Ethanol acts synergistically with a D2 dopamine agonist to cause translocation of protein kinase C.

Ethanol and other drugs of abuse increase synaptic dopamine levels; however, little is known about how ethanol alters dopaminergic signaling. We have reported that ethanol induces translocation of delta and epsilon protein kinase C (PKC) in neural cells in culture. Using NG108-15 and Chinese hamster ovary cell lines that express the dopamine D2 receptor (D2R), we show here that the D2R agonist R(-)-2,10,11-trihydroxy-N-propyl-noraporphine hydrobromide (NPA) also causes translocation of delta and epsilon PKC to the same sites as ethanol-induced translocation. D2R agonist and ethanol-induced translocation of delta and epsilon PKC share a common pathway that is blocked by pertussis toxin and requires phospholipase C (PLC) activity. These data suggest that both D2R agonists and ethanol activate PLC via a trimeric G protein leading to production of diacylglycerol with subsequent activation and translocation of delta and epsilon PKC. Moreover, ethanol and NPA, when present together at low concentrations that alone are ineffective, act synergistically to cause translocation of delta and epsilon PKC. Our data suggest that ethanol causes translocation of delta and epsilon PKC but cells expressing the D2R, such as neurons in the nucleus accumbens, may be particularly sensitive to low concentrations of ethanol.

Localization of a peripheral membrane protein: Gbetagamma targets Galpha(Z).

To explore the relative roles of protein-binding partners vs. lipid modifications in controlling membrane targeting of a typical peripheral membrane protein, Galpha(z), we directed its binding partner, betagamma, to mislocalize on mitochondria. Mislocalized betagamma directed wild-type Galpha(z) and a palmitate-lacking Galpha(z) mutant to mitochondria but did not alter localization of a Galpha(z) mutant lacking both myristate and palmitate. Thus, in this paradigm, a protein-protein interaction controls targeting of a peripheral membrane protein to the proper compartment, whereas lipid modifications stabilize interactions of proteins with membranes and with other proteins.

Gbetagamma and palmitate target newly synthesized Galphaz to the plasma membrane.

The subcellular location of a signaling protein determines its ability to transmit messages accurately and efficiently. Three different lipid modifications tether heterotrimeric G proteins to membranes: alpha subunits are myristoylated and/or palmitoylated, and gamma subunits are prenylated. In a previous study, we examined the role of lipid modifications in maintaining the membrane attachment of a G protein alpha subunit, alphaz, which is myristoylated and palmitoylated (Morales, J., Fishburn, C. S., Wilson, P. T., and Bourne, H. R. (1998) Mol. Biol. Cell 9, 1-14). Now we extend this analysis by characterizing the mechanisms that target newly synthesized alphaz to the plasma membrane (PM) and analyze the role of lipid modifications in this process. In comparison with newly synthesized alphas, which is palmitoylated but not myristoylated, alphaz moves more rapidly to the membrane fraction following synthesis in the cytosol. Newly synthesized alphaz associates randomly with cellular membranes, but with time accumulates at the PM. Palmitoylated alphaz is present only in PM-enriched fractions, whereas a nonpalmitoylated mutant of alphaz (alphazC3A) associates less stably with the PM than does wild-type alphaz. Expression of a C-terminal fragment of the beta-adrenoreceptor kinase, which sequesters free betagamma, impairs association of both alphaz and alphazC3A with the PM, suggesting that the alpha subunit must bind betagamma in order to localize at the PM. Based on these findings, we propose a model in which, following synthesis on soluble ribosomes, myristoylated alphaz associates randomly and reversibly with membranes; upon association with the PM, alphaz binds betagamma, which promotes its palmitoylation, thus securing it in the proper place for transmitting the hormonal signal.

Adenylyl cyclase interaction with the D2 dopamine receptor family; differential coupling to Gi, Gz, and Gs.

1. The D2-type dopamine receptors are thought to inhibit adenylyl cyclase (AC), via coupling to pertussis toxin (PTX)-sensitive G proteins of the Gi family. We examined whether and to what extent the various D2 receptors (D2S, D2L, D3S, D3L, and D4) couple to the PTX-insensitive G protein Gz, to produce inhibition of AC activity. 2. COS-7 cells were transiently transfected with the individual murine dopamine receptors alone, as well as together with the alpha subunit of Gz. PTX treatment was employed to inactivate endogenous alpha i, and coupling to Gi and Gz was estimated by measuring the inhibition of cAMP accumulation induced by quinpirole, in forskolin-stimulated cells. 3. D2S or D2L receptors can couple to the same extent to Gi and to Gz. The D4 dopamine receptor couples preferably to Gz, resulting in about 60% quinpirole-induced inhibition of cAMP accumulation. The D3S and D3L receptor isoforms couple slightly to Gz and result in 15 and 30% inhibition of cAMP accumulation, respectively. 4. We have demonstrated for the first time that the two D3 receptor isoforms, and not any of the other D2 receptor subtypes, also couple to Gs in both COS-7 and CHO transfected cells, in the presence of PTX. 5. Thus, the differential coupling of the D2 dopamine receptor subtypes to various G proteins may add another aspect to the diversity of dopamine receptor function.

Human D3 dopamine receptor in the medulloblastoma TE671 cell line: cross-talk between D1 and D3 receptors.

In search of a cell line in which the D3 dopamine receptor is expressed endogenously, we found that the neuron-derived human medulloblastoma cell line TE671 expresses the human D3 (hD3) and D1 (hD1) receptor, but neither the D2 or D4 receptors. Exposure of TE671 cells to the D3 agonist 7-OH-DPAT (DPAT), or to the D1 agonist SKF-38393 (SKF) increased the expression of hD3 or hD1 mRNA, respectively. Moreover, whereas DPAT had no effect on hD1 mRNA levels, stimulating the cells with SKF caused an increase in both hD1 and hD3 transcript levels. These results suggest (i) that following ligand stimulation, hD3 and hD1 receptors are upregulated to enhance their own receptor expression, and (ii) that upregulation of hD1 receptor transcripts leads to a stimulation of the hD3 dopamine receptor transcripts.

Plasma membrane localization of G alpha z requires two signals.

Three covalent attachments anchor heterotrimeric G proteins to cellular membranes: the alpha subunits are myristoylated and/or palmitoylated, whereas the gamma chain is prenylated. Despite the essential role of these modifications in membrane attachment, it is not clear how they cooperate to specify G protein localization at the plasma membrane, where the G protein relays signals from cell surface receptors to intracellular effector molecules. To explore this question, we studied the effects of mutations that prevent myristoylation and/or palmitoylation of an epitope-labeled alpha subunit, alpha z. Wild-type alpha z (alpha z-WT) localizes specifically at the plasma membrane. A mutant that incorporates only myristate is mistargeted to intracellular membranes, in addition to the plasma membrane, but transduces hormonal signals as well as does alpha z-WT. Removal of the myristoylation site produced a mutant alpha z that is located in the cytosol, is not efficiently palmitoylated, and does not relay the hormonal signal. Coexpression of beta gamma with this myristoylation defective mutant transfers it to the plasma membrane, promotes its palmitoylation, and enables it to transmit hormonal signals. Pulse-chase experiments show that the palmitate attached to this myristoylation-defective mutant turns over much more rapidly than does palmitate on alpha z-WT, and that the rate of turnover is further accelerated by receptor activation. In contrast, receptor activation does not increase the slow rate of palmitate turnover on alpha z-WT. Together these results suggest that myristate and beta gamma promote stable association with membranes not only by providing hydrophobicity, but also by stabilizing attachment of palmitate. Moreover, palmitoylation confers on alpha z specific localization at the plasma membrane.

Functional expression of the murine D2, D3, and D4 dopamine receptors in Xenopus laevis oocytes.

The different murine D2-type dopamine receptors (D2L, D2S, D3L, D3S, and D4) were expressed in Xenopus laevis oocytes. The D2-type receptors were all similarly and efficiently expressed in Xenopus oocytes and were shown to bind the D2 antagonist [125I]sulpride. They were all shown to activate Cl- influx upon agonist stimulation. Using the diagnostic inhibitor bumetanide, we were able to separate the Na+/K+/2Cl- cotransporter component of the Cl- influx from the total unidirectional Cl- influx. The D3L subtype was found to operate exclusively through the bumetanide-insensitive Cl- influx whereas the other D2-type receptors acted on the Na+/K+/2Cl- cotransporter as well. The pertussis toxin sensitivity of the receptor-activated chloride influx via the Na+/K+/2Cl- cotransporter varied between the various D2-type receptors showing that they may couple to different G proteins, and activate different second messenger systems.

A targeted mutation of the D3 dopamine receptor gene is associated with hyperactivity in mice.

While most effects of dopamine in the brain are mediated by the D1 and D2 receptor subtypes, other members of this G protein-coupled receptor family have potentially important functions. D3 receptors belong to the D2-like subclass of dopamine receptors, activation of which inhibits adenylyl cyclase. Using targeted mutagenesis in mouse embryonic stem cells, we have generated mice lacking functional D3 receptors. A premature chain-termination mutation was introduced in the D3 receptor gene after residue Arg-148 in the second intracellular loop of the predicted protein sequence. Binding of the dopamine antagonist [125I]iodosulpride to D3 receptors was absent in mice homozygous for the mutation and greatly reduced in heterozygous mice. Behavioral analysis of mutant mice showed that this mutation is associated with hyperactivity in an exploratory test. Homozygous mice lacking D3 receptors display increased locomotor activity and rearing behavior. Mice heterozygous for the D3 receptor mutation show similar, albeit less pronounced, behavioral alterations. Our findings indicate that D3 receptors play an inhibitory role in the control of certain behaviors.

Early expression of D3 dopamine receptors in murine embryonic development.

In order to determine whether the D2 and D3 dopamine receptors may have a role in prenatal development, we have studied the mRNA expression and distribution of these receptors during murine embryonic development. Using RT-PCR on RNA from embryos taken at progressive stages of development, we have shown that the D3 receptor is expressed significantly earlier than the D2 receptor, being detectable at day 9.5 post-conception (p.c.) compared with day 13.5 p.c. for the D2 subtype. We have also examined the mRNA distribution of the two receptors by whole mount in situ hybridisation. In agreement with the PCR assays, the D3 receptor was expressed earlier than the D2 subtype. D3 receptor transcripts were first detected at day 9.5 p.c. in the ventral aspect of the anterior neural tube, whereas D2 receptor transcripts first appeared a day later. By day 10.5-11.5 p.c. both D3 and D2 receptor transcripts were present in the developing forebrain, and later also in the branchial arches and along the prospective vertebral column. The early appearance of the D3 subtype in murine development and its predominance over the D2 subtype suggest that the D3 receptor may have a functional role in prenatal development.

Differential glycosylation and intracellular trafficking for the long and short isoforms of the D2 dopamine receptor.

The D2 dopamine receptor exists in two alternatively spliced isoforms, "long" and "short" (D2L and D2S), which differ by 29 amino acids in the third cytoplasmic domain. The functional differences between these two isoforms are still obscure. We have performed pulse-chase studies on the D2L and D2S receptors expressed in CHO cells in order to follow the post-translational processing of the two isoforms. Both isoforms are present in three post-translational states: a newly synthesized protein, a partially glycosylated product, and a fully glycosylated mature 70-kDa receptor. However, the processing to the mature receptor differs between the two isoforms. First, the D2S receptor is processed to the mature 70-kDa species faster than the D2L receptor. Second, at 20 degrees C the D2S isoform is fully processed to the 70-kDa species, whereas the D2L isoform persists in its partially processed 45-kDa state. Finally, a significant portion of the D2L receptor remains in its partially processed form in an intracellular compartment and does not reach the plasma membrane. These results give rise to the suggestion that the difference observed between the two alternatively spliced isoforms of the D2 receptor may lie in their post-translational processing and intracellular trafficking.

Molecular cloning and characterisation of the gene encoding the murine D4 dopamine receptor.

The murine D4 dopamine receptor was isolated from a murine genomic DNA library. The receptor's entire coding region was contained within a 6 kb EcoRI genomic fragment, indicating that the murine D4 receptor gene is significantly smaller than the corresponding D2 and D3 receptor genes, the coding regions of which each stretch over 30 kb. The murine D4 receptor gene has three introns and four exons, in common with the rat and human D4 receptor genes. RT-PCR on mRNA from different brain regions shows that the D4 receptor mRNA is expressed in various areas of the brain, with some differences from the rat and human receptor homologues.

Structural organization of the murine D3 dopamine receptor gene.

We have cloned the gene encoding the murine D3 dopamine receptor and have analyzed its intron-exon structural organization, to gain a better understanding of the detailed architecture of the D2 dopamine receptor genes. Restriction and sequence analysis reveal the presence of six introns, in contrast to the five introns previously reported for the rat D3 receptor. The extra intron is located in the receptor's putative third cytoplasmic loop and generates an intron-exon organization directly analogous to that found in the D2 receptor gene. In addition, we have sequenced the 5' and 3' nontranslated sequences flanking the coding region and have identified a putative poly(A) adenylation signal. These sequences are found to have a far lower homology with the corresponding rat nontranslated sequences than is found for the D2 receptor, suggesting that the control of D3 receptor expression may vary more between species than the control of D2 receptor expression.

In vitro translation of D2 dopamine receptors and their chimaeras: analysis by subtype-specific antibodies.

We have developed a range of specific anti-peptide antibodies directed against the D2 and D3 dopamine receptors, and their alternatively spliced isoforms. To demonstrate the subtype- and isoform-specificities of these antibodies we have expressed the receptors in an in vitro translation system and have immunoprecipitated the expressed proteins. We have shown that the D2 and D3 receptors unexpectedly exhibit different migration properties on SDS-PAGE, in addition to displaying a significant difference between their predicted and apparent molecular weights. We have used D2/D3 chimaeric receptors to show that the differential migration between the subtypes can be attributed to the N-terminal half of the receptor and propose that the highly hydrophobic nature of the receptor proteins underlies their anomalous migration properties.

The effect of haloperidol on D2 dopamine receptor subtype mRNA levels in the brain.

Chronic neuroleptic treatment induces an increase in the density of D2 dopamine receptors in the striatum. The effect of prolonged administration of haloperidol on mRNA levels of the short (D2S) and long (D2L) isoforms of the D2 dopamine receptor and the D3 dopamine receptor in different brain regions was examined. Haloperidol caused a significant rise in both D2L and D2S mRNA levels in the striatum, but had no effect on D3 mRNA levels. This rise was restricted to the striatum, showing that the effect of haloperidol on dopamine receptor mRNA is both subtype- and tissue-specific.

Synthesis and processing of D2 dopamine receptors.

Dopamine receptors belong to a superfamily of neurotransmitter receptors that are functionally coupled to guanine nucleotide binding proteins. In this study, we have used Chinese hamster ovary (CHO) cells stably transfected with the rat D2L receptor, in conjunction with specific anti-peptide antibodies that we have developed, in order to visualize this protein and the course of its synthesis. The newly synthesized receptor exists as a 45-kDa protein which undergoes further processing to a 75-kDa glycosylated receptor in the CHO cells. In pulse-chase experiments it was noticed that a 35-kDa precursor was present which disappeared after 30 min. In order to determine whether this 35-kDa protein represents an unprocessed form of the receptor, we have employed an in vitro translation system with cDNA constructs coding for both the murine D2 and D3 dopamine receptor isoforms. In the absence of processing, the D2 and D3 receptors have an apparent molecular mass of 35 kDa. The translated proteins were shown to be the full length receptors by immunoprecipitation with various anti-peptide antibodies and by the demonstration that they can undergo glycosylation to apparent molecular masses of approximately 45 kDa in an in vitro system.

A novel short isoform of the D3 dopamine receptor generated by alternative splicing in the third cytoplasmic loop.

The mouse D3 dopamine receptor has been cloned from olfactory tubercle cDNA using polymerase chain reaction and has been found to exist in two alternatively spliced forms. These two mRNA isoforms differ by the presence or absence of 63 base pairs (bp), which encode 21 amino acids in the putative third cytoplasmic loop of the receptor. The longer form corresponds to the previously reported rat D3 dopamine receptor, to which it bears sequence homology of 94%. Northern blot analysis shows the mouse D3 receptor to be most abundant in the olfactory tubercle. Expression studies show the novel short D3 isoform to bind dopaminergic ligands with a D3-like pharmacological profile. Polymerase chain reaction analysis on different mouse brain regions shows the long and short D3 receptors to be present in the same tissues, the longer form invariably being the predominant one. Analysis of the gene for the mouse D3 dopamine receptor shows that no separate exon encodes the 63-bp stretch and reveals the presence of a consensus sequence for an acceptor site at the 3' end of the 63-bp stretch. This suggests that an internal acceptor site in the exon coding for the distal part of the third cytoplasmic loop directs alternative splicing of the D3 dopamine receptor.

Degradation of plasma proteins by the trypsin-like enzyme of Porphyromonas gingivalis and inhibition of protease activity by a serine protease inhibitor of human plasma.

The interaction between Porphyromonas gingivalis culture supernatant and human serum was examined. Hydrolysis of the major serum proteins was thiol-dependent and correlated with the trypsin-like activity of the sample. Transferrin and IgG light chains were less susceptible to degradation than albumin and IgG heavy chains and partially degraded IgG retained antigen-binding capability. Serum inhibited the trypsin-like activity in a fluorimetric assay. The inhibition was shown to be independent of the level of IgG antibody reactive with whole cells of P. gingivalis. Purified preparations of antithrombin III, a serine protease inhibitor, but not alpha 1-antitrypsin nor alpha 2-macroglobulin inhibited the trypsin-like activity in the fluorometric assay.