Prominence of the dopamine D2 short isoform in dopaminergic pathways

Abstract

As a result of alternative splicing, the D2 gene of the dopamine receptor family exists in two isoforms. The D2 long is characterized by the insertion of 29 amino acids in the third cytoplasmic loop, which is absent in the short isoform. We have produced subtype-specific antibodies against both the D2 short and D2 long isoforms and found a unique compartmentalization between these two isoforms in the primate brain. The D2 short predominates in the cell bodies and projection axons of the dopaminergic cell groups of the mesencephalon and hypothalamus, whereas the D2 long is more strongly expressed by neurons in the striatum and nucleus accumbens, structures targeted by dopaminergic fibers. These results show that the splice variants of the dopamine D2 receptor are differentially distributed and possess distinct functions. The strategic localization of the D2 short isoform in dopaminergic cell bodies and axons strongly suggests that this isoform is the likely dopamine autoreceptor, whereas the D2 long isoform is primarily a postsynaptic receptor.Among all the neurotransmitter receptors cloned to date, the D2 subtype of dopamine receptors has been the one most closely linked to the positive symptoms of schizophrenia (1, 2) and often implicated in extrapyramidal side effects (3, 4). Neuroanatomical (5, 6) and physiological (7,8) studies indicate that this receptor has both autoreceptor and postsynaptic functions. Further, recent evidence of loss of autoreceptor function in D2-deficient mice (8) but sparing of this function in D3 mutant mice (9) strongly suggests that the D2 subtype is the only autoreceptor within the dopamine system. However, the D2 receptor exists in two isoforms (10, 11), and it is not known if both isoforms contribute to autoreceptor function. With the use of recently developed subtype-specific antibodies to dopamine D2 short (D2S) and D2 long (D2L) isoforms, we have found prevalent labeling of the D2S isoform in the cell bodies and axons of brain stem dopamine neurons, suggesting that this isoform is the autoreceptor of the dopamine system. METHODSPreparation of Antibodies. The D2S peptide TPLKDAAR and D2L peptide SNGSFPVNRRRM corresponding to residues 238-245 and 259-270 (10, 11), respectively, were derived from the third cytoplasmic loop of the receptor. The D2S peptide was arranged by adopting four amino acids from each side of the insertion site to differentiate between D2S and D2L isoforms. A similar procedure has previously been used for the preparation of antibodies to ␥-aminobutyric acid (GABA) receptors (12). The peptides were coupled to keyhole limpet hemocyanin (KLH) protein. Peptide-KLH conjugate (100 g) emulsified in complete Freund's adjuvant was injected into rabbits for antibody development. Affinity purification of the antisera was as described elsewhere (12). In brief, peptide (5 mg) was coupled to 1 g of activated thiopropyl-Sepharose 6B (Pharmacia LKB). Phosphate-buffered saline (PBS) diluted antiserum (1:5) was circulated through the column. After washing with PBS, th...

Evolution and expression of D2 and D3 dopamine receptor genes in zebrafish

“…The functional significance of the fifth exon in D2 and D3 receptors is not well understood. In mammals, the D2S receptor exhibits a predominant presynaptic localization, whereas the D2L receptor is postsynaptically situated (Khan et al, 1998). Recent experiments suggest that the fifth exon may contribute to dopamine receptor trafficking.…”

Cholinergic interneuron characteristics and nicotinic properties in the striatum

“…The D 2 gene itself can be alternatively spliced. Evidence indicates that the short form of D 2 (D 2 S) often serves as the presynaptic autoreceptor that inhibits DA release; whereas the long form (D 2 L) is more likely to be found postsynaptically (Khan et al, 1998;Usiello et al, 2000). Both D 1 -like and D 2 -like receptors are widely expressed in the brain with the relative abundance in the order of (Boyson et al, 1986;Richfield et al, 1989;Levey et al, 1993;Missale et al, 1998).…”

Kappa-Opioid Agonist U69593 Potentiates Locomotor Sensitization to the D2/D3 Agonist Quinpirole: Pre- and Postsynaptic Mechanisms

“…The typical autoreceptor effects, such as near total inhibition of spontaneous cell firing by midbrain dopamine neurons, can be produced with doses of QNP as low as 0.03 mg/kg (Mottola et al, 2002;Pitts et al, 1995). Moreover, strong support for the claim that this effect of QNP is produced by stimulation of dopamine autoreceptors is provided by findings that QNP is ineffective in D2 receptor null mice but continues to inhibit dopamine neuron activity in knockout mice lacking the D2Long isoform (Centonze et al, 2002), the isoform characteristic of the postsynaptic dopamine receptor (Khan et al, 1998;Usiello et al, 2000). Second, neurochemical studies show that similarly low doses of dopamine agonists, including QNP, are effective in the inhibition of dopamine release mediated by D2-like presynaptic receptors (Acri et al, 2001;Imperato et al, 1988;Shilliam and Heidbreder, 2003).…”