Loss of Octarepeats in two processed prion pseudogenes in the red squirrel, Sciurus vulgaris.

The N-terminal region of the mammalian prion protein (PrP) contains an 'octapeptide' repeat which is involved in copper binding. This eight- or nine-residue peptide is repeated four to seven times, depending on the species, and polymorphisms in repeat number do occur. Alleles with three repeats are very rare in humans and goats, and deduced PrP sequences with two repeats have only been reported in two lemur species and in the red squirrel, Sciurus vulgaris. We here describe that the red squirrel two-repeat PrP sequence actually represents a retroposed pseudogene, and that an additional and older processed pseudogene with three repeats also occurs in this species as well as in ground squirrels. We argue that repeat numbers may tend to contract rather than expand in prion retropseudogenes, and that functional prion genes with two repeats may not be viable.

Evolution of closely linked gene pairs in vertebrate genomes.

The orientation of closely linked genes in mammalian genomes is not random: there are more head-to-head (h2h) gene pairs than expected. To understand the origin of this enrichment in h2h gene pairs, we have analyzed the phylogenetic distribution of gene pairs separated by less than 600 bp of intergenic DNA (gene duos). We show here that a lack of head-to-tail (h2t) gene duos is an even more distinctive characteristic of mammalian genomes, with the platypus genome as the only exception. In nonmammalian vertebrate and in nonvertebrate genomes, the frequency of h2h, h2t, and tail-to-tail (t2t) gene duos is close to random. In tetrapod genomes, the h2t and t2t gene duos are more likely to be part of a larger gene cluster of closely spaced genes than h2h gene duos; in fish and urochordate genomes, the reverse is seen. In human and mouse tissues, the expression profiles of gene duos were skewed toward positive coexpression, irrespective of orientation. The organization of orthologs of both members of about 40% of the human gene duos could be traced in other species, enabling a prediction of the organization at the branch points of gnathostomes, tetrapods, amniotes, and euarchontoglires. The accumulation of h2h gene duos started in tetrapods, whereas that of h2t and t2t gene duos only started in amniotes. The apparent lack of evolutionary conservation of h2t and t2t gene duos relative to that of h2h gene duos is thus a result of their relatively late origin in the lineage leading to mammals; we show that once they are formed h2t and t2t gene duos are as stable as h2h gene duos.

Molecular phylogeny and divergence times of Malagasy tenrecs: influence of data partitioning and taxon sampling on dating analyses.

BACKGROUND: Malagasy tenrecs belong to the Afrotherian clade of placental mammals and comprise three subfamilies divided in eight genera (Tenrecinae: Tenrec, Echinops, Setifer and Hemicentetes; Oryzorictinae: Oryzorictes, Limnogale and Microgale; Geogalinae:Geogale). The diversity of their morphology and incomplete taxon sampling made it difficult until now to resolve phylogenies based on either morphology or molecular data for this group. Therefore, in order to delineate the evolutionary history of this family, phylogenetic and dating analyses were performed on a four nuclear genes dataset (ADRA2B, AR, GHR and vWF) including all Malagasy tenrec genera. Moreover, the influence of both taxon sampling and data partitioning on the accuracy of the estimated ages were assessed. RESULTS: Within Afrotheria the vast majority of the nodes received a high support, including the grouping of hyrax with sea cow and the monophyly of both Afroinsectivora (Macroscelidea + Afrosoricida) and Afroinsectiphillia (Tubulidentata + Afroinsectivora). Strongly supported relationships were also recovered among all tenrec genera, allowing us to firmly establish the grouping of Geogale with Oryzorictinae, and to confirm the previously hypothesized nesting of Limnogale within the genus Microgale. The timeline of Malagasy tenrec diversification does not reflect a fast adaptive radiation after the arrival on Madagascar, indicating that morphological specializations have appeared over the whole evolutionary history of the family, and not just in a short period after colonization. In our analysis, age estimates at the root of a clade became older with increased taxon sampling of that clade. Moreover an augmentation of data partitions resulted in older age estimates as well, whereas standard deviations increased when more extreme partition schemes were used. CONCLUSION: Our results provide as yet the best resolved gene tree comprising all Malagasy tenrec genera, and may lead to a revision of tenrec taxonomy. A timeframe of tenrec evolution built on the basis of this solid phylogenetic framework showed that morphological specializations of the tenrecs may have been affected by environmental changes caused by climatic and/or subsequent colonization events. Analyses including various taxon sampling and data partitions allow us to point out some possible pitfalls that may lead to biased results in molecular dating; however, further analyses are needed to corroborate these observations.

Site-specific transamidation and deamidation of the small heat-shock protein Hsp20 by tissue transglutaminase.

Crosslinking of small heat-shock proteins (sHsps) by tissue transglutaminase (tTG) is enhanced by stress and under pathological conditions. We here used hexapeptide probes to determine the amine donor (K) and acceptor (Q) sites for tTG in Hsp20. Mass spectrometric peptide mass fingerprinting and peptide fragmentation established that Q31 and the C-terminal K162 are involved in inter- and intramolecular crosslinking (transamidation). Q31 is a conserved glutamine in sHsps where the neighboring residue determines its reactivity. Moreover, we detected highly efficient simultaneous deamidation of Q66, which suggests that tTG-catalyzed transamidation and deamidation is specific for different glutamine residues.

Testis-specific human small heat shock protein HSPB9 is a cancer/testis antigen, and potentially interacts with the dynein subunit TCTEL1.

Searching EST databases for new members of the human small heat shock protein family, we recently identified HSPB9, which is expressed exclusively in testis as determined by Northern blotting (Kappé et al., Biochim. Biophys. Acta 1520, 1-6, 2001). Here we confirm this testis-specific expression pattern by RT-PCR in a larger series of normal tissues. Interestingly, while screening HSPB9 ESTs, we also noted expression in tumours, which could be verified by RT-PCR. Protein expression of HSPB9 was also detected in normal human testis and various tumour samples using immunohistochemical staining. We thus conclude that HSPB9 belongs to the steadily growing number of cancer/testis antigens. To get a better understanding of the function of HSPB9, we performed a yeast two-hybrid screen to search for HSPB9-interacting proteins. TCTEL1, a light chain component of cytoplasmic and flagellar dynein, interacted in both the yeast two-hybrid system and in immunoprecipitation experiments with HSPB9. Additionally, immunohistochemical staining showed co-expression of HSPB9 and TCTEL1 in similar stages of spermatogenesis and in tumour cells. The possible functional significance of this interaction is discussed.

Tsp36, a tapeworm small heat-shock protein with a duplicated alpha-crystallin domain, forms dimers and tetramers with good chaperone-like activity.

Small heat shock proteins (sHSPs), which range in monomer size between 12 and 42 kDa, are characterized by a conserved C-terminal alpha-crystallin domain of 80-100 residues. They generally form large homo- or heteromeric complexes, and typically have in vitro chaperone-like activity, keeping unfolding proteins in solution. A special type of sHSP, with a duplicated alpha-crystallin domain, is present in parasitic flatworms (Platyhelminthes). Considering that an alpha-crystallin domain is essential for the oligomerization and chaperone-like properties of sHSPs, we characterized Tsp36 from the tapeworm Taenia saginata. Both wild-type Tsp36 and a mutant (Tsp36C-->R) in which the single cysteine has been replaced by arginine were expressed and purified. Far-UV CD measurements of Tsp36 were in agreement with secondary structure predictions, which indicated alpha-helical structure in the N-terminal region and the expected beta-sandwich structure for the two alpha-crystallin domains. Gel permeation chromatography and nano-ESI-MS showed that wild type Tsp36 forms dimers in a reducing environment, and tetramers in a non-reducing environment. The tetramers are stabilized by disulfide bridges involving a large proportion of the Tsp36 monomers. Tsp36C-->R exclusively occurs as dimers according to gel permeation chromatography, while the nondisulfide bonded fraction of wild type Tsp36 dissociates from tetramers into dimers under nonreducing conditions at increased temperature (43 degrees C). The tetrameric form of Tsp36 has a greater chaperone-like activity than the dimeric form.

Transglutaminase catalyzes differential crosslinking of small heat shock proteins and amyloid-beta.

Crosslinking of proteins by tissue transglutaminase (tTG) is enhanced in amyloid (Abeta) deposits characteristic of Alzheimer's disease and sporadic inclusion body myositis. Small heat shock proteins (sHsps) also occur in amyloid deposits. We here report the substrate characteristics for tTG of six sHsps. Hsp27, Hsp20 and HspB8 are both lysine- and glutamine-donors, alphaB-crystallin only is a lysine-donor, HspB2 a glutamine-donor, and HspB3 no substrate at all. Close interaction of proteins stimulates crosslinking efficiency as crosslinking between different sHsps only takes place within the same heteromeric complex. We also observed that alphaB-crystallin, Hsp27 and Hsp20 associate with Abeta in vitro, and can be readily crosslinked by tTG.

Differential effect of simvastatin on activation of Rac(1) vs. activation of the heat shock protein 27-mediated pathway upon oxidative stress, in human smooth muscle cells.

In the present study, we have analyzed the response of human smooth muscle cell (SMC)s to oxidative stress, in terms of recruitment of key elements of the stress-activated protein kinase (SAPK) pathway, such as Rac(1), p38, and the small heat shock protein (HSP)27. The level of expression of three small HSPs, alphaB-crystallin, HSP20, HSP27, as well as the phosphorylation levels of HSP27 and p38, were higher in cultured, asynchronously growing SMCs originating from left interior mammary artery (LIMA) than those originating from aorta, saphenous vein, and umbilical vein, validating the choice of SMCs from LIMA as a model system in our study. In synchronized, quiescent SMCs from LIMA, oxidative stress (H(2)O(2) stimulation)-induced membrane translocation of Rac(1), p38 phosphorylation, membrane translocation, and phosphorylation of HSP27. In these cells, simvastatin (S), an HMG-CoA reductase inhibitor, blocked, in a mevalonate-dependent way, oxidative stress-induced membrane translocation of Rac(1). However, S pretreatment prior to oxidative stress increased the levels of p38 phosphorylation, HSP27 membrane translocation/phosphorylation, actin polymerization, and apoptosis in these cells, in a mevalonate-dependent way. These results establish that S pretreatment has a stimulatory effect on the stress-activated p38/HSP27 pathway, despite its blocking effect on Rac(1) activation.

Influence of hormones and growth factors on lens protein composition: the effect of dexamethasone and PDGF-AA.

PURPOSE: To investigate the effect of hormones and ocular growth factors on the expression of alpha-, beta-, and gamma-crystallins in rat lens epithelial and fiber cells. METHODS: PDGF-AA, EGF, NGF, M-CSF, BMP-2, BMP-4, dexamethasone, and estrogen were tested for their ability to alter the spectrum of crystallins in explanted newborn rat lens epithelial cells or in vitro differentiating newborn rat lens fiber cells. The accumulation of alphaA-, aB-, betaA3/1-, betaB2-, and gamma-crystallin was measured by western blot and dot blot analysis. The morphology of the rat lens explants after culture was examined by hematoxylin-eosin staining, while crystallins were localized by immunofluorescence. RESULTS: Only dexamethasone and PDGF-AA showed an effect on relative crystallin levels. In the presence of dexamethasone the amount of alphaB-crystallin was increased in lens epithelial cells, but dexamethasone did not affect the crystallin spectrum in fiber cells. In rat lens epithelial explants cultured with PDGF-AA an increase in beta- and gamma-crystallin expression was seen. The spectrum of beta- and gamma-crystallins synthesized differed from that present in lens fiber cells. The cells expressing beta- and gamma-crystallin after culture with PDGF-AA were scattered in the epithelial cell layer and retained an epithelial morphology. PDGF-AA did not change the spectrum of crystallins synthesized in lens fiber cells but did enhance the rate of fiber cell differentiation, in agreement with results of others. CONCLUSIONS: Both dexamethasone and PDGF-AA influence crystallin gene expression in cultured rat lens epithelial cells. Dexamethasone enhances the expression of alphaB-crystallin while culturing in the presence of PDGF-AA caused an increase in beta- as well as gamma-crystallin synthesis. Since at least the gamma-crystallin genes are known to be silenced in epithelial cells by DNA methylation, PDGF-AA may be able to induce one of the steps towards fiber cell differentiation in some epithelial cells.

The human genome encodes 10 alpha-crystallin-related small heat shock proteins: HspB1-10.

To obtain an inventory of all human genes that code for alpha-crystallin-related small heat shock proteins (sHsps), the databases available from the public International Human Genome Sequencing Consortium (IHGSC) and the private Celera human genome project were exhaustively searched. Using the human Hsp27 protein sequence as a query in the protein databases, which are derived from the predicted genes in the human genome, 10 sHsp-like proteins were retrieved, including Hsp27 itself. Repeating the search procedure with all 10 proteins and with a variety of more distantly related animal sHsps, no further human sHsps were detected, as was the case when searches were performed at deoxyribonucleic acid level. The 10 retrieved proteins comprised the 9 earlier recognized human sHsps (Hsp27/HspB1, HspB2, HspB3, alphaA-crystallin/HspB4, alphaB-crystallin/HspB5, Hsp20/HspB6, cvHsp/HspB7, H11/HspB8, and HspB9) and a sperm tail protein known since 1993 as outer dense fiber protein 1 (ODF1). Although this latter protein probably serves a structural role and has a high cysteine content (14%), it clearly contains an alpha-crystallin domain that is characteristic for sHsps. ODF1 can as such be designated as HspB10. The expression of all 10 human sHsp genes was confirmed by expressed sequence tag (EST) searches. For Hsp27/HspB1, 2 retropseudogenes were detected. The HspB1-10 genes are dispersed over 9 chromosomes, reflecting their ancient origin. Two of the genes (HspB3 and HspB9) are intronless, and the others have 1 or 2 introns at various positions. The transcripts of several sHsp genes, notably HspB7, display low levels of alternative splicing, as supported by EST evidence, which may result in minor amounts of isoforms at the protein level.

Why proteins without an alpha-crystallin domain should not be included in the human small heat shock protein family HSPB.

The presence of an alpha-crystallin domain documents the evolutionary relatedness of the ubiquitous family of small heat shock proteins. Sequence and three-dimensional structure provide no evidence for the presence of such a domain in HSPC034, recently proposed as the 11th member of the human HSPB family. Also, phylogenetic analyses detect no relationship between HSPC034 and the human HSPB1-10 sequences. Arguments are provided as to why inclusion in the HSPB family of proteins like HSPC034, which resemble small heat shock proteins in being heat-inducible and having chaperone-like properties and a low monomeric mass, but are evolutionarily unrelated, is misleading and confusing.

The small heat-shock proteins HSPB2 and HSPB3 form well-defined heterooligomers in a unique 3 to 1 subunit ratio.

Various mammalian small heat-shock proteins (sHSPs) can interact with one another to form large polydisperse assemblies. In muscle cells, HSPB2/MKBP (myotonic dystrophy protein kinase-binding protein) and HSPB3 have been shown to form an independent complex. To date, the biochemical properties of this complex have not been thoroughly characterized. In this study, we show that recombinant HSPB2 and HSPB3 can be successfully purified from Escherichia coli cells co-expressing both proteins. Nanoelectrospray ionization mass spectrometry and sedimentation velocity analytical ultracentrifugation analysis showed that HSPB2/B3 forms a series of well defined hetero-oligomers, consisting of 4, 8, 12, 16, 20 and 24 subunits, each maintaining a strict 3:1 HSPB2/HSPB3 subunit ratio. These complexes are thermally stable up to 40 degrees C, as determined by far-UV circular dichroism spectroscopy. Surprisingly, HSPB2/B3 exerted a poor chaperone-like and thermoprotective activity, which is likely related to the low surface hydrophobicity, as revealed by its interaction with the hydrophobic probe 1-anilino-8-naphthalenesulfonic acid. Co-immunoprecipitation experiments demonstrated that the HSPB2/B3 oligomer cannot interact with HSP20, HSP27 or alphaB-crystallin, whereas the homomeric form of HSPB2, thus not in complex with HSPB3, could associate efficiently with HSP20. Taken altogether, this study provides evidence that, despite the high level of sequence homology within the sHSP family the biochemical properties of the HSPB2/B3 complex are distinctly different from those of other sHSPs, indicating that the HSPB2/B3 assembly is likely to possess cellular functions other than those of its family members.

Tissue transglutaminase catalyzes the deamidation of glutamines in lens betaB(2)- and betaB(3)-crystallins.

Tissue transglutaminase (tTG) is a Ca(2+)-dependent enzyme catalyzing the formation of covalent crosslinks between peptide-bound glutamine and lysine residues. Lens crystallins, including alphaB-crystallin and several beta-crystallins, are in vitro substrates for tTG. In both human and bovine fetal lens extracts treated with commercially available guinea pig liver tTG we detected the formation of high molecular weight (HMW) aggregates containing crosslinked betaB(2)- and betaA(3)-crystallin. More interestingly, 2D-gel electrophoresis combined with mass spectrometry analysis revealed that glutamines present in the N-terminal arms of betaB(2)- and betaB(3)-crystallins deamidate readily in the presence of tTG. We found that both tTG-catalyzed crosslinking and deamidation disrupt the beta-crystallin complex, suggesting that these tTG-catalyzed modifications can influence the macromolecular assembly of lens crystallins. These data together suggest that tTG can contribute to the age-related deamidation of glutamine residues of lens crystallins.

Arrival and diversification of caviomorph rodents and platyrrhine primates in South America.

Platyrrhine primates and caviomorph rodents are clades of mammals that colonized South America during its period of isolation from the other continents, between 100 and 3 million years ago (Mya). Until now, no molecular study investigated the timing of the South American colonization by these two lineages with the same molecular data set. Using sequences from three nuclear genes (ADRA2B, vWF, and IRBP, both separate and combined) from 60 species, and eight fossil calibration constraints, we estimated the times of origin and diversification of platyrrhines and caviomorphs via a Bayesian relaxed molecular clock approach. To account for the possible effect of an accelerated rate of evolution of the IRBP gene along the branch leading to the anthropoids, we performed the datings with and without IRBP (3768 sites and 2469 sites, respectively). The time window for the colonization of South America by primates and by rodents is demarcated by the dates of origin (upper bound) and radiation (lower bound) of platyrrhines and caviomorphs. According to this approach, platyrrhine primates colonized South America between 37.0 +/- 3.0 Mya (or 38.9 +/- 4.0 Mya without IRBP) and 16.8 +/- 2.3 (or 20.1 +/- 3.3) Mya, and caviomorph rodents between 45.4 +/- 4.1 (or 43.7 +/- 4.8) Mya and 36.7 +/- 3.7 (or 35.8 +/- 4.3) Mya. Considering both the fossil record and these molecular datings, the favored scenarios are a trans-Atlantic migration of primates from Africa at the end of the Eocene or beginning of the Oligocene, and a colonization of South America by rodents during the Middle or Late Eocene. Based on our nuclear DNA data, we cannot rule out the possibility of a concomitant arrival of primates and rodents in South America. The caviomorphs radiated soon after their arrival, before the Oligocene glaciations, and these early caviomorph lineages persisted until the present. By contrast, few platyrrhine fossils are known in the Oligocene, and the present-day taxa are the result of a quite recent, Early Miocene diversification.

The platypus is in its place: nuclear genes and indels confirm the sister group relation of monotremes and Therians.

Morphological data supports monotremes as the sister group of Theria (extant marsupials + eutherians), but phylogenetic analyses of 12 mitochondrial protein-coding genes have strongly supported the grouping of monotremes with marsupials: the Marsupionta hypothesis. Various nuclear genes tend to support Theria, but a comprehensive study of long concatenated sequences and broad taxon sampling is lacking. We therefore determined sequences from six nuclear genes and obtained additional sequences from the databases to create two large and independent nuclear data sets. One (data set I) emphasized taxon sampling and comprised five genes, with a concatenated length of 2,793 bp, from 21 species (two monotremes, six marsupials, nine placentals, and four outgroups). The other (data set II) emphasized gene sampling and comprised eight genes and three proteins, with a concatenated length of 10,773 bp or 3,669 amino acids, from five taxa (a monotreme, a marsupial, a rodent, human, and chicken). Both data sets were analyzed by parsimony, minimum evolution, maximum likelihood, and Bayesian methods using various models and data partitions. Data set I gave bootstrap support values for Theria between 55% and 100%, while support for Marsupionta was at most 12.3%. Taking base compositional bias into account generally increased the support for Theria. Data set II exclusively supported Theria, with the highest possible values and significantly rejected Marsupionta. Independent phylogenetic evidence in support of Theria was obtained from two single amino acid deletions and one insertion, while no supporting insertions and deletions were found for Marsupionta. On the basis of our data sets, the time of divergence between Monotremata and Theria was estimated at 231-217 MYA and between Marsupialia and Eutheria at 193-186 MYA. The morphological evidence for a basal position of Monotremata, well separated from Theria, is thus fully supported by the available molecular data from nuclear genes.

Asynchronous colonization of Madagascar by the four endemic clades of primates, tenrecs, carnivores, and rodents as inferred from nuclear genes.

Madagascar harbors four large adaptive radiations of endemic terrestrial mammals: lemurs, tenrecs, carnivorans, and rodents. These rank among the most spectacular examples of evolutionary diversification, but their monophyly and origins are debated. The lack of Tertiary fossils from Madagascar leaves molecular studies as most promising to solve these controversies. We provide a simultaneous reconstruction of phylogeny and age of the four radiations based on a 3.5-kb data set from three nuclear genes (ADRA2B, vWF, and AR). The analysis supports each as a monophyletic clade, sister to African taxa, and thereby identifies four events of colonization out of Africa. To infer the time windows for colonization, we take into account both the divergence from the closest non-insular sister group and the initial intra-insular radiation, which is a novel but conservative approach in studies of the colonization history of Madagascar. We estimate that lemurs colonized Madagascar between 60 million years ago (Mya) (split from lorises) and 50 Mya (lemur radiation) (70-41 Mya taking 95% credibility intervals into account), tenrecs between 42 and 25 Mya (50-20 Mya), carnivorans between 26 and 19 Mya (33-14 Mya), and rodents between 24 and 20 Mya (30-15 Mya). These datings suggest at least two asynchronous colonization events: by lemurs in the Late Cretaceous-Middle Eocene, and by carnivorans and rodents in the Early Oligocene-Early Miocene. The colonization by tenrecs may have taken place simultaneously with either of these two events, or in a third event in the Late Eocene-Oligocene. Colonization by at least lemurs, rodents, and carnivorans appears to have occurred by overseas rafting rather than via a land bridge hypothesized to have existed between 45 and 26 Mya, but the second scenario cannot be ruled out if credibility intervals are taken into account.

Nuclear import of {alpha}B-crystallin is phosphorylation-dependent and hampered by hyperphosphorylation of the myopathy-related mutant R120G.

Phosphorylation modulates the functioning of alphaB-crystallin as a molecular chaperone. We here explore the role of phosphorylation in the nuclear import and cellular localization of alphaB-crystallin in HeLa cells. Inhibition of nuclear export demonstrated that phosphorylation of alphaB-crystallin is required for import into the nucleus. As revealed by mutant analysis, phosphorylation at Ser-59 is crucial for nuclear import, and phosphorylation at Ser-45 is required for speckle localization. Co-immunoprecipitation experiments suggested that the import of alphaB-crystallin is possibly regulated by its phosphorylation-dependent interaction with the survival motor neuron (SMN) protein, an important factor in small nuclear ribonucleoprotein nuclear import and assembly. This interaction was supported by co-localization of endogenous phosphorylated alphaB-crystallin with SMN in nuclear structures. The cardiomyopathy-causing alphaB-crystallin mutant R120G was found to be excessively phosphorylated, which disturbed SMN interaction and nuclear import, and resulted in the formation of cytoplasmic inclusions. Like for other protein aggregation disorders, hyperphosphorylation appears as an important aspect of the pathogenicity of alphaB-crystallin R120G.

Molecules consolidate the placental mammal tree.

Deciphering relationships among the orders of placental mammals remains an important problem in evolutionary biology and has implications for understanding patterns of morphological character evolution, reconstructing the ancestral placental genome, and evaluating the role of plate tectonics and dispersal in the biogeographic history of this group. Until recently, both molecular and morphological studies provided only a limited and questionable resolution of placental relationships. Studies based on larger and more diverse molecular datasets, and using an array of methodological approaches, are now converging on a stable tree topology with four major groups of placental mammals. The emerging tree has revealed numerous instances of convergent evolution and suggests a role for plate tectonics in the early evolutionary history of placental mammals. The reconstruction of mammalian phylogeny illustrates both the pitfalls and the powers of molecular systematics.

Molecular evolution of the mammalian alpha 2B adrenergic receptor.

The alpha 2B adrenergic receptor (A2AB) is a heptahelical G protein-coupled receptor for catecholamines. We compared the almost complete coding region (about 1,175 bp) of the A2AB gene from 48 mammalian species, including eight newly determined sequences, representing all the 18 eutherian and two marsupial orders. Comparison of the encoded proteins reveals that residues thought to be involved in agonist binding are highly conserved, as are the regions playing a role in G protein-coupling. The three extracellular loops are generally more variable than the transmembrane domains and two of the intracellular loops, indicating a lower functional constraint. However, the greatest variation is observed in the very long, third intracellular loop, where only a few residues and a polyglutamyl tract are preserved. Although this polyglutamyl domain displays a great variation in length, its presence in all described A2ABs confirms its proposed role in agonist-dependent phosphorylation of the third intracellular loop. Phylogenetic analyses of the A2AB data set, including Bayesian methods, recognized the superordinal clades Afrotheria, Laurasiatheria, and Euarchontoglires, in agreement with recent molecular evidence, albeit with lower support. Within Afrotheria, A2AB strongly supports the paenungulate clade and the association of the continental African otter shrew with Malagasy tenrecs. Among Laurasiatheria, A2AB confirms the nesting of whales within the artiodactyls, as a sister group to hippopotamus. Within the Euarchontoglires, there is constant support for rodent monophyly.