Evolution of the Neuropeptide Y Receptor Family: Gene and Chromosome Duplications Deduced from the Cloning and Mapping of the Five Receptor Subtype Genes in Pig

The endogenous ligands of neuropeptide Y (NPY) receptors are a family of structurally related peptides that includes NPY, peptide YY (PYY), and pancreatic polypeptide (PP). Each ligand consists of 36 amino acids that adopt a hairpin-like structure called a PP-fold (Fuhlendorff et al. 1990). NPY and PYY are the ancestral peptides, whereas the PP gene is a later duplication of PYY (Hort et al. 1995; Larhammar 1996). NPY is involved in the regulation of many physiological activities, such as food intake, blood pressure, and circadian rhythms. PYY and PP are mainly localized to endocrine cells and influence gastric and pancreatic secretion, and gastric and intestinal motility (Gehlert 1998). NPY receptors belong to class A of the G protein-coupled receptor superfamily, that is, the rhodopsin-like receptors. The intracellular loops of these receptors interact with G proteins, which evoke a variety of second messenger responses (Bockaert and Pin 1999). There are currently five cloned NPY receptor subtypes in mammals, Y1 (NPY1R), Y2 (NPY2R), Y4 (NPY4R or PPYR1), Y5 (NPY5R), and y6 (NPY6R) (Blomqvist and Herzog 1997; Michel et al. 1998). The Y3 receptor has not been cloned but has been identified on the basis of its unique binding profile in vivo (Lee and Miller 1998). Early reports that a receptor clone displayed Y3-like binding (Rimland et al. 1991) were shown to be incorrect (Herzog et al. 1993b; Jazin et al. 1993). This receptor was later found to be a chemokine receptor, now designated CXCR4, which binds the ligand SDF-1 (Loetscher et al. 1994; Feng et al. 1996; Oberlin et al. 1996). NPY and PYY display high affinity for Y1, Y2, and Y5 receptor subtypes, which show very low sequence identity to each other (∼30%), suggesting that they are quite ancient duplications (Larhammar et al. 1998). PP preferentially binds to the Y4 receptor, whereas y6 (written with a lower case y as it has no physiological correlate) is a pseudogene in human and has very different pharmacological properties between mouse and rabbit (Gregor et al. 1996; Matsumoto et al. 1996). The human (HSA) genes NPY1R, NPY2R, and NPY5R are clustered on HSA4q31–4q32 (Herzog et al. 1993a; Gerald et al. 1996; Lutz et al. 1997a). In the mouse (MMU), the corresponding region is split over the two chromosomes, MMU3 and MMU8 (Lutz et al. 1997a, 1997b). The NPY4R and NPY6R genes are most similar to NPY1R, but the human genes are localized to two other chromosomes, HSA10q11.2–q21 and HSA5q31 (Gregor et al. 1996; Lutz et al. 1997a, 1997b; Rose et al. 1997). The mouse NPY4R and NPY6R genes also localize to two other chromosomes, MMU14 and MMU18, respectively (Lutz et al. 1997b). Gene families may expand by various duplication mechanisms, and these duplications provide opportunities for subsequent functional differentiation. It has been proposed that early in vertebrate evolution, the ancestral genome underwent large-scale duplications, either genome doublings (tetraploidizations) or at least extensive duplications of chromosomal segments (Lundin 1993; Holland et al. 1994; Sidow 1996). This is supported by the presence of three to four copies of several large gene clusters in the mammalian genome, for example the homeobox (HOX) clusters are localized on human chromosomes HSA2, HSA7, HSA12, and HSA17 (Schughart et al. 1989; Holland et al. 1994; Ruddle et al. 1994). These large-scale duplications presumably facilitated the evolution of the complex anatomical organization of vertebrates, particularly the gnathostomes (jawed vertebrates) (Holland et al. 1994; Sidow 1996). These and several other examples show that information about chromosomal localization is an important complement to sequence-based analyses to deduce gene relationships and species phylogenies. For example, NPY and PYY have arisen from a common ancestor through a chromosomal duplication, with the NPY gene (HSA7q15.1) close to the HOXA cluster (HSA7p15–p14), whereas PYY (HSA17q21.1) is close to the HOXB cluster (HSA17q21–q22) (Larhammar et al. 1997). The third member, PP, arose through a local and more recent tandem duplication of PYY (Hort et al. 1995; Larhammar 1996). These relationships are not obvious from overall sequence identity, as the most recent member, PP, has the highest replacement rate (Larhammar 1996). The NPY receptor genes display modest sequence identity between subtypes (31%–51%) (Michel et al. 1998), have widely differing substitution rates between subtypes (Lundell et al. 1996), and display different chromosomal localizations between human and mouse. Together, these features have hindered investigations into their evolution. We decided to use a representative from a third order of mammals, the domestic pig (Sus scrofa) to enhance investigations into the evolution of this receptor family. The pig gene map is relatively dense (Yerle et al. 1997) compared with many other mammals (excluding human or mouse) (O'Brien et al. 1999), it is well characterized with respect to the role of NPY in cardiovascular regulation (Malmstrom et al. 1998) and is interesting for studying the affects of the NPY system in feeding and metabolism. We report here the isolation of all currently identified NPY receptor genes from the pig, the receptor sequences, and their chromosomal localization within the genome. (We have reported previously the Y5 mapping; Tornsten et al. 1998.) This has enabled us to deduce the ancestral organization for the NPY receptor genes and to trace the evolution of three large chromosomal segments.