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The serotonin (5-hydroxytryptamine (5-HT)) 1B and 1D receptor subtypes share a high amino acid sequence identity and have similar ligand binding properties. In this study, we demonstrate that both receptor subtypes exist as monomers and homodimers when expressed alone and as monomers and heterodimers when co-expressed. Gene expression studies have shown that there are brain regions where the 5-HT1B and 5-HT1D receptors are co-localized and where heterodimerization may occur physiologically. This is the first direct visualization of the physical association between G protein-coupled receptors of different subtypes.

Following the cloning of the dopamine receptors we continued a search of the human genome for related genes. We searched an EST data base and discovered cDNA fragments encoding novel G protein-coupled receptor genes. The available GenBank sequence of one of these EST fragments showed that it encoded a receptor with closest similarity to the D2 dopamine and adrenergic receptors. This cDNA was used to isolate the gene (GPR19), and the encoded receptor also demonstrated similarity with the neuropeptide Y receptor. The gene was mapped to chromosome 12, in region p13.2-12.3. Northern blot analysis revealed expression of GPR19 in peripheral regions, and brain regions significantly overlapping with the D2 receptor gene expression. A sequence of the rat orthologue of GPR19 was obtained and in situ hybridization analysis demonstrated a very abundant expression in rat brain.

An approach based on the polymerase chain reaction (PCR) was used to isolate additional members of the G-linked receptor family from a rat striatal lambda gtII cDNA library. Priming with one degenerate probe corresponding to highly conserved consensus sequences in the third transmembrane (TM) domain of 15 G-linked receptors and sequences in the phage vector resulted in one clone (G-13) encoding a dopamine D2 receptor variant with a 29 amino acid insert in the third cytoplasmic loop. In addition, the amino acid sequence encoded by clone G-36 contained conserved sequences characteristic of the G-linked class of receptors and displayed sequence homology in TM domains with the beta 2-adrenergic receptor (48%). Two conserved serine residues in TM5 postulated to be part of a ligand binding site in the adrenergic receptor, suggests that G-36 encodes a catecholaminergic receptor. Northern blot analysis confirmed the expression of G-36 in rat brain, but not in kidney, heart and lung. Several strong hybridizing bands to G-36 were obtained in both human and rat genomic DNA. The general PCR strategy employed here should prove to be extremely useful for the isolation of other members of the G-linked receptor family.