Serine- and threonine-rich domain regulates internalization of muscarinic cholinergic receptors.

Upon agonist exposure, most membrane receptors internalize into the cell as part of an adaptation process. Receptor domains that mediate internalization have been defined for several receptor classes, e.g.

Mapping the ligand binding pocket of the human muscarinic cholinergic receptor Hm1: contribution of tyrosine-82.

The ligand binding pocket of many G protein-coupled receptors is thought to be located within the core formed by their seven transmembrane domains (TMDs). Previous results suggested that muscarinic antagonists bind to a pocket located toward the extracellular region of the TMDs, primarily at TMDs 2, 3, 6, and 7. Tyrosine-82 (Y82) is located in TMD2 only one helical turn from the presumed membrane surface of Hm1, whereas a phenylalanine (F124) is found in the equivalent position of the closely related Hm3.

Mutational analysis of third cytoplasmic loop domains in G-protein coupling of the HM1 muscarinic receptor.

We measured dose-response curves for carbachol stimulation of phosphatidyl inositol (PI) turnover with mutants of the Hm1 muscarinic cholinergic receptor having various deletions from amino acids 219 to 358 of the large third intracellular (i3) loop (208 to 366). These deletions had only small or no effects on the ability of Hm1 transfected into HEK 293 cells to stimulate PI turnover. This result indicates that only small regions of 9 to 11 amino acids adjacent to trans-membrane domains (TMDs) 5 and 6 can be directly involved in G protein coupling.

Interaction among mu-opioid receptors and alpha 2-adrenoceptors on SH-SY5Y human neuroblastoma cells.

The clonal human neuroblastoma cell line SK-N-SH-SY5Y was previously shown to express mu-opioid and alpha 2-adrenoceptors which are both negatively coupled to adenylyl cyclase. Because of the potential use of alpha 2-agonists in the treatment of narcotic dependence, we tested the interactions among he alpha 2-agonists, clonidine and norepinephrine, and morphine on AC in SH-SY5Y cells. Pretreatment with retinoic acid resulting in partial neuronal differentiation greatly enhanced the cells' sensitivity towards adenylyl cyclase stimulation by prostaglandin E1, and its inhibition by morphine and alpha 2-agonists.

Hm1 muscarinic cholinergic receptor internalization requires a domain in the third cytoplasmic loop.

Selected regions of the Hm1 muscarinic cholinergic receptor were mutated to analyze the molecular mechanisms of agonist-induced receptor internalization (or sequestration). The wild-type and mutant Hm1 genes were expressed, using pSG5, in U293 human kidney cells. Whereas surface receptor density measured with the polar tracer N-[3H]methylscopolamine was rapidly reduced by carbachol exposure, total receptor content measured with [3H]quinuclidinyl benzilate did not decline for at least 24 h, indicating the absence of extensive receptor down-regulation in U293 cells.

Characterization of the RDC1 gene which encodes the canine homolog of a proposed human VIP receptor. Expression does not correlate with an increase in VIP binding sites.

We have isolated a portion of the canine gene encoding the orphan receptor RDC1 [1]. The complete coding sequence is contained in a single exon, and an intron divides the 5' untranslated region of RDC1 mRNA. The RDC1 protein is 94% homologous to the gene product of GPRN1, which has been proposed to serve as a VIP receptor when expressed in CHO-K1 and COS-7 cells (Sreedharan, S.

Characterization of three T-lymphoid cell lines with distinct sensitivities to deoxyadenosine plus deoxycoformycin.

Three established human T-cell lines, HPB-MLT, HPB-ALL and PEER, were characterized and tested for their sensitivity to deoxyadenosine (dAdo) plus deoxycoformycin (dCoF). Phenotypic characterizations showed that all three cell lines had receptors for peanut agglutinin (PNA) and soybean agglutinin (SBA) while HPB-MLT and HPB-ALL, but not PEER, expressed the cortical thymocyte-specific marker, CD1. The majority of HPB-MLT cells (88%) expressed only CD4 but not CD8 antigen while most HPB-ALL cells (81%) co-expressed CD8 and CD4 antigens.

125I-beta-endorphin binding to neuroblastoma X glioma NG108-15 cells: distribution of delta opioid receptors.

The mouse neuroblastoma x rat glioma hybrid NG108-15 was previously shown to express delta opioid receptors. Because neuroblastoma cells display different phenotypes and cloned cell lines are heterogenous, we studied the characteristics and distribution of human 125I-beta-endorphin (125I-beta E) binding sites in cultures of NG108-15 cells with the use of micro-autoradiography and light microscopy. 125I-beta E labeled delta sites in NG108-15 in the presence of the non-opioid blocking peptide, beta-endorphin (6-31) (beta E (6-31)).

Rapid agonist-induced loss of 125I-beta-endorphin opioid receptor sites in NG108-15, but not SK-N-SH neuroblastoma cells.

We have measured mu and delta opioid receptor sites on intact SK-N-SH and NG108-15 neuroblastoma cells, respectively, in culture. Use of 125I-beta-endorphin (beta E) as a tracer, together with beta E(6-31) to block high-affinity non-opioid binding in both cell lines, permitted the measurement of cell surface mu and delta opioid receptor sites. Labeling was at delta sites in NG108-15 cells and predominantly at mu sites in SK-N-SH cells.

Structure and function of G protein coupled receptors.

The G protein coupled receptors (GPC-Rs) comprise a large superfamily of genes encoding numerous receptors which all show common structural features, e.g., seven putative membrane spanning domains.

Regulation of cyclic AMP by the mu-opioid receptor in human neuroblastoma SH-SY5Y cells.

The human neuroblastoma clonal cell line SH-SY5Y expresses both mu- and delta-opioid receptors (ratio approximately 4.5:1). Differentiation with retinoic acid (RA) was previously shown to enhance the inhibition of adenylyl cyclase (AC) by mu-opioid agonists.

Internalization of the Hm1 muscarinic cholinergic receptor involves the third cytoplasmic loop.

The m1 muscarinic receptor was previously shown to stimulate phosphatidyl inositol (PI) turnover and to internalize rapidly upon agonist activation. Three receptor mutants with large deletions of the third cytoplasmic loop (i3) of human Hm1, leaving only 11 and 8 amino acids at the amino and carboxy terminal junctions of i3, respectively, retained full ability to stimulate PI turnover, when expressed in U293 cells, but receptor internalization was greatly reduced in two mutants with deletions reaching close to the NH2 terminal of i3. We propose that a receptor domain located toward the amino terminal junction of i3 plays a role in Hm1 internalization.

Mechanisms of 2'-deoxyguanosine toxicity in mouse T-lymphoma cells with purine nucleoside phosphorylase deficiency and resistance to inhibition of ribonucleotide reductase by dGTP.

Purine nucleoside phosphorylase (PNP; EC 2.4.2.

Cytotoxicity of lymphokine-activated killer cells against human neuroblastoma cells: modulation by neuroblast differentiation.

The cytolytic activity of lymphokine-activated killer (LAK) cells against human neuroblastoma (NB) cells was investigated using the continuous NB cell lines, IMR-32, Kelly, and two subclones of SK-N-SH, SH-SY5Y (neuroblastic phenotype), and SH-EP (non-neuronal phenotype). NB cells were found to be sensitive targets of LAK. Of the SK-N-SH subclones, the neuroblasts, SH-SY5Y, were more susceptible to LAK killing than were the non-neuronal cells, SH-EP.

Retinoic acid enhances VIP receptor expression and responsiveness in human neuroblastoma cell, SH-SY5Y.

Retinoic acid (RA) induces partial differentiation of neuroblastoma (NB) cells in vitro. In the human NB line, SH-SY5Y (a neuroblastic subclone of SK-N-SH), RA was previously shown to enhance the stimulatory (PGE1) and inhibitory (opioid) regulation of adenylyl cyclase. Since these cells are also sensitive to cAMP stimulation by vasoactive intestinal peptide (VIP), we have tested the effects of RA on VIP receptor expression and function.

Functional vasoactive intestinal polypeptide (VIP) receptors in human neuroblastoma subclones that contain VIP precursor mRNA and release VIP-like substances.

Three phenotypically distinct subclones (SH-SY-5Y, SH-EP, SH-IN) of the human neuroblastoma cell line SK-N-SH were found to possess vasoactive intestinal polypeptide (VIP) precursor mRNA, release immunoreactive VIP, and express high-affinity VIP receptors coupled to adenylate cyclase. The apparent molecular mass for the receptor polypeptide, as determined by covalent cross-linking of 125I-VIP, was 49 kDa. After 2 days in culture, a concentration of immunoreactive VIP equivalent to the binding affinity of VIP to its receptor was found in the medium in two of these clones (SH-IN and SH-EP).

[Biocytin13]dynorphin A 1-13 amide: a potential probe for the kappa-opioid receptor.

A dynorphin A 1-13 amide (DYN) derivative biotinylated in position lysine 13 (B-DYN) has been prepared by automated solid-phase peptide synthesis. The derivative retained its ability to bind to avidin, and B-DYN-avidin complex showed a dissociated half-life of 10 hr at 37 degrees C. Opioid receptor binding was measured in membrane preparations of rat brain (mu), NG-108-15 neuroblastoma-glioma hybrid cells (delta), and guinea pig cerebellum (kappa).

Differentiation of human neuroblastoma cells: marked potentiation of prostaglandin E-stimulated accumulation of cyclic AMP by retinoic acid.

Neuroblastoma cells in culture contain low levels of cyclic AMP, a second messenger which plays a major role in neuronal maturation. In this study, human neuroblastoma cells, SK-N-SH-SY5Y, were induced to differentiate by treatment with either nerve growth factor (50 ng/ml), retinoic acid (10 microM), dibutyryl cyclic AMP (1 mM), or 12-O-tetradecanoylphorbol-13-acetate (0.1 microM), and the ability of several neurotransmitters or hormones to stimulate adenylyl cyclase was tested.

Neuronal cell differentiation of human neuroblastoma cells by retinoic acid plus herbimycin A.

We investigated the effect of retinoic acid (RA) and herbimycin A (herb-A) on cell growth, cell differentiation, and colony formation of human neuroblastoma cell lines. The neuroblastoma line SK-N-SH expressed both neuroblast and nonneuronal phenotypes, whereas its subclone SH-SY5Y and the Kelly cell line were predominantly neuroblastic. Both herb-A and RA, given alone, moderately reduced cell growth and colony formation of the neuroblastic cell lines.

Guanine for DNA synthesis. A compulsory route through ribonucleotide reductase.

Two alternative pathways for the synthesis of dGTP and its incorporation into DNA were studied: guanine (Gua)----GMP----GDP----dGDP----dGTP----DNA and dG----dGMP----dGDP----dGTP----DNA. To determine the contribution of each pathway to DNA synthesis independently of each other, [14C]Gua and [3H]dG tracer experiments were performed in a double-mutant S-49 mouse T-lymphoma cell line, dGuo-L, with purine nucleoside phosphorylase (EC 2.4.

Modulation of 6-thioguanine activity by guanine in human promyelocytic leukemia HL-60 cells.

The effects of guanine coadministration on the metabolism and biological activity of 6-thioguanine (6-TG) were studied in human promyelocytic leukemia cells (HL-60). Cell growth, cytotoxicity (cloning assay), and cell differentiation were measured, along with nucleotide metabolism. Guanine was efficiently salvaged by HL-60 cells; at 200 microM, guanine suppressed the formation of 6-TG mononucleotides and abolished 6-TG incorporation into nucleic acids.

A biotin-avidin-based enzyme immunoassay for beta h-endorphin.

An avidin-biotin enzyme-linked immunosorbent assay (ELISA) is described for beta h-endorphin (beta h-EP). Microtiter plates coated with commercially available antibodies were used together with beta h-EP tracer derivatives that were biotinylated in positions 24, 28, and 29 via a C6 spacer arm. Nonspecific binding of biotinylated derivatives to the microtiter plates was blocked with a mixture of 1% casein and 10% ethanolamine in 0.