by protein kinases C and A. The effects of activators are often highly synergistic or conditional, suggesting function of these enyzmes as coincidence detectors. The plethora of receptors, G proteins, and adenylyl cyclases permits assembly of very complex signaling systems with a wide variety of integrative characteristics.

Adenylyl Cyclases↗

PubMed1995

Ethanol potentiates interleukin-1 beta-stimulated inducible nitric oxide synthase expression in cultured vascular smooth muscle cells.

Experiments were performed to examine the effect of ethanol on the production of nitric oxide from interleukin-1 beta (IL-1 beta)-treated cultured rat aortic smooth muscle cells. Incubation of vascular smooth muscle cells with IL-1 beta resulted in the release of nitrite and in the intracellular accumulation of L-citrulline. In parallel with this, IL-1 beta increased inducible nitric oxide synthase (iNOS) mRNA and protein. Ethanol (6.5-650 mM) potentiated the IL-1 beta-mediated stimulation of iNOS mRNA production, the appearance of iNOS protein and the generation of nitrite and L-citrulline from smooth muscle cells in a concentration-dependent manner. In the absence of IL-1 beta, ethanol failed to induce iNOS expression. These results demonstrate that pharmacologically relevant concentrations of ethanol enhance the IL-1 beta-induced expression of the iNOS gene in vascular smooth muscle. The ability of ethanol to augment the release of the platelet inhibitor and vasodilator nitric oxide may, in part, contribute to the beneficial cardiovascular effects associated with moderate alcohol consumption.

Amino Acid Oxidoreductases↗

PubMed1994

Receptor-receptor link in membranes revealed by ligand competition: example for dopamine D1 and D2 receptors.

An interaction or link between dopamine D1 receptors and dopamine D2 receptors was found by a ligand competition method, using [3H]raclopride to label dopamine D2 receptors and SCH 23390 to block dopamine D1 receptors. In the presence of endogenous or exogenous dopamine, SCH 23390 increased the binding of [3H]raclopride in post-mortem human striata homogenates or in tissue culture cells containing human dopamine D1 and D2 receptors. In order to reveal such intramembrane receptor-receptor interactions in general, therefore, it appears essential to add two agonists, one for each receptor, and then to block one of the receptors when measuring the binding of a ligand to the second receptor.

Animals↗

PubMed1993

Localization of D1 and D2 dopamine receptors in brain with subtype-specific antibodies.

Five or more dopamine receptor genes are expressed in brain. However, the pharmacological similarities of the encoded D1-D5 receptors have hindered studies of the localization and functions of the subtypes. To better understand the roles of the individual receptors, antibodies were raised against recombinant D1 and D2 proteins and were shown to bind to the receptor subtypes specifically in Western blot and immunoprecipitation studies. Each antibody reacted selectively with the respective receptor protein expressed both in cells transfected with the cDNAs and in brain. By immunocytochemistry, D1 and D2 had similar regional distributions in rat, monkey, and human brain, with the most intense staining in striatum, olfactory bulb, and substantia nigra. Within each region, however, the precise distributions of each subtype were distinct and often complementary. D1 and D2 were differentially enriched in striatal patch and matrix compartments, in selective layers of the olfactory bulb, and in either substantia nigra pars compacta or reticulata. Electron microscopy demonstrated that D1 and D2 also had highly selective subcellular distributions. In the rat neostriatum, the majority of D1 and D2 immunoreactivity was localized in postsynaptic sites in subsets of spiny dendrites and spine heads in rat neostriatum. Presynaptic D1 and D2 receptors were also observed, indicating both subtypes may regulate neurotransmitter release. D1 was also present in axon terminals in the substantia nigra. These results provide a morphological substrate for understanding the pre- and postsynaptic functions of the genetically defined D1 and D2 receptors in discrete neuronal circuits in mammalian brain.

Antibody Specificity↗

PubMed1993

Serotonin receptor cDNA cloned from Lymnaea stagnalis.

Serotonin (5-HT) is a major neurotransmitter that influences various behaviors, neuronal plasticity, learning, and memory in molluscs. Although the physiology of 5-HT transmission in molluscs is well studied, little is known about the pharmacology and diversity of the 5-HT receptor system. Based on the high homology of genes coding for guanine nucleotide-binding protein (G protein)-coupled receptors, we have cloned a gene for the Lymnaea stagnalis 5-HT (5HTlym) receptor. The putative receptor protein, 509 amino acids long, has highest homology with the Drosophila 5-HT receptors and mammalian 5HT1 receptors. As revealed by RNA blot-hybridization analysis, two mRNA species of 2.3 and 3.2 kb are detected in the central nervous system of Lymnaea. Transient expression of 5HTlym in COS-7 cells showed saturable [3H]lysergic acid diethylamide binding with an estimated dissociation constant of 0.9 nM. The 5HTlym receptor exhibited a mixed 5HT-like pharmacology that cannot be precisely categorized with existing mammalian classification nomenclature. However, the 5HTlym receptor does display some characteristics that have been attributed to the putative mammalian vascular 5HT1-like receptor.

Amino Acid Sequence↗

PubMed1993

Schizophrenia: dopamine D1 receptor sequence is normal, but has DNA polymorphisms.

Genes that regulate dopamine (DA) receptors may underlie the overactive DA system in schizophrenia. Since it is known that there is an abnormally reduced or absent regulation of the DA D2 receptor by the DA D1 receptor in the postmortem schizophrenia brain, the human DA D1 receptor gene was sequenced from genomic deoxyribonucleic acid (DNA) of seven schizophrenic individuals. The tissues from two schizophrenics had previously been found to have a reduced link between DA D1 and D2 receptors. The D1 receptor genes were amplified by the polymerase chain reaction, subcloned, and sequenced. Although three DNA polymorphisms were found, the deduced amino acid sequence of the DA D1 receptor was normal in these tissues.

Adult↗

PubMed1993

Exclusion of close linkage of Tourette's syndrome to D1 dopamine receptor.

OBJECTIVE: The authors' goal was to establish if a mutation in D1 dopamine receptor locus (DRD1), or one genetically close to it, could cause Gilles de la Tourette's syndrome. METHOD: DRD1 and linked markers (D5S36, D5S61, and D5S62) were studied in a large Mennonite Tourette's syndrome kindred. Only individuals with the full Tourette's syndrome were considered to be affected in one series of analyses; in another series the diagnostic spectrum was broadened to include chronic multiple tics. Liability classes were defined to take into account age at onset and sex differences; dominant inheritance was assumed. The authors' version of the LINKMAP program of the LINKAGE package modified to run under distributed parallel processing (Linda LINKMAP) was used for the multipoint linkage analysis. RESULTS: Complete (theta = 0.0) linkage of Tourette's syndrome with DRD1 was ruled out (lod score of -10.1) when the disease was defined narrowly. The area of exclusion of linkage (lod score between -2 and -10.5) extended from map position -0.10 to map position 0.50. The authors conducted an additional (centromeric) multipoint analysis with D5S36 as well as glucocorticoid receptor (GRL) and D5S22, resulting in an overlapping area of exclusion to map position -0.30 when the disease was defined narrowly. CONCLUSIONS: This result provides strong evidence against linkage of the DRD1 D1 dopamine receptor locus with Tourette's syndrome. This exclusion extends the authors' earlier work with the dopamine system in Tourette's syndrome to exclude the two best characterized dopamine receptors from linkage with Tourette's syndrome.

Adolescent↗

PubMed1993

Dopamine receptors and antipsychotic drug response.

Dopamine receptors have been divided into two major types--D1 and D2--based primarily on pharmacological and biochemical criteria. Recent advances in the molecular biology of the dopamine receptor system have allowed the identification and characterisation of at least five distinct neuronal dopamine receptor genes (D1 to D5). These genes encode dopamine receptors belonging to the D1 receptor family, termed D1 and D5, and three D2-like receptors, termed D2, D3 and D4. These receptors are distinguished on the basis of their primary structure, chromosomal location, mRNA size and tissue distribution, and biochemical and pharmacological differences. Although individually these receptor subtypes may not be directly and exclusively involved in the maintenance or expression of schizophrenia, alterations of any of the receptors may contribute to the perturbation or instability of dopaminergic homeostasis in the brain. What was once thought to be a simple two-receptor system seems to have emerged as an intricate and interactive entity. This review summarises what is currently understood about dopamine receptors, their role in antipsychotic drug action, and their association with psychosis.

Animals↗

PubMed1993

Cloning and expression of a human somatostatin-14-selective receptor variant (somatostatin receptor 4) located on chromosome 20.

Based on pharmacological, biochemical, and molecular criteria, multiple somatostatin receptor (SSTR) subtypes selective for somatostatin (SST)-14 and -28 have been postulated to exist in both the brain and periphery. We report here on the cloning and characterization of a human gene encoding a new member of the guanine nucleotide-binding protein-linked SSTR family, termed human (h)SSTR4. The 388-amino acid protein, with a predicted molecular mass of approximately 42 kDa, displays sequence similarity, particularly within putative transmembrane domains, with the recently cloned hSSTR1 (69%), hSSTR2 (56%), and hSSTR3 (58%). Membranes prepared from COS-7 cells transiently expressing the hSSTR4 gene bound 125I-[Leu8,D-Trp22,Tyr25]SST-28 in a saturable manner with high affinity (approximately 60 pM) and with a pharmacological profile and rank order of potency ([D-Trp8]SST-14 > SST-14 > SMS 201-995 > SST-28 > MK-678) indicative of a SST-14-selective receptor. Ki values for the inhibition of 125I-[Leu8,D-Trp22,Tyr25]SST-28 binding to the expressed receptor by these somatostatinergic peptides were 0.3, 1.1, 1.4, 2.2, and 6.5 nM, respectively. High affinity agonist binding to hSSTR4 was significantly reduced by GTP and pertussis toxin, indicating association of the expressed receptor with pertussis toxin-sensitive guanine nucleotide-binding proteins. Northern blot analysis revealed the presence of an SSTR4 mRNA species of approximately 4 kilobases in select regions of the monkey brain, including the hippocampus, hypothalamus, cortex, and striatum, with little or no receptor mRNA detected in either the olfactory tubercle, medulla, cerebellum, or amygdala. The SSTR4 gene maps to human chromosome 20. These findings document the existence of a novel human SSTR gene. Although the hSSTR4 displays an overall deduced amino acid homology of 86% with the recently reported rat homolog [Proc. Natl. Acad. Sci. USA 89:11151-11155 (1992)], the two gene products possess distinctive pharmacological profiles and affinities for the SST agonists SMS 201-995 and MK-678.

Amino Acid Sequence↗