Dan Larhammar

Evolution of neuropeptide Y, peptide YY and pancreatic polypeptide

The neuropeptide Y family of peptides consists of neuropeptide Y (NPY), which is expressed in the central and peripheral nervous systems, and peptide YY (PYY) and pancreatic polypeptide (PP) which are gut endocrine peptides. All three peptides are 36 amino acids long and act on G-protein-coupled receptors. NPY and PYY are present in all vertebrates, whereas PP probably arose as a copy of PYY in an early tetrapod ancestor. NPY is one of the most conserved peptides during evolution and no gnathostome (jawed) species differs from the ancestral gnathostome sequence at more than five positions. PYY is more variable, particularly in mammals which have nine differences to the gnathostome ancestor. PP may be the most rapidly evolving neuroendocrine peptide among tetrapods with only 50% identity between mammals, birds, and amphibians. Ancestral gnathostome NPY and PYY seem to have differed at only four positions, suggesting that the gene duplication occurred shortly before the appearance of the gnathostomes. The two peptides differ from one another at 9-12 positions in tetrapod species and share at least two receptor subtypes in mammals. In bony and cartilaginous fishes, NPY and PYY have only 5-6 differences which, together with more extensive neuronal localization of PYY, indicate an even greater functional overlap between the two peptides in these animal groups. The emergence of sequence information for several receptor subtypes from various species will shed additional light on the evolution of the functions of the NPY-family peptides.

Structural diversity of receptors for neuropeptide Y, peptide YY and pancreatic polypeptide

The NPY (neuropeptide Y) family of neuroendocrine peptides consists of NPY, PYY (peptide YY) and PP (pancreatic polypeptide). Several receptors have been characterized pharmacologically of which three have now been cloned. All three belong to the superfamily of receptors that couple to G proteins and all three cause inhibition of cAMP accumulation. Receptor subtypes Y1 and Y2 bind both NPY and PYY. Surprisingly, Y1 and Y2 share only 31% overall sequence identity, the lowest percentage reported for receptors that bind the same peptide ligand. Nevertheless, each subtype is 94% identical between human and rat, suggesting a slow rate of change. These observations suggest that Y1 and Y2 started to diverge from one another very long ago, possibly before the origin of vertebrates. The PP receptor, called PP1 or Y4, is 42% identical to the Y1 receptor (57% in the transmembrane regions) and is one of the most rapidly evolving receptors with only 75% overall identity between man and rat. Interestingly, this receptors preferred ligand, PP, also evolves extremely rapidly. The PP receptor also differs between man and rat in tissue distribution and binding properties. The Y1 and PP receptors bind to both termini of their ligands whereas Y2 mainly interacts with the C-terminal part. Thus, within the same family there are highly conserved receptors and peptide ligands as well as one rapidly evolving receptor and ligand.

Mutations and selection in the generation of class II histocompatibility antigen polymorphism.

A comparison of seven human DR and DC class II histocompatibility antigen beta‐chain amino acid sequences indicates that the allelic variation is of comparable magnitude within the DR and DC beta‐chain genes. Silent and replacement nucleotide substitutions in six DR and DC beta‐chain sequences, as well as in seven murine class II sequences (three I‐A beta and four I‐A alpha alleles) were analyzed. The results suggest that the mutation rates are of a comparable magnitude in the nucleotide sequences encoding the first and second external domains of the class II molecules. Nevertheless, the allelic amino acid replacements are predominantly located in the first domains. We conclude that a conservative selective pressure acts on the second domains, whereas in many positions in the first domains replacement substitutions are selectively neutral or maybe even favoured. Thus, the difference between the first and second domains as regards the number of amino acid replacements is mainly due to selection.

Molecular evolution of NPY receptor subtypes

The neuropeptide Y (NPY) system consists in mammals of three peptides and 4-5 G-protein-coupled receptors called Y receptors that are involved in a variety of physiological functions such as appetite regulation, circadian rhythm and anxiety. Both the receptor family and the peptide family display unexpected evolutionary complexity and flexibility as shown by information from different classes of vertebrates. The vertebrate ancestor most likely had a single peptide gene and three Y receptor genes, the progenitors of the Y1, Y2 and Y5 subfamilies. The receptor genes were probably located in the same chromosomal segment. Additional gene copies arose through the chromosome quadruplication that took place before the emergence of jawed vertebrates (gnathostomes) whereupon differential losses of the gene copies ensued. The inferred ancestral gnathostome gene repertoire most likely consisted of two peptide genes, NPY and PYY, and no less than seven Y receptor genes: four Y1-like (Y1, Y4/a, Y6, and Yb), two Y2-like (Y2 and Y7), and a single Y5 gene. Whereas additional peptide genes have arisen in various lineages, the most common trend among the Y receptor genes has been further losses. Mammals have lost Yb and Y7 (the latter still exists in frogs) and Y6 is a pseudogene in several mammalian species but appears to be still functional in some. One challenge is to find out if mammals have been deprived of any functions through these gene losses. Teleost fishes like zebrafish and pufferfish, on the other hand, have lost the two major appetite-stimulating receptors Y1 and Y5. Nevertheless, teleost fishes seem to respond to NPY with increased feeding why some other subtype probably mediates this effect. Another challenge is to deduce how Y2 and Y4 came to evolve an inhibitory effect on appetite. Changes in anatomical distribution of receptor expression may have played an important part in such functional switching along with changes in receptor structures and ligand preferences.

Both alpha and beta chains of HLA-DC class II histocompatibility antigens display extensive polymorphism in their amino-terminal domains.

At least three class II antigens, all composed of an alpha and a beta subunit, are encoded in the human major histocompatibility complex, i.e., DR, DC and SB. Two cDNA clones, encoding a DC alpha and a DC beta chain, respectively, were isolated from a cDNA library of the lymphoblastoid cell line Raji (DR3,w6). The two polypeptides predicted from the nucleotide sequences of these clones are each composed of a signal peptide, two extracellular domains, a hydrophobic transmembrane region and a short cytoplasmic tail. Comparison of the DC alpha sequence with two previously published partial sequences shows that the majority of the differences is located in the amino‐terminal domain. The differences are not randomly distributed; a cluster of replacements is present in the central portion of the amino‐terminal domain. Likewise, the allelic polymorphism of the DC beta chains occurs preferentially in the amino‐terminal domain, where three minor clusters of replacements can be discerned. The non‐random distribution of the variability of DC alpha and beta chains may be due to phenotypic selection against replacement substitutions in the second domains of the polypeptides.

Alpha chain of HLA-DR transplantation antigens is a member of the same protein superfamily as the immunoglobulins

Four cDNA clones, pDR-alpha-1, pDR-alpha-2, pDR-alpha-3 and pDR-alpha-4, corresponding to the alpha chain of HLA-DR antigens, have been sequenced. Restriction maps and sequences suggest that all clones are identical apart from a single-base substitution present in pDR-alpha-1. Amino acid sequence data, together with the nucleotide sequence data, allowed the complete amino acid sequence to be predicted. The alpha chain is composed of 229 amino acids, of which 191 are exposed on the outside of the plasma membrane. The membrane-embedded portion of the chain consists of 23 hydrophobic amino acids. The succeeding 15 amino acids form the cytoplasmically localized hydrophilic tail. The extracellular portion, with carbohydrate moieties linked to Asn78 and Asn118, seems to be organized into two domains. The second domain, which contains the only disulfide bond of the alpha chain, displays amino acid sequence homology to immunoglobulin constant regions, to the second domain of the beta chain of a class II antigen, to the third domain of heavy chains of class I antigens and to beta 2-microglobulin. Thus the subunits of immunoglobulins, class I antigens and class II antigens are related evolutionarily.

Structure of the murine immune response I-Aβ locus: Sequence of the I-Aβ gene and an adjacent β-chain second domain exon

Abstract The murine major histocompatibility complex I region encodes two class II antigens, I-A and I-E. From a mouse spleen DNA cosmid library of the b haplotype, we isolated a clone containing the entire I-A β gene and a separate exon encoding a β-chain second domain (A β2 ). The A β gene, encompassing more than 6 kb, is encoded by six exons corresponding to the different domains of the A β polypeptide. The translated A β amino acid sequence displays 73% homology to human DC β chains; homologies to other subsets of human β-chains are lower, establishing that I-A corresponds structurally to DC. The A β2 exon is about 20 kb centromeric to the A β gene. Its translated amino acid sequence includes all the conserved amino acids of other class II β-chain second domains. It shows about 60% homology to each of three subsets of human β-chains available for comparison, and to the A β chain. No A β2 first domain exon has been detected with A β or DC β probes.

Numerous groups of chromosomal regional paralogies strongly indicate two genome doublings at the root of the vertebrates

The appearance of the vertebrates demarcates some of the most far-reaching changes of structure and function seen during the evolution of the metazoans. These drastic changes of body plan and expansion of the central nervous system among other organs coincide with increased gene numbers. The presence of several groups of paralogous chromosomal regions in the human genome is a reflection of this increase. The simplest explanation for the existence of these paralogies would be two genome doublings with subsequent silencing of many genes. It is argued that gene localization data and the delineation of paralogous chromosomal regions give more reliable information about these types of events than dendrograms of gene families as gene relationships are often obscured by uneven replacement rates as well as other factors. Furthermore, the topographical relations of some paralogy groups are discussed.

Origins of the many NPY-family receptors in mammals

The NPY system has a multitude of effects and is particularly well known for its role in appetite regulation. We have found that the five presently known receptors in mammals arose very early in vertebrate evolution before the appearance of jawed vertebrates 400 million years ago. The genes Y(1), Y(2) and Y(5) arose by local duplications and are still present on the same chromosome in human and pig. Duplications of this chromosome led to the Y(1)-like genes Y(4) and y(6). We find evidence for two occasions where receptor subtypes probably arose before peptide genes were duplicated. These observations pertain to the discussion whether ligands or receptors tend to appear first in evolution. The roles of Y(1) and Y(5) in feeding may differ between species demonstrating the importance of performing functional studies in additional mammals to mouse and rat.

Neuropeptide expression in rat paraventricular hypothalamic neurons that project to the spinal cord

The paraventricular hypothalamic nucleus (PVH) exerts many of its regulatory functions through projections to spinal cord neurons that control autonomic and sensory functions. By using in situ hybridization histochemistry in combination with retrograde tract tracing, we analyzed the peptide expression among neurons in the rat PVH that send axons to the spinal cord. Projection neurons were labeled by immunohistochemical detection of retrogradely transported cholera toxin subunit B, and radiolabeled long riboprobes were used to identify neurons containing dynorphin, enkephalin, or oxytocin mRNA. Of the spinally projecting neurons in the PVH, approximately 40% expressed dynorphin mRNA, 40% expressed oxytocin mRNA, and 20% expressed enkephalin mRNA. Taken together with our previous findings on the distribution of vasopressin‐expressing neurons in the PVH (Hallbeck and Blomqvist [1999] J. Comp. Neurol. 411:201–211), the results demonstrated that the different PVH subdivisions display distinct peptide expression patterns among the spinal cord–projecting neurons. Thus, the lateral parvocellular subdivision contained large numbers of spinal cord–projecting neurons that express any of the four investigated peptides, whereas the ventral part of the medial parvocellular subdivision displayed a strong preponderance for dynorphin‐ and vasopressin‐expressing cells. The dorsal parvocellular subdivision almost exclusively contained dynorphin‐ and oxytocin‐expressing spinal cord–projecting neurons. This parcellation of the peptide‐expressing neurons suggested a functional diversity among the spinal cord–projecting subdivisions of the PVH that provide an anatomic basis for its various and distinct influences on autonomic and sensory processing at the spinal level. J. Comp. Neurol. 433:222–238, 2001.