Hirofumi Tsuga

Effects of short-term toluene exposure on ligand binding to muscarinic acetylcholine receptors in the rat frontal cortex and hippocampus.

Changes in the binding affinity of the muscarinic acetylcholine receptor agonist carbamylcholine were determined in membranes isolated from the brains of rats exposed to toluene at concentrations of 500-2,000 ppm for 6 h. Membrane fractions of the frontal cortex and hippocampus were prepared and agonist-binding affinities were determined by measuring the displacement of [3H]N-methyl scopolamine-binding activity by carbamylcholine. In the frontal cortex, the affinity of high-affinity carbamylcholine binding was reduced following exposure to toluene at a concentration of 1000 ppm or higher. However, in the hippocampus, the affinity of high-affinity binding of carbamylcholine was increased following exposure to toluene. These observations suggest that toluene exposure affects binding affinity of carbamylcholine, and the effect differs by brain region.

Effects of toluene on regulation of adenylyl cyclase by stimulation of G-protein-coupled receptors expressed in CHO cells.

We determined the effects of toluene exposure on activation or inhibition of adenylyl cyclase by stimulating human beta2-adrenergic receptors (beta2-AR) and muscarinic acetylcholine receptor (mAChR) m2 subtypes, respectively, expressed in CHO cells. The formation of cAMP via beta2-AR stimulation was slightly but not significantly facilitated in the presence of 3.7 microM toluene. On the other hand, the inhibition of adenylyl cyclase by 10 microM of carbamylcholine stimulation of mAChR m2 subtypes was attenuated in the presence of toluene. These results strongly suggest that toluene affects activation of Gi rather than Gs.

G PROTEIN-COUPLED RECEPTOR KINASE : PHOSPHORYLATION OF MUSCARINIC RECEPTORS AND FACILITATION OF RECEPTOR SEQUESTRATION

Publisher Summary Agonist-bound receptors interact with G proteins composed of an αβγ -trimer and facilitate the exchange of GDP and GTP, thereby causing dissociation of α GDP βγ into α GTP - and βγ -subunits. Dissociated α GTP -subunits interact with and activate or inhibit effectors such adenylyl cyclase, phospholipase CB, or ion channels. The phosphorylation is considered to cause desensitization of the reaction induced by agonist-bound receptors. Three different processes of desensitization are known-uncoupling of receptors from G proteins, sequestration (or internalization) of receptors from the cell surface, and decrease in the numbers of receptors. Kinases that phosphorylate G protein-coupled receptors in an agonist-dependent manner are known as G protein-coupled receptor kinases (GRKs). At present, six different kinds of GRKs have been cloned. This chapter describes phosphorylation of muscarinic acetylcholine receptor m2 subtypes (m2 receptors) by GRKs belonging to the GRK2/GRK3 subgroup. The chapter focuses on synergistic activation of GRK2 by G protein βγ -subunits and agonist-bound receptors and on facilitation of m2 receptor sequestration that results from phosphorylation. The partially purified kinase, which will be termed mAChR kinase, was found to phosphorylate muscarinic receptors purified from porcine brain. The atrial m2 receptors were better substrates than brain receptors and have been used in most of the following experiments. The mAChR kinase showed essentially the same enzymatic properties as those reported for β-adrenergic receptor kinases, which include inhibition by heparin or salts and independence of second messengers or Ca 2+ ion.

Carbachol-induced desensitization of rat basophilic leukemia (RBL-2H3) cells transfected with human m3 muscarinic acetylcholine receptors

1. Carbachol-induced homologous desensitization of the secretory response was investigated by transfecting RBL-2H3 cells with cDNA encoding the human m3 muscarinic acetylcholine receptor (RBL-m3). 2. Exposure of RBL-m3 cells to 100 microM carbachol for 30 min in Ca2+-free medium inhibited the secretion induced by the subsequent addition of 10 microM carbachol plus Ca2+. 3. Desensitized cells bound [3H]quinuclidinyl benzilate with a similar Bmax and Kd to those of control cells. 4. The carbachol-induced transient increase in levels of inositol 1,4,5-trisphosphate was not changed by desensitization. 5. Homologous desensitization persisted when desensitized cells were permeabilized with Staphylococcal alpha-toxin.

Regulation of G protein-coupled receptor kinase 2

Publisher Summary G protein-coupled receptor kinase 2 (GRK2) was originally termed a “β-adrenergic receptor kinase” (βARK) because it was purified as a kinase of the adrenergic β 2 receptor, and the adrenergic β 2 receptor was the only known substrate of GRK2. GRK2 is now known to phosphorylate different kinds of G protein-coupled receptors (GPCRs), including rhodopsin, muscarinic acetylcholine receptors, and adrenergic β2 receptors when they are stimulated by light absorption or agonist binding. There is no appreciable homology among amino acid sequences around phosphorylation sites in these GPCRs, except that they are flanked by acidic amino acid residues. The substrate specificity of GRK2, however, is strict in a sense that only a few proteins are known to be substrates of GRK2 besides agonist bound GPCRs. One of the reasons why agonist-bound GPCRs are phosphorylated by GRK2 is that GRK2 is activated by agonist-bound receptors. Very recently, tubulin, synuclein, and phosducin have been reported to be phosphorylated by GRK2, but it is not known whether they serve as activators of GRK2 and why they are substrates of GRK2 unless they are activators.