A human synaptic vesicle monoamine transporter cDNA predicts posttranslational modifications, reveals chromosome 10 gene localization and identifies TaqI RFLPs.

A human vesicular monoamine transporter cDNA has been identified by screening a human brainstem library using sequences from the rat brain synaptic vesicle monoamine transporter (SVMT) [(1992) Cell 70, 539-551; (1992) Proc. Natl. Acad. Sci. USA 89, 10993-10997]. The hSVMT shares 92% amino acid identity with the rat sequence, but displays one less consensus site for asparagine N-linked glycosylation and one more consensus site for phosphorylation by protein kinase C. The human SVMT gene maps to chromosome 10q25 using Southern blotting analysis of human/rodent hybrid cell lines and fluorescent in situ hybridization approaches. The cDNA, and a subclone, recognize TaqI polymorphisms that may prove useful to assess this gene's involvement in neuropsychiatric disorders involving monoaminergic brain systems.

Control of mu opioid receptor expression by modification of cDNA 5'- and 3'-noncoding regions.

Removal of a 712 base pair (bp) sequence following the coding region of a human micro opioid receptor (hmuOR) cDNA unexpectedly increased expression of the receptor protein. A series of 3'-noncoding region deletion mutants revealed that at least three discrete regions following the stop codon influenced receptor expression levels. Deletion of the 205-bp 5'-noncoding region immediately preceding the Kozak sequence doubled receptor expression relative to wild type, and simultaneous deletion of 5'- and 3'-noncoding regions increased expression several fold. The hmuOR noncoding regions may participate in a regulatory mechanism that controls the number of cell surface receptors.

Mutation of human mu opioid receptor extracellular "disulfide cysteine" residues alters ligand binding but does not prevent receptor targeting to the cell plasma membrane.

The mu opioid receptor, a primary site of action in the brain for opioid neuropeptides and opiate drugs of abuse, is a member of the seven transmembrane, G protein-coupled receptor (GPCR) superfamily. Two cysteine residues, one in each of the first two of three extracellular loops (ECLs), are highly conserved among GPCRs, and there is direct or circumstantial evidence that the residues form a disulfide bond in many of these receptors. Such a bond would dramatically govern the topology of the ECLs, and possibly affect the position of the membrane-spanning domains. Recent findings from several laboratories indicate the importance of the ECLs for opioid ligand selectivity. These conserved cysteine residues in the mu opioid receptor were studied using site-directed mutagenesis. Little or no specific binding of radiolabled opiate alkaloid or opioid peptide agonists or antagonists was observed for receptors mutated at either "disulfide cysteine" residue. Each mutant mu opioid receptor was expressed in both transiently- and stably-transfected cells, in some cases at levels comparable to the wild type receptor. The two point mutants possessing serine-for-cysteine substitutions were also observed to successfully reach the cell plasma membrane, as evidenced by electron microscopy. Consistent with related work with other GPCRs, the mu opioid receptor apparently also employs the extracellular disulfide bond. This information now permits accurate molecular modeling of extracellular aspects of the receptor, including plausible scenarios of mu receptor docking of opioid ligands known to require specific extracellular loop features for high affinity binding.

Dopaminergic gene expression during amphetamine withdrawal.

Animals and humans display a constellation of behavioral and neurochemical signs after termination of psychostimulant administration. Amphetamine withdrawal could involve the dopaminergic systems that are thought to underlie psychostimulant rewarding effects, and may thus conceivably alter expression of key genes for dopaminergic transmission, including those encoding tyrosine hydroxylase (TH), the membrane dopamine transporter (DAT) and the synaptic vesicle amine transporter (SVAT). Withdrawal from 7.5 mg kg-1 i.p. amphetamine (b.i.d. for a two week duration) yields no significant changes in rat DAT mRNA. TH mRNA levels are modestly enhanced over the same week of withdrawal, during which dopamine levels and behavioral novelty responses are both depressed. SVAT expression is significantly blunted following chronic amphetamine treatment. Altered TH and/or SVAT gene expression might contribute to restoring normal function to neurons "withdrawing" from amphetamine treatments.

Properties of mu 3 opiate alkaloid receptors in macrophages, astrocytes, and HL-60 human promyelocytic leukemia cells.

An opiate alkaloid-selective receptor, designated mu(3), mediates inhibition by morphine of activation of human peripheral blood monocytes and granulocytes. The mu(3) receptor is present on several macrophage cell types including microglia, on cultured astrocytes, and in brain and retina. Murine macrophage cell lines and human HL-60 leukemia cells contain high concentrations of these receptors. Binding of 3H-morphine to the receptor is displaced by morphine, etorphine, naloxone, diprenorphine and morphine 6-glucuronide, but not by morphine 3-glucuronide, fentanyl, benzomorphans, enkephalins, dynorphin, beta-endorphin, endomorphin-1, other opioid peptides or nociceptin (orphanin FQ). The mu(3) receptor appears to be much more sensitive to inactivation by reduced glutathione than are classical mu, delta and kappa receptors. Evidence is also presented for G protein-coupling of these receptors. These and other data raise the possibility that the mu(3) receptor is a member of a chemokine or of another related receptor family, rather than the opioid receptor family. The affinity for morphine of mu(3) receptors of granulocytic-differentiated HL-60 cells is markedly enhanced in the presence of levorphanol and certain benzomorphans. In contrast, receptors of monocytes, macrophage cell lines, microglia, macrophage-differentiated HL-60 cells and astrocytes are not affected by levorphanol or benzomorphans. It is concluded that mu(3) receptors of granulocytic and promyelocytic cells differ from those of macrophage and astrocyte cell types, possibly due to differences in receptor subtype or to the presence of an additional component in the granulocytic and promyelocytic cells.

Spontaneous cleavage of RNA in ternary complexes of Escherichia coli RNA polymerase and its significance for the mechanism of transcription.

Ternary complexes of RNA polymerase, bearing the nascent RNA transcript, are intermediates in the synthesis of all RNAs and are regulatory targets of factors that control RNA chain elongation and termination. To study the catalytic and regulatory properties of RNA polymerases during elongation, we have developed methods for the preparation of these intermediates halted at defined positions along a DNA template. To our surprise, some of these halted complexes undergo a reaction in which the RNA transcript is cleaved up to 10 nucleotides from its 3'-terminal growing point. The 5'-terminal fragment, bearing a free 3'-OH residue, remains bound to the RNA polymerase-DNA complex and can resume elongation, whereas the 3'-terminal oligonucleotide of 2-10 nucleotides, bearing a 5'-phosphate, is released. RNA cleavage occurs only in the ternary complex and requires a divalent metal ion such as Mg2+. Since RNA polymerases are believed to have a single catalytic site for nucleotide addition, this reaction is unlikely to be due to hydrolysis catalyzed by this site comparable to the 3'----5' exonuclease activity associated with the catalytic center found for some DNA polymerases. Nor is this reaction easily explained by models for transcription elongation that postulate a 12-base-pair DNA.RNA hybrid as intermediate. Instead, we suggest that this is an unusual kind of protein-facilitated reaction in which tight binding of the RNA product to the enzyme strains the RNA phosphodiester linkage, resulting in cleavage of the RNA well away from the catalytic center. By this model, the nascent RNA enters a product binding site beginning 3 or 4 nucleotides from the growing point at the 3' terminus. This RNA binding site extends for up to 16 nucleotides along the protein surface. The stress brought about by this binding appears to vary considerably for different ternary complexes and may play a role in driving the translocation of the RNA polymerase along the DNA.

Calmodulin regulation of basal and agonist-stimulated G protein coupling by the mu-opioid receptor (OP(3)) in morphine-pretreated cell.

Calmodulin (CaM) has been shown to suppress basal G protein coupling and attenuate agonist-stimulated G protein coupling of the mu-opioid receptor (OP(3)) through direct interaction with the third intracellular (i3) loop of the receptor. Here we have investigated the role of CaM in regulating changes in OP(3)-G protein coupling during morphine treatment, shown to result in CaM release from plasma membranes. Basal and agonist-stimulated G protein coupling by OP(3) was measured before and after morphine pretreatment by incorporation of guanosine 5'-O-(3-[(35)S]thiotriphosphate) into membranes, obtained from HEK 293 cells transfected with human OP(3) cDNA. The opioid antagonist beta-chlornaltrexamine fully suppressed basal G protein coupling of OP(3), providing a direct measure of basal signaling. Pretreatment of the cells with morphine enhanced basal G protein coupling (sensitization). In contrast, agonist-stimulated coupling was diminished (desensitization), resulting in a substantially flattened morphine dose-response curve. To test whether CaM is involved in these changes, we constructed OP(3)-i3 loop mutants with reduced affinity for CaM (K273A, R275A, and K273A/R275A). Basal signaling of these mutant OP(3) receptors was higher than that of the wild-type receptor and, moreover, unaffected by morphine pretreatment, whereas desensitization to agonist stimulation was only slightly attenuated. Therefore, CaM-OP(3) interactions appear to play only a minor role in the desensitization of OP(3). In contrast, release of CaM from the plasma membrane appears to enhance the inherent basal G protein coupling of OP(3), thereby resolving the paradox that OP(3) displays both desensitization and sensitization during morphine treatment.

Construction and processing of transfer RNA precursor models.

Several "dimeric" tRNA molecules were constructed as potential substrates for ribonuclease P (RNase P) and for M1 RNA, the catalytic subunit of RNase P. Construction was affected by the T4 RNA ligase-mediated coupling of a mature Escherichia coli tRNA (acceptor substrate) and nucleotides 1-36 of yeast tRNAPhe (donor substrate), followed by annealing of the 3'-half of yeast tRNAPhe (nucleotides 38-76). E. coli RNase P and M1 RNA were both found to cleave the dimeric tRNA precursor model constructed from E. coli tRNAPhe (5'-tRNA) and yeast tRNAPhe (3'-tRNA) in a reaction that was dependent on the presence of the annealed 3'-half molecule derived from yeast tRNAPhe, or on some conformation imposed by the presence of this species; the product had the same mobility as authentic E. coli tRNAPhe on a polyacrylamide gel. By utilizing tRNA precursor models radiolabeled at phosphodiesters immediately preceding or following the putative site of processing, cleavage of the substrate by both M1 RNA and the holoenzyme was demonstrated to occur at the expected phosphate ester linkage. The results obtained here suggest that the endonucleolytic separation of two tRNAs by RNase P is dependent on one or more structural features in the 3'-half of the 3'-tRNA, and thus are consistent with the report of McClain et al. (McClain, W. H., Guerrier-Takada, C., and Altman, S. (1987) Science 238, 527-530) that identifies the T stem and loop as a possible recognition site.

Metal ion and substrate structure dependence of the processing of tRNA precursors by RNase P and M1 RNA.

A synthetic tRNA precursor analog containing the structural elements of Escherichia coli tRNA(Phe) was characterized as a substrate for E. coli ribonuclease P and for M1 RNA, the catalytic RNA subunit. Processing of the synthetic precursor exhibited a Mg2+ dependence quite similar to that of natural tRNA precursors such as E. coli tRNA(Tyr) precursor. It was found that Sr2+, Ca2+, and Ba2+ ions promoted processing of the dimeric precursor at Mg2+ concentrations otherwise insufficient to support processing; very similar behavior was noted for E. coli tRNA(Tyr). As noted previously for natural tRNA precursors, the absence of the 3'-terminal CA sequence in the synthetic precursor diminished the facility of processing of this substrate by RNase P and M1 RNA. A study of the Mg2+ dependence of processing of the synthetic tRNA dimeric substrate radiolabeled between C75 and A76 provided unequivocal evidence for an alteration in the actual site of processing by E. coli RNase P as a function of Mg2+ concentration. This property was subsequently demonstrated to obtain (Carter, B. J., Vold, B.S., and Hecht, S. M. (1990) J. Biol. Chem. 265, 7100-7103) for a mutant Bacillus subtilis tRNAHis precursor containing a potential A-C base pair at the end of the acceptor stem.

Preparation of misacylated aminoacyl-tRNA(Phe)'s useful as probes of the ribosomal acceptor site.

Several pyroglutamylaminoacyl-tRNA's were prepared by T4 RNA ligase mediated condensation of synthetic pyroglutamylaminoacyl-pCpA's with tRNA's from which the last two nucleotides at the 3'-end had been removed. The derived pyroglutamylaminoacyl-tRNA's were incubated in the presence of calf liver pyroglutamate aminopeptidase, which effected their conversion to free aminoacyl-tRNA's. The lack of contaminating esterase activities in the pyroglutamate aminopeptidase was verified by direct assay for the presence of the aminoacyl moieties in the formed aminoacyl-tRNA's and by the use of the deblocked aminoacyl-tRNA's as acceptors in the peptidyltransferase reaction using an Escherichia coli ribosomal system. These findings provide the wherewithal for a detailed investigation of the substrate specificity of the peptidyltransferase center and for the elaboration of polypeptides containing modified amino acids at predetermined sites.

-mu opiate receptor. Charged transmembrane domain amino acids are critical for agonist recognition and intrinsic activity.

The mu opiate receptor is a principal brain site for activities of morphine, other opiate drugs, and opioid peptides in modulating pain and altering mood. Recent cloning of cDNAs encoding rat and human mu receptors reveals charged amino acid residues within putative transmembrane domains (TMs) II, III, and VI, a substantial N-terminal extracellular domain, and a C-terminal intracellular domain. Deletion of 64 N-terminal amino acids produced little effect on receptor function (Wang, J.B., Imai, Y., Eppler, C.M., Gregor, P., Spivak, C.E., and Uhl, G.R. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 10230-10234). Further deletion of 33 C-terminal amino acids yielded a receptor at which morphine, but not the substituted enkephalin DAMGO ([D-Ala2,MePhe4,Glyol5]enkephalin), inhibited adenylate cyclase. Alanine substitution for each charged TM residue in the N-terminally deleted receptor reduced affinities for morphine, DAMGO, and the opiate antagonist naloxone. Replacement of TM II Asp114 with asparagine or glutamic acid increased mu receptor affinity for naloxone. TM II and TM III glutamic acid substitutions for Asp114 and Asp147 reduced agonist binding affinities but allowed full inhibition of adenylate cyclase at high agonist concentrations. TM VI histidine substitution with alanine yielded a receptor that produced almost twice the cyclase inhibition displayed by the wild type receptor in parallel transient expression assays. These findings underscore the importance of charged residues in TM II, III, and VI for different receptor functions and the modest involvement of extensive portions of N- and C-terminal receptor domains in these processes.

Sodium- and chloride-dependent transporters in brain, kidney, and gut: lessons from complementary DNA cloning and structure-function studies.

The family of Na(+)- and Cl(-)-dependent, 12 transmembrane domain transporter proteins now includes transporters for neurotransmitter molecules in the brain and for substances important in extraneuronal tissues, including adrenal, kidney, and gut. Transported substrates include monoamine and amino acid neurotransmitters and nonperturbing osmolytes. A common protein topology is predicted and features intracellular N- and C-termini possessing phosphorylation sites and at least one large extramembranous loop with N-linked glycosylation. Using the rat dopamine transporter as a template, molecular modeling of putative transmembrane domains coupled with amino acid sequence conservation analysis indicates amino acid residues potentially involved in substrate and/or ion recognition. Targeting such residues with site-directed mutagenesis will help clarify substrate and ion binding sites and should facilitate rational design of therapeutics to combat depression, locomotor disorders, and substance abuse.

Role for the C-terminus in agonist-induced mu opioid receptor phosphorylation and desensitization.

Determining which domains and amino acid residues of the mu opioid receptor are phosphorylated is critical for understanding the mechanism of mu opioid receptor phosphorylation. The role of the C-terminus of the receptor was investigated by examining the C-terminally truncated or point-mutated mu opioid receptors in receptor phosphorylation and desensitization. Both wild-type and mutated receptors were stably expressed in Chinese hamster ovary (CHO) cells. The receptor expression was confirmed by receptor radioligand binding and immunoblottting. After exposure to 5 microM of DAMGO, phosphorylation of the C-terminally truncated receptor and the mutant receptor T394A was reduced to 40 and 10% of that of the wild-type receptor, respectively. Mutation effects on agonist-induced desensitization were studied using adenylyl cyclase inhibition assays. The C-terminally truncated receptor and mutant receptor T394A both showed complete loss of DAMGO-induced desensitization, while the mutant T/S-7A receptor only lost part of its ability to desensitize. Taken together, these results suggest that the C-terminus of the mu opioid receptor participates in receptor phosphorylation and desensitization with threonine 394, a crucial residue for both features. DAMGO-induced mu opioid receptor phosphorylation and desensitization are associated and appear to involve both the mu opioid receptor C-terminus and other domains of the receptor.

Naloxone activation of mu-opioid receptors mutated at a histidine residue lining the opioid binding cavity.

The mu-opioid receptor is the principal site of action in the brain by which morphine, other opiate drugs of abuse, and endogenous opioid peptides effect analgesia and alter mood. A member of the seven-transmembrane domain (TM) G protein-coupled receptor (GPCR) superfamily, the mu-opioid receptor modulates ion channels and second messenger effectors in an opioid agonist-dependent fashion that is reversible by the classic opiate antagonist naloxone. Mutation of a histidine residue (His297) in TM 6 afforded agonist-like G protein-coupled signal transduction mediated by naloxone and other alkaloid antagonists and enhanced the intrinsic activity of documented alkaloid partial agonists, including buprenorphine. The intrinsic activities of all opioid peptide agonists and antagonists tested were not altered at the His297 mutant receptors. Consistent with a role for the TM 6 histidine in maintaining high affinity binding sites for opioid agonists and antagonists, opioid ligand-dependent protection of this residue from a histidine-specific alkylating agent indicated that the His297 side chain is positioned in or very near the binding cavity. The TM 6 His297 mutants identify a discrete region of the receptor critical for determining whether a specific drug pharmacophore triggers receptor activation. Because many GPCRs possess a similarly positioned TM histidine residue, our findings with the mu-opioid receptor may extend to these receptors and potentially serve as a model for rational design of therapeutic GPCR partial agonists and antagonists.