Tatsuya Haga

The cholinergic neurons have long been a model for biochemical studies of neurotransmission. The components responsible for cholinergic neurotransmission, such as choline acetyltransferase, vesicular acetylcholine transporter, nicotinic and muscarinic acetylcholine receptors, and acetylcholine esterase, have long been defined as functional units and then identified as molecular entities. Another essential component in the cholinergic synapses is the one responsible for choline uptake from the synaptic cleft, which is thought to be the rate-limiting step in acetylcholine synthesis. A choline uptake system with a high affinity for choline has long been assumed to be present in cholinergic neurons. Very recently, the molecular entity for the high-affinity choline transporter was identified and is designated CHT1. CHT1 mediates Na+-and Cl--dependent choline uptake with high sensitivity to hemicholinium-3. CHT1 has been characterized both at the molecular and functional levels and was confirmed to be specifically expressed in cholinergic neurons.

G-protein-coupled receptor kinase 2 (GRK2) is known to specifically phosphorylate the agonist-bound forms of G-protein-coupled receptors (GPCRs). This strict specificity is due at least partly to activation of GRK2 by agonist-bound GPCRs, in which basic residues in intracellular regions adjacent to transmembrane segments are thought to be involved. Tubulin was found to be phosphorylated by GRK2, but it remains unknown if tubulin can also serve as both a substrate and an activator for GRK2. Purified tubulin, phosphorylated by GRK2, was subjected to biochemical analysis, and the phosphorylation sites in beta-tubulin were determined to be Thr409 and Ser420. In addition, the Ser444 in beta(III) -tubulin was also indicated to be phosphorylated by GRK2. The phosphorylation sites in tubulin for GRK2 reside in the C-terminal domain of beta-tubulin, which is on the outer surface of microtubules. Pretreatment of tubulin with protein phosphatase type-2A (PP2A) resulted in a twofold increase in the phosphorylation of tubulin by GRK2. These results suggest that tubulin is phosphorylated in situ probably by GRK2 and that the phosphorylation may affect the interaction of microtubules with microtubule-associated proteins. A GST fusion protein of a C-terminal region of beta(I) -tubulin (393-445 residues), containing 19 acidic residues but only one basic residue, was found to be a good substrate for GRK2, like full-length beta-tubulin. These results, together with the finding that GRK2 may phosphorylate synuclein and phosducin in their acidic domains, indicate that some proteins with very acidic regions but without basic activation domains could serve as substrates for GRK2.

G-protein-coupled receptor kinase 2 (GRK2) is known to specifically phosphorylate the agonist-bound forms of G-protein-coupled receptors (GPCRs). This strict specificity is due at least partly to activation of GRK2 by agonist-bound GPCRs, in which basic residues in intracellular regions adjacent to transmembrane segments are thought to be involved. Tubulin was found to be phosphorylated by GRK2, but it remains unknown if tubulin can also serve as both a substrate and an activator for GRK2. Purified tubulin, phosphorylated by GRK2, was subjected to biochemical analysis, and the phosphorylation sites in beta-tubulin were determined to be Thr409 and Ser420. In addition, the Ser444 in beta(III) -tubulin was also indicated to be phosphorylated by GRK2. The phosphorylation sites in tubulin for GRK2 reside in the C-terminal domain of beta-tubulin, which is on the outer surface of microtubules. Pretreatment of tubulin with protein phosphatase type-2A (PP2A) resulted in a twofold increase in the phosphorylation of tubulin by GRK2. These results suggest that tubulin is phosphorylated in situ probably by GRK2 and that the phosphorylation may affect the interaction of microtubules with microtubule-associated proteins. A GST fusion protein of a C-terminal region of beta(I) -tubulin (393-445 residues), containing 19 acidic residues but only one basic residue, was found to be a good substrate for GRK2, like full-length beta-tubulin. These results, together with the finding that GRK2 may phosphorylate synuclein and phosducin in their acidic domains, indicate that some proteins with very acidic regions but without basic activation domains could serve as substrates for GRK2.

High affinity choline uptake plays a critical role in the regulation of acetylcholine synthesis in cholinergic neurons. Recently, we succeeded in molecular cloning of the high affinity choline transporter (CHT1), which is specifically expressed in cholinergic neurons. Here we demonstrate the presence of functionally relevant, nonsynonymous single nucleotide polymorphism in the human CHT1 gene by comprehensive sequence analysis of the exons and the intron/exon boundaries including the transcription start site. The deduced amino acid change for the polymorphism is isoleucine to valine at amino acid 89 (I89V) located within the third transmembrane domain of the protein. The allele frequency of I89V was 6% for Ashkenazi Jews. Functional assessment of the I89V transporter in mammalian cell lines revealed a 40-50% decrease in V-max for choline uptake rate compared with the wild type, whereas there was no alteration in the apparent affinities for choline, sodium, chloride, and the specific inhibitor hemicholinum-3. There also was no change in the specific hemicholinum-3 binding activity. The decreased choline uptake was not associated with the surface expression level of the protein as assessed by biotinylation assay. These results suggest an impaired substrate translocation in the I89V transporter. The Caenorhabditis elegans ortholog of CHT1 has a valine residue at the corresponding position and a single replacement from valine to isoleucine caused a decrease in the choline uptake rate by 40%, suggesting that this hydrophobic residue is generally critical in the choline transport rate in CHT1. This polymorphism in the allelic CHT1 gene may represent a predisposing factor for cholinergic dysfunction.

High affinity choline uptake plays a critical role in the regulation of acetylcholine synthesis in cholinergic neurons. Recently, we succeeded in molecular cloning of the high affinity choline transporter (CHT1), which is specifically expressed in cholinergic neurons. Here we demonstrate the presence of functionally relevant, nonsynonymous single nucleotide polymorphism in the human CHT1 gene by comprehensive sequence analysis of the exons and the intron/exon boundaries including the transcription start site. The deduced amino acid change for the polymorphism is isoleucine to valine at amino acid 89 (I89V) located within the third transmembrane domain of the protein. The allele frequency of I89V was 6% for Ashkenazi Jews. Functional assessment of the I89V transporter in mammalian cell lines revealed a 40-50% decrease in V-max for choline uptake rate compared with the wild type, whereas there was no alteration in the apparent affinities for choline, sodium, chloride, and the specific inhibitor hemicholinum-3. There also was no change in the specific hemicholinum-3 binding activity. The decreased choline uptake was not associated with the surface expression level of the protein as assessed by biotinylation assay. These results suggest an impaired substrate translocation in the I89V transporter. The Caenorhabditis elegans ortholog of CHT1 has a valine residue at the corresponding position and a single replacement from valine to isoleucine caused a decrease in the choline uptake rate by 40%, suggesting that this hydrophobic residue is generally critical in the choline transport rate in CHT1. This polymorphism in the allelic CHT1 gene may represent a predisposing factor for cholinergic dysfunction.

β1-Adrenergic receptor (β1AR) shows the resistance to agonist-induced internalization. However, β1AR can internalize as G protein-coupled receptor kinase 2 (GRK2) is fused to its carboxyl terminus. Internalization of the β1AR and GRK2 fusion protein (β1AR/GRK2) is dependent on dynamin but independent of β-arrestin and phosphorylation. The β1AR/GRK2 fusion protein internalizes via clathrin-coated pits and is found to co-localize with the endosome that contains transferrin. The fusion proteins consisting of β1AR and various portions of GRK2 reveal that the residues 498-502 in the carboxyl-terminal domain of GRK2 are critical to promote internalization of the fusion proteins. This domain contains a consensus sequence of a clathrin-binding motif defined as a clathrin box. In vitro binding assays show that the residues 498-502 of GRK2 bind the amino-terminal domain of clathrin heavy chain to almost the same extent as β-arrestin1. The mutation of the clathrin box in the carboxyl-terminal domain of GRK2 results in the loss of the ability to promote internalization of the fusion protein. GRK2 activity increases and then decreases as the concentration of clathrin heavy chain increases. Taken together, these results imply that GRK2 contains a functional clathrin box and directly interacts with clathrin to modulate its function.

β1-Adrenergic receptor (β1AR) shows the resistance to agonist-induced internalization. However, β1AR can internalize as G protein-coupled receptor kinase 2 (GRK2) is fused to its carboxyl terminus. Internalization of the β1AR and GRK2 fusion protein (β1AR/GRK2) is dependent on dynamin but independent of β-arrestin and phosphorylation. The β1AR/GRK2 fusion protein internalizes via clathrin-coated pits and is found to co-localize with the endosome that contains transferrin. The fusion proteins consisting of β1AR and various portions of GRK2 reveal that the residues 498-502 in the carboxyl-terminal domain of GRK2 are critical to promote internalization of the fusion proteins. This domain contains a consensus sequence of a clathrin-binding motif defined as a clathrin box. In vitro binding assays show that the residues 498-502 of GRK2 bind the amino-terminal domain of clathrin heavy chain to almost the same extent as β-arrestin1. The mutation of the clathrin box in the carboxyl-terminal domain of GRK2 results in the loss of the ability to promote internalization of the fusion protein. GRK2 activity increases and then decreases as the concentration of clathrin heavy chain increases. Taken together, these results imply that GRK2 contains a functional clathrin box and directly interacts with clathrin to modulate its function.

MJ0968 has been proposed to be an ancestor of P-type ATPase, because its primary structure is highly homologous to that of the core catalytic domain of P-type ATPase, However it completely lacks amino acid sequences that possibly constitute transmembrane domains. To examine if MJ0968 is indeed a P-type ATPase, it was overexpressed in Escherichia coli and purified. It did show ATPase activity, autophosphorylation and inhibition by vanadate, All these properties support the idea that MJ0968 is indeed a soluble P-type ATPase. (C) 2000 Federation of European Biochemical Societies.

1. The cardiac muscarinic receptor-K+ channel system was reconstructed in Chinese hamster ovary (CHO) cells by transfecting the cells with the various components of the system. The activity of the muscarinic K+ channel was measured with the cell-attached configuration of the patch clamp technique. 2. In CHO cells transfected with the channel (Kir3.1/Kir3.4), receptor (hm2) and receptor kinase (GRK2), on exposure to agonist, there was a decline in channel activity a a result of desensitization, similar to that in atrial cells. 3. Whereas the desensitization was almost abolished by not transfecting with the receptor kinase or by transfecting with a mutant receptor lacking phosphorylation sites, it was only reduced (by similar to 39%) by transfecting with a mutant receptor kinase with little kinase activity. 4. These results suggest that the receptor kinase is responsible for desensitization of the muscarinic K+ channel and that this involves phosphorylation-dependent and -independent mechanisms.

The a subunits of G(q) family G proteins, G(L1)alpha(G(14)alpha), G(L2)alpha(G(11)alpha), and G(q) alpha were expressed with G protein beta(1) and gamma(2) subunits in insect cells using a baculovirus system, The trimeric forms of G proteins, G(L1) (G(L1)alpha beta gamma), G(L2) (C(L2)alpha beta gamma), and G(q) (G(q) alpha beta gamma), were solubilized by 1% sodium cholate and purified by sequential chromatography on three kinds of columns, G(L1), G(L2), and G(q) activated phospholipase C-beta purified from bovine brain in the presence of aluminum fluoride to the same extent, Muscarinic acetylcholine receptor m1 subtype stimulated the guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) binding to G(L1), G(L2) and G(q) in the presence of similar concentrations of carbamylcholine. When m1 receptor, G protein, and phospholipase C-beta were reconstituted in lipid vesicles, each subtype of G(q) family G proteins mediated the activation of phospholipase C-beta by carbamylcholine in the presence of either 1 mu M GTP gamma S or 1 mM GTP. Phospholipase C-beta stimulated the GTPase activity of G(L1), G(L2), and G(q) in the presence of mi receptor and carbamylcholine but did not stimulate the GTPase activity of G(o). Protein kinase C phosphorylated m1 receptor and phospholipase C-beta, but the phosphorylation did not significantly affect the ability of the m1 receptor to stimulate phospholipase C-beta in the reconstitution system of purified proteins.