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.

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.

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.

Modulation of anti-acetylcholine receptor antibody specificities and of experimental autoimmune myasthenia gravis by synthetic peptides.

Synthetic peptides corresponding to selected sequences from the nicotinic acetylcholine receptor (AChR) were employed to identify possible antigenic determinants within the receptor which can modulate the anti-AChR response and experimental autoimmune myasthenia gravis (EAMG). Immunization of rabbits with peptides T alpha 73-89, T alpha 351-368, T delta 354-367 and H alpha 351-368, prior to AChR inoculation, affected the course of EAMG in six out of eight rabbits. These six protected rabbits survived three inoculations of AChR and survived for at least five months after the third injection with AChR, whereas control rabbits died following one or two injections of AChR. The survival of peptide-preimmunized rabbits injected with AChR seemed to correlate with the antibody specificities in immunoblots. Following AChR inoculation there was a shift in reactivity, from a subunit-restricted response, to reactivity with all subunits of the receptor. This shift was delayed in protected rabbits. This may indicate that the reactivity with the entire Torpedo receptor molecule represents a loss of tolerance to AChR which culminates in the autoimmune disease, EAMG.

Differences between embryonic and adult Torpedo acetylcholine receptor gamma subunit.

Antibodies to a synthetic peptide corresponding to residues 346-359 of the Torpedo acetylcholine receptor (AChR) gamma subunit, were employed to compare the adult and embryonic receptor. This peptide contains a consensus phosphorylation site for cAMP-dependent protein kinase (PKA). The anti-peptide antibodies discriminated between adult and embryonic AChRs, and reacted preferentially with the adult gamma form. These observed immunological differences did not seem to stem from different phosphorylation states. Our results suggest that the embryonic Torpedo AChR may have a gamma-like subunit that differs from the known adult form of this subunit, at least in the specific region that contains the phosphorylation site for PKA.