D2like receptors

The dopamine D2 receptor The gene encoding the D2 subtype was the first dopamine receptor gene to be identified. The rat cDNA was cloned by homology with the hamster p2 adrenergic receptor (Bunzow et al. 1988). Soon after the publication of the rat dopamine D2 receptor sequence, several studies reported the existence of a longer form of the receptor, generated by alternative splicing of the same gene (Chio et al. 1990; Dal Toso et al. 1989; Giros et al. 1989; Grandy et al. 1989; Miller et al. 1989; Monsma et al. 1989; O'Malley et al. 1990; Rao et al. 1990; Selbie et al. 1989). The two isoforms of the dopamine D2 receptors were termed D2s (short) and D2l (long). The longer isoform contains a 29 amino acid insert in the third intracellular loop. In vivo, D2l is thought to be the predominant form, although some variability in the proportions of the two isoforms occurs among different brain regions (Neve etal. 1991; Snyder etal. 1991).

The human dopamine D2 receptor gene has been localized to chromosome 11q22-q23 (Grandy et al. 1989) and contains eight exons (Gandelman et al. 1992), with alternative splicing occuring at exon 6, yielding mRNAs that differ by 87 nucleotides (generating D2s and D2l ). The sequence of that exon is well-conserved across species, indicating an important function for the region (Fryxell 1994).

The rat dopamine D2s and D2l receptors are 415 and 444 residue proteins (Bunzow et al. 1988; Monsma et al. 1989), with a Mr ~47,000 and 50,900, respectively. The human counterparts contain only 414 and 443 amino acids, respectively, due to the absence of an isoleucine within the C-terminal half of the third intracellular loop of the human receptor. Dopamine D2 receptors identified in different species (e.g. murine, Xenopus and bovine (Chio et al. 1990; Montmayeur et al. 1991; Martens et al. 1991)) show a high degree of sequence homology with the rat and human D2 receptors.

Like the D1 receptors, the mammalian D2 receptors have consensus sites for N-linked glycosylation, including Asn-5, Asn-17, and Asn-23. Several potential sites of phosphorylation by PKA have been identified, such as Ser-147 and 148 in the second cytoplasmic loop, Ser-229, -296, -354, and -364 in the third cytoplasmic loop.

The dopamine D3 receptor The cloning of the cDNA encoding the dopamine D3 receptor was reported in 1990. Sokoloff etal. (1990) used a protocol based on sequence homology with the existing dopamine D2 receptor to identify this new dopamine receptor. The sequence of the receptor encoded by rat D3 cDNA was found to share an overall 52 per cent amino acid identity with the D2 receptor, with >70 per cent identity within the transmembrane regions. The cDNA's encoding human receptors have been also cloned and characterized (MacKenzie et al. 1994; Pilon et al. 1994) and the human dopamine D3 receptor gene mapped to chromosome 3q13.3 (Le Corniat etal. 1991). The dopamine D3 receptor genes from various species possess several exons and introns in their sequences. The murine and human D3 receptor genes have seven exons and six introns (Park et al. 1995), whereas the rat gene possesses only five introns (Giros et al. 1991).

The rat D3 receptor is a 446 amino acid protein, with ~90 per cent and 97 per cent overall amino acid sequence homology with the human and murine forms of the receptor, respectively. However, the human D3 receptor is 46 amino acid shorter than the rat D3 receptor (Giros etal. 1991). Two murine isoforms of D3 receptors resulting from alternative splicing of the gene have been identified. The long form (D3l) corresponds to the rat D3 receptor (446 amino acids), whereas the murine D3s lacks 21 amino acids compared to rat and human D3 receptors (Giros etal. 1991; Park etal. 1995).

A truncated form of dopamine D3 receptor, which lacks the sixth and the seventh transmembrane segments found in the full-length D3 receptor was identified and named D3nf (Schmauss et al. 1993; Liu et al. 1994; Nimchinsky et al. 1997). The function of the D3nf is not entirely clear, but recent evidence shows that it may act as a dominant-negative regulator of D3 receptor activity (Karpa etal. 2000).

Potential sites for N-linked glycosylation on the dopamine D3 receptors from different species include the Asn-12, -19, and -97 residues, while a potential site for phosphorylation by PKA, corresponding to the Ser-348 of the rat receptor has been identified. In addition, several further phosphorylation sites within the third cytoplasmic loop have been postulated.

The dopamine D4 receptor The cloning of the DNA encoding the dopamine D4 receptor was first reported by Van Tol et al. in early 1991 (Van Tol et al. 1991). As with the discovery of other dopamine receptor subtypes, the dopamine D4 receptor was identified by homology sequence with the dopamine D2 receptor. However, initial cloning of the full-length cDNA appeared unsuccessful, and this first description of dopamine D4 receptor was based on a gene/cDNA hybrid. The dopamine D4 receptor was found to share 41 per cent sequence homology with the D2 receptor and 39 per cent with the D3 receptor, with higher percentage of homologies observed within the transmembrane regions. The rat and the murine dopamine D4 genes were cloned independently of the human receptor (O'Malley et al. 1992; Fishburn et al. 1995). The gene encoding the human dopamine D4 receptor was found to be located on chromosome 11p15.5 (Gelernter et al. 1992; Petronis et al. 1993). This gene has five exons and is contained within 5 kb of DNA, whereas the rat D4 gene is a 3.5 kb, with four exons (Van Tol et al. 1991; O'Malley et al. 1992).

Shortly after the description of the D4 receptor it was found that in some species, the receptor has a striking structural feature, consisting of the presence of a 16-amino acid direct repeat in the third cytoplasmic loop. In humans, up to 19 different repeat units present in 2-10 copies have been described (the 9 repeat variant is not seen). This polymorphism has also been described in nonhuman primates (Livak et al. 1995; Matsumoto et al. 1995), but does not exist in the rat D4 receptor. The most common allele found in humans has 4 direct repeats (D4.4) (Lichter et al. 1993), which is a 449 amino acid residues protein, sharing 73 per cent amino acid identity with the rat D4 receptor. Potential sites for N-linked glysosylation and phosphorylation by PKA of the human dopamine D4 receptor are located on Asn-3 and Ser-234, respectively.

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