Molecular Evolution of the Mammalian Alpha 2B Adrenergic Receptor

Ole Madsen*, Diederik Willemsen*, Björn M. Ursing{dagger},1, Ulfur Arnason{dagger} and Wilfried W. de Jong*{ddagger},2

*Department of Biochemistry 161, University of Nijmegen, The Netherlands;
{dagger}Department of Genetics, Division of Evolutionary Molecular Systematics, University of Lund, Sweden;
{ddagger}Institute for Biodiversity and Ecosystem Dynamics, Amsterdam, The Netherlands


    Abstract
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Acknowledgements
 References
 
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.


    Introduction
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Acknowledgements
 References
 
The gene for the alpha 2B adrenergic receptor (SWISS-PROT name A2AB) has extensively been used in recent studies on mammalian phylogeny (Springer et al. 1997Citation ; Stanhope et al. 1998Citation ; Madsen et al. 2001Citation ; Murphy et al. 2001bCitation ). A2AB has a length of about 450 amino acid residues and is encoded by a single copy gene (Lomasney et al. 1990Citation ). The gene is intronless, so the coding sequence can almost completely be amplified and sequenced from genomic DNA. Thus, an analysis of the deduced protein sequences can provide a fairly complete impression of the evolution of the protein.

A2AB is a representative of the biomedically highly important superfamily of G protein–coupled receptors (GPCRs) (Strader et al. 1995Citation ). With at least 616 members in the human genome, it is one of the largest known gene families (Venter et al. 2001Citation ) and can be divided into six groups: class A to E and the frizzled-smoothened family (Bockaert and Pin 1999Citation ; Horn, Vriend, and Cohen 2001Citation ; see http://www.gpcr.org/7tm/). These membrane receptors mediate signals induced by extracellular stimuli to the intracellular environment by way of guanyl nucleotide–binding proteins (G proteins). GPCRs are characterized by a bundle of seven transmembrane (TM) helices (TM1–7), which are connected by three extra- and three intracellular loops (EL1–3 and IL1–3, respectively), and have an extracellular N-terminus and an intracellular C-terminus (Baldwin 1993Citation ). The only known GPCR crystal structure is that of rhodopsin, which is a class-A receptor (Palczewski et al. 2000Citation ). A2AB is also a class-A receptor and is one of the adrenoceptors (adrenergic receptors) in the subfamily of amine receptors. Three types of mammalian adrenoceptors can be distinguished: alpha 1, alpha 2, and beta, each again having at least three subtypes. Class-A receptors bind mostly small ligands inside the helical bundle and are characterized by a conserved aspartate in TM2, important for monovalent cation regulation, and a tripeptide DRY or ERW at the intracellular end of TM3, involved in G-protein coupling (Ceresa and Limbird 1994Citation ; Oliveira et al. 1994Citation ; Strader et al. 1995Citation ; Scheer et al. 1996Citation ).

A2AB is a receptor for catecholamines, such as adrenaline and nonadrenaline, and mainly mediates the inhibition of adenylyl cyclase by way of coupling to Gi/o-proteins (Limbird 1988Citation ), but stimulation of adenylyl cyclase by way of coupling to Gs-proteins has also been reported (Eason et al. 1992Citation ). A2AB has a distinct expression in various tissues, mostly in the periphery, with highest expression in the kidney (e.g., Eason and Liggett 1993Citation ; Link et al. 1996Citation ). Knockout studies in mouse have shown that lack of the A2AB gene influences viability, the response to salt-induced hypertension, and blood pressure responses to agonists (Link et al. 1996Citation ; Makaritsis et al. 2000Citation ). Special features of A2AB are the absence of N-linked glycosylation sites in the extracellular domains and the presence of a very long, third intracellular loop, which contains a unique glutamate repeat. The third intracellular loop is necessary for microtubule sorting of A2AB to the cell surface (Saunders and Limbird 2000Citation ), whereas the polyglutamate domain is required for short-term agonist-promoted phosphorylation and consequent desensitization of A2AB (Jewell-Motz and Liggett 1995Citation ; Small et al. 2001Citation ). As with many other GPCRs (Pierce and Lefkowitz 2001Citation ), desensitization and internalization of A2AB is mediated by arrestins (DeGraff et al. 1999Citation ).

Little attention has generally been given to the molecular evolutionary information of genes used in phylogenetic studies. In this article we analyze the evolution of the structure and function of A2AB from 48 mammalian species. In addition, we also use a subset of 41 A2AB sequences to compare the phylogenetic relationships supported by the A2AB sequences with those obtained from concatenated data sets (e.g., Madsen et al. 2001Citation ; Murphy et al. 2001bCitation ). Combined data sets are required to get a robust phylogenetic tree, but it remains important to know the congruence with results obtained from single genes.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Acknowledgements
 References
 
Eight new A2AB sequences are reported in this study and combined with 43 A2AB sequences extracted from the EMBL databank (release 68, September 2001) (table 1 ). The new A2AB sequences were obtained as described elsewhere (Springer et al. 1997Citation ; Madsen et al. 2001Citation ). In short, the primers A2ABFOR (5'-asccctactcngtgcaggcnacng-3') and A2ABREV (5'-ctgttgcagtagccdatccaraaraaraaytg-3') were used for PCR amplification on genomic DNA with the Expand High Fidelity PCR system (Boehringer Mannheim). PCR products were directly sequenced on both strands with internal primers or cloned into a pGEM-T vector (Promega), or both. If a sequence was determined from cloned DNA only, clones from at least two independent PCRs were sequenced to detect ambiguity caused by the PCR or allelic variations, or both. Sequencing was done with the Thermo Sequenase fluorescent-labeled primer or Thermo Sequenase Cy5 Dye Terminator cycle sequencing kits (Amersham Pharmacia Biotech).


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Table 1 Species Names and Corresponding Accession Numbers of Sequences Used in this Study

 
Amino acid sequence alignments were made with GCG PILEUP (Wisconsin Package Version 10.0, GCG, Madison) and further optimized by eye. The nucleotide alignment was adjusted to correspond with the amino acid alignment.

To reduce computing time, seven of the nine bat species were excluded from the phylogenetic analyses because the relationships within bats (as based on A2AB) has recently been shown (Springer et al. 2001Citation ). Furthermore, we only used one of the alleles from human, alpaca, and guinea pig. Phylogenetic analyses were done on a 39-taxon (eutherians only) and a 41-taxon data set (eutherians plus out group marsupials) (see Results). Because great variability in base composition is observed at third codon positions (data not shown), phylogenetic analyses were performed with third codon positions unweighted and as transversions only, and without third codon positions. We further used the logdet model of sequence evolution (e.g., Lake 1994Citation ) in distance analyses to compensate for any possible heterogeneity in base composition among species. The glutamic acid domain in IL3 was excluded in phylogenetic analyses because it is difficult to align.

The following methods were used to obtain phylogenetic trees: maximum parsimony on DNA and amino acids, minimum evolution on DNA with logdet distances (e.g., Lake 1994Citation ), bio–neighbor joining (Gascuel 1997Citation ) on protein with JTT matrix distances (Jones, Taylor, and Thornton 1992Citation ), maximum likelihood on DNA, and Bayesian posterior probability on DNA. PAUP4.0b2–4 (Swofford 1998Citation ) was used for parsimony, distance on DNA, and maximum likelihood analyses. The programs SEQBOOT, PROTDIST, and CONSENSE from the PHYLIP package (Felsenstein 2001Citation ), and BIONJ (Gascuel 1997Citation ) were used for distance analyses on amino acids.

Parsimony analyses included first, second, and third codon position unweighted; first and second position unweighted, with third position transversions only; first and second position unweighted; and amino acid unweighted. Ten random input orders of sequences were used, with gaps scored as missing, and in all PAUP analyses, the tree bisection–reconnection branch swapping option was used to swap branches. Bootstrap analyses included 500 or 100 replicates for DNA (parsimony-distance and maximum likelihood, respectively) and 250 replicates for amino acid sequences.

Maximum likelihood analyses were done with the HKY85 (Hasegawa, Kishino, and Yano 1985Citation ) model of sequence evolution. Transition to transversion ratios were calculated on minimum evolution trees obtained with HKY85 distances. These values were subsequently used for maximum likelihood calculations.

Bayesian phylogenetic analyses were performed with MRBAYES 2.1 (Huelsenbeck and Ronquist 2001Citation ). First, modeltest 3.06 (Posada and Crandall 1998Citation ) was used to determine which model of sequence evolution best fits the data under the maximum likelihood assumption. The best ML model of sequence evolution was subsequently used in Bayesian analyses, and the Metropolis-coupled Markov chain Monte Carlo sampling approach was used to calculate posterior probabilities. Initial probabilities for all trees were equal, and starting trees were random. Four Markov chains were run simultaneously 200,000, 350,000, and 500,000 times (to check consistency of results), tree sampling was done every 10 generations, and burn-in values were determined from the likelihood values.

Partitioned phylogenetic analyses included the following four partitions: extracellular (positions 14–24, 61–97, 138–181, and 395–422 in fig. 1 ), intracellular (25–60, 98–137, 182–298, and 320–394), intracellular without the variable parts of IL3 (25–60, 98–137, 182–202, and 372–394), and the variable parts of IL3 (203–298; and 320–371). Analyses on partitioned data were on protein with JTT matrix distances only, and the number of bootstrap replicates were 100 (see above for details).



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Fig. 1.—Sequence variation in mammalian A2AB. The observed variation is given above the majority consensus sequence (positions 14–422), as deduced from an alignment of A2AB sequences from 46 eutherian and two marsupial species. Because of the localization of the PCR primers, all but four of the 48 sequences miss the nucleotides coding for the 13 N-terminal and 42 C-terminal residues of the protein. The sequence from positions 1 to 13 and from 423 to 464 is human and includes the positions of the two primers used for PCR (arrows). Gaps are indicated by –. Residues involved in ligand binding in other adrenergic receptors (B2AR, A1AB, and A2AA; SWISS-PROT names (Ballesteros, Shi, and Javitch 2001Citation )) are indicated by *, and ¥ indicates the conserved D58 important in monovalent cation regulation of GPCRs (Ceresa and Limbird 1994Citation ). G-protein coupling regions predicted in the three alpha 2 adrenoreceptor subtypes A2AA, A2AB, and A2AC (Wade et al. 1994Citation ; Eason and Liggett 1995Citation ; Saunders and Limbird 2000Citation ) are indicated by %. The secondary structure of A2AB, as predicted from the crystal structure of bovine rhodopsin (Palczewski et al. 2000Citation ), shows the positions of TM regions (TM1–7), intracellular loops (IL1–3), and extracellular loops (EL1–3). To predict the secondary structure, highly conserved TM positions (#) were used to align A2AB with bovine rhodopsin (Ballesteros, Shi, and Javitch 2001Citation ). Side chains in the TM domains predicted to face the lipid bilayer are marked in gray; those facing inward in the TM bundle are in black; and those unassigned are in white. For the glutamic acid domain in IL3, only the consensus sequence is given, and the variability of this domain in different species is presented in figure 2

 
The rate of nonsynonymous nucleotide substitutions (NNS) per site between human and mouse A2AB was calculated with MEGA 2.0 (Kumar et al. 2001Citation ), using the method of Li, Wu, and Luo (1985)Citation . The divergence between human and mouse was set to 80 Myr (to make NNS of A2AB comparable with that of Graur and Li 2000Citation ), and the rate unit is per site per billion years. This unit was used to facilitate the comparison of the NNS rate of A2AB with that of other mammalian protein-coding genes. The part of the human and mouse A2AB used to calculate the NNS corresponds to positions 14–405 in the human sequence, and the glutamic acid domain in IL3 was included in this calculation.

To detect whether the A2AB data set contains any significantly long branches, we performed RASA analyses using the program RASA 3.01 (Lyons-Weiler 2001Citation ).


    Results and Discussion
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Acknowledgements
 References
 
Molecular Evolution of A2AB
A2AB sequences, including the eight newly determined ones, are now available for 48 mammalian species. These represent all the 18 eutherian orders and the major subordinal groups, as well as two species from divergent marsupial orders. Figure 1 relates the amino acid sequence variation deduced from the amplified part of the A2AB genes to the structural features of the protein. This composite sequence immediately reveals that most residues in the TM domains, in the intracellular loops IL1 and IL2, and in the extracellular loop EL1 are strictly conserved. It also emphasizes the huge sequence variability in the long IL3 and in EL2, and less so in EL3. In IL3, as many as nine different residues can be found at corresponding positions in different A2AB sequences, and it is the only region where indels occur. The observed sequence conservation and variability can be interpreted as follows.

Transmembrane Domains
Residues in TM3, TM5, and TM6 that are thought to be involved in ligand binding (* in fig. 1 ) are all conserved. The same is true for the intracellular ends of TM3 and TM6 that participate in G-protein coupling (% in fig. 1 ). The distribution of variable positions in the TM domains is interesting. Residues facing inward into the TM bundle are better conserved than those facing outward to the lipid bilayer (black and gray, respectively, in fig. 1 ). Of the former, only 9% are variable, against 34% of the lipid-facing positions, which also allow multiple replacements more often. Six of the seven inward-facing replacements are conserved with respect to side chain size, whereas residues oriented toward the lipid layer show greater size variation. Inward-facing residues are clearly more constrained than outward-facing ones in terms of size and chemical properties, reflecting their closer packing and tighter interaction with other residues in the TM bundle.

Extracellular Loops
Despite considerable sequence variation, it is noteworthy that all three extracellular loops have conserved their lengths. This suggests that the length of these loops is important for maintaining the proper conformation of the receptor. In EL1, only one position (81) is variable, whereas the other five form a conserved "GYWYF motif" (positions 75–80). The sequence YWYF is also retained in other alpha 2 adrenergic receptors (A2AA and A2AC), whereas W78 is conserved in all amine receptors but not in other class-A (rhodopsin-like) receptors (http://www.gpcr.org/7tm/). In the crystal structure of rhodopsin, EL1 as well as EL3 run along the periphery of the receptor (Palczewski et al. 2000Citation ). If this also applies to A2AB, EL1 may be in direct contact with other extracellular parts (EL2, EL3, distal parts of TM helices) thus stabilizing the conformation of the receptor. A functional role for W78 is supported by the finding that mutation to alanine or phenylalanine results in a 40-fold decrease in the binding of acetylcholine in the muscarinic m1 receptor (Matsui, Lazareno, and Birdsall 1995Citation ). Such a function of W78 would imply that EL1 is situated in the center of the receptor, close to or as a lid above the ligand-binding pocket, taking part in ligand binding or selection.

In EL2, 12 of the 19 residues are variable or even hypervariable. The conserved C164, found in most GPCRs, forms a structurally important disulfide bond with C85 at the extracellular end of TM3 (Savarese, Wang, and Fraser 1992Citation ). The adjacent proline (P162) might be conserved to allow EL2 to bend back into the helix bundle when forming the C85–C164 disulfide bond. In rhodopsin, two antiparallel ß-strands are present in EL2, corresponding to positions ~154–158 and ~163–166 in A2AB, of which the latter forms a part of the ligand-binding pocket (Palczewski et al. 2000Citation ). It seems unlikely that similar ß-strands are present in A2AB because these positions are quite variable in this protein. The hypervariable nature of position 165 also makes it difficult to imagine that this region is a part of the binding-site crevice in A2AB. In rhodopsin, the residues corresponding to Y150 and N167 in A2AB are near each other, which, considering their conservation, may also be the case in A2AB. In class-A receptors, the N-terminal residues of EL2 are possibly important, either structurally or for ligand binding (Javitch et al. 2000Citation ). This could explain the relative conservation of this region in A2AB (positions 150–153).

Five out of the eight residues are conserved in EL3. Two of these, C415 and P418, are retained in other alpha-2 adrenergic receptors, but not in the alpha-1 and beta-adrenergic receptor families, indicating a possible role in the former (http://www.gpcr.org/7tm/). As mentioned, EL3 runs along the periphery of the receptor in rhodopsin. EL swapping experiments between beta-3 and alpha-1a adrenergic receptors have shown that EL3 plays a role in controlling receptor and G-protein affinity, probably by influencing the helical packing (Zhao, Gaivin, and Perez 1998Citation ). The same probably applies for EL3 in A2AB.

Intracellular Loops IL1 and IL2
The strict conservation of IL1 seems difficult to explain because no particular role has been suggested for this loop. Indeed, mutation in A1AB of the two basic residues in IL1 (corresponding to positions 41 and 44 in A2AB) does not have any effect on receptor activation and G-protein coupling (Greasley et al. 2001Citation ). On the other hand, IL1 in rhodopsin has a rigid organization (Palczewski et al. 2000Citation ) which may demand specific types of residues; the same may apply to A2AB, considering that the basic amino acids R41 (K in rhodopsin) and R44, as well as L43 are conserved between rhodopsin and A2AB.

IL2 forms a part of the site for selective coupling of the G-protein complex (e.g., Ostrowski et al. 1992Citation ; Palczewski et al. 2000Citation ). Only three positions are conservatively variable, indicating that only few changes are tolerated to maintain subtype-specific G-protein coupling. One of these variable sites, S122, may play a role together with R116 and A113 in the difference in dose-response–induced cAMP production between A2AB and A2AA in Sf9 cells (Nasman, Jansson, and Akerman 1997Citation ). Interestingly, although all placentals have A113, this is S113 in the marsupial A2AB, as in the A2AA sequence at this position. This may suggest some differences in dose-response–induced cAMP production between placental and marsupial A2AB.

IL3 and the Polyglutamate Repeat
IL3 is extremely variable in A2AB, including many indels. However, the length is maintained, between 155 and 177 residues, and thus seems functionally important. In agreement with their role in G-protein coupling, the very N- and C-terminal ends of IL3 are relatively more conserved. In A2AA, both the N- and C-terminal ends are required for Gs coupling, whereas either one is sufficient for Gi coupling (Eason and Liggett 1996Citation ). The maintenance of two such Gi-coupling domains in one receptor is considered indicative of a strong evolutionary constraint. If A2AB also has redundant Gi-coupling domains, the greater conservation of the N- than the C-terminal end could reflect that they preferably couple different Gi proteins and therefore are under different evolutionary pressure to maintain optimal coupling.

In the hypervariable part of IL3, only very few residues and regions are conserved, notably E248, P335, positions ~346–~353, and the polyglutamate tract from position 299 to 319, shown in figure 1 . All sequenced A2ABs possess such an acidic domain, but the length—and thus net acidity—varies considerably between species (fig. 2 ). The longest sequences are found in various cetferungulates (tapir, horse, pangolin, alpaca). Length polymorphisms within a species also occur. In humans, a three-residue deletion has been found (Baldwin et al. 1999Citation ; Heinonen et al. 1999Citation ; Small et al. 2001Citation ; Snapir et al. 2001Citation ), which has a frequency of 0.31 in Caucasians and 0.12 in African-Americans (Small et al. 2001Citation ). Among the 44 A2AB sequences that we determined, there was one case of length heterozygosity in the acid domain; in alpaca, two alleles were found, of which one had an 18 bp tandem repeat, coding for the sequence AAEEEE (alpaca 1, in fig. 2 ). Also, the guinea pig sample that we sequenced differed from the entry in the database in having one less Glu in the repeat (guinea pig 1, in fig. 2 ), in addition to five other amino acids differences.



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Fig. 2.—Glutamic acid domain in IL3 of 48 mammalian A2AB sequences. To better reveal the length variation, alignment of the polyE tracts is not optimized. Codon usage for glutamic acid in the polyE domains is indicated by black for GAG and gray for GAA. At the bottom, the allelic variation in human, guinea pig, and alpaca is given at the DNA level. For full species names, see Materials and Methods. Pangolin as. and pangolin af. refer to pangolin with Asian (Manis sp.) and African (Manis tetradactyla) origin, respectively

 
Length variation of such poly–amino acid tracts is caused by unequal crossing-over and replication slippage, and is stabilized by point mutations (e.g., Smith 1976Citation ; Levinson and Gutman 1987Citation ; Jeffreys, Neil, and Neumann 1998Citation ; Alba, Santibanez-Koref, and Hancock 1999Citation ). Length variation by replication slippage is characterized by identical codon repeats, whereas mixed codon usage may indicate unequal crossover, replication slippage followed by point mutations, or a combinations of these processes. As seen in figure 2 , GAG (black) is the most frequently used Glu codon, but it is often interrupted by one or more GAA codons (gray) or codons for other amino acids. The latter are apparently under selective control, considering that next to E and D, almost exclusively A, V, and G seem to be allowed at positions 3–5 in the eutherian polyE tract. Whereas replication slippage is likely to occur in the GAG repeats, unequal crossing-over is more probably involved in the length polymorphisms in alpaca and humans (bottom, fig. 2 ).

Functional studies have shown that the acid domain is necessary for optimal agonist-promoted phosphorylation and the consequent desensitization of human A2AB (Jewell-Motz and Liggett 1995Citation ; Small et al. 2001Citation ). The shorter polyE allele in humans is associated with reduced metabolic rate in obese subjects and is a genetic risk factor for acute coronary events (Heinonen et al. 1999Citation ; Snapir et al. 2001Citation ). Also, in combination with a W64R polymorphism in the beta-3 adrenoceptor, an effect on fat mass is observed (Dionne et al. 2001Citation ). A full-length acid stretch is thus necessary for optimal receptor function of human A2AB. It has been suggested that the polyE tract provides the acid milieu that is required for the activity of the G protein–coupled receptor kinases (GRK) 2 and 3, which mediate the agonist-promoted phosphorylation of A2AB (Jewell-Motz and Liggett 1995Citation ). Such a functional role for acidic tracts might be more general because they are found in several other GPCRs (e.g., ACM1 and B1AR) and generally in their cytoplasmic domains. However, alignments of these GPCR sequences from different species (from http://www.gpcr.org/7tm/) reveal little length variations in their acid domains. Thus, in the case of A2AB, the conspicuous interspecies variation in length and acidity of the acid domain might possibly relate to the degree of phosphorylation needed in different species for desensitization and internalization of their A2AB receptors by way of beta-arrestin or reflect the conditions needed for optimal activity of GRKs in different species. A mutual adaptation between the level of desensitization needed for regulating activity, i.e., number of cell membrane receptors, and phosphorylation of different A2ABs can thus be envisaged (Daunt et al. 1997Citation ; DeGraff et al. 1999Citation ; Schramm and Limbird 1999Citation ).

IL3 is involved in microtubule-dependent sorting of A2AB (Saunders and Limbird 2000Citation ). The conservation of positions 346–353 may be important in this process to interact with proteins that bind to IL3, such as spinophilin and 14-3-3-{zeta} (Prezeau et al. 1999Citation ; Richman et al. 2001Citation ). Binding of arrestin to this domain is less likely because this is hyperphosphorylation dependent (Small et al. 2001Citation ), and there is only one phosphorylatable Thr in this domain. The conservation of E248 and P336 is difficult to explain. One might speculate that P336 allows IL3 to bend and bring the acid domain close to the serines and threonines that must be phosphorylated upon ligand binding and that E248 contributes to creating the proper acid milieu for GRK activity or is needed for phosphorylation of specific residues. Mutation studies are needed to confirm these speculations.

Evolutionary Rate of A2AB
Compared with other members of the adrenergic receptor family, A2AB seems to have one of the highest rates of evolutionary change. Whereas most adrenergic receptors, such as A1AB and B1AB, show identity scores around or above 90% between mouse-rat and humans (www.cmbi.kun.nl/7tm/), there is 82% identity in the case of A2AB. This is likely due to the great variability in the long IL3 in A2AB. Furthermore, the rate of NNS between mouse and human A2AB is 1.13 substitutions/site/billion years, which is in the higher range as compared with most other types of proteins (Graur and Li 2000Citation , pp. 102–103). Insulin, for example, has a NNS rate of 0.20, myoglobin of 0.57, and prion protein of 0.29.

The A2AB Gene Tree
Different evolutionary constraints may act on different parts of a protein as a consequence of interactions with other cellular components (for GPCRs, see Donnelly, Findlay, and Blundell 1994Citation ). If this were the case for A2AB, different domains of the protein might provide different trees, which would be interesting from a functional point of view but would hamper the phylogenetic inferences. We therefore performed separate analyses on the extra- and intracellular domains of A2AB, with and without exclusion of the variable regions of IL3, and on this variable region itself (see Materials and Methods). Bootstrapped phylogenetic analyses of these four, partitioned A2AB data sets revealed no significantly supported differences in tree topologies, indicating that any possible distinct evolutionary constraints acting on the different partitions were not apparent in this manner (data not shown). A2AB was thus taken to evolve as a single genetic unit, making it useful for analysis of species phylogeny.

Phylogenetic analyses of concatenations of many genes, including A2AB, have been necessary to achieve a well-supported resolution of the eutherian tree (Murphy et al. 2001bCitation ). It nevertheless remains important to assess individual gene trees; strongly supported deviations from the combined tree might reveal peculiar mutational features in certain evolutionary lineages of that particular gene or might indicate the inclusion of paralogs. We therefore performed phylogenetic analyses on A2AB, both unrooted (eutherians only) and rooted (with marsupial out-groups). Unrooted analyses may detect relationships that are less pronounced in rooted analyses because out-group taxa may influence in-group relationships due to long branch attraction and sequence saturation (see Scally et al. 2001Citation for further explanation). A rooted maximum likelihood tree is shown in figure 3 , and support values from various analyses are shown in table 2 . Although certain lineages appear to evolve faster than others (fig. 3 ; e.g., murid rodents, tenrecs), RASA analyses showed that these branches are not significantly longer than the others.



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Fig. 3.—Rooted maximum likelihood tree of eutherian A2AB sequences. The tree is based on first and second codon positions of a 41-taxon data set (-ln L = 6756.46) using the HKY85 model of sequence evolution, with transition to transversion ratio set to 1.46. See Materials and Methods for details. Branch lengths are proportional to the number of DNA changes, and the bar corresponds to 0.1 nucleotide change per site. Values in rectangles give average bootstrap-Bayesian support for nodes that are supported by more than 80% or 0.80, respectively, in all analyses described in table 2 . Other nodes, supported by 50% or more in half of the analyses, are denoted a to n, and support values are shown in table 2 . Nodes not supported by 50% or more in half of the analyses are marked with the average bootstrap-Bayesian support and the number of times that the node is supported (top and bottom values, respectively). Unmarked nodes are supported in less than half of the phylogenetic analyses. For species names, see Materials and Methods. Megabat is Cynopterus sphinx and microbat is Macrotus californicus. Pangolin as. and pangolin af. refer to pangolin with Asian (Manis sp.) and African (Manis tetradactyla) origin, respectively

 

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Table 2 Bootstrap and Bayesian Probability Support for Branches a to n in Figure 3

 
In no instance does the A2AB tree deviate significantly from the recently emerged eutherian tree (Madsen et al. 2001Citation ; Murphy et al. 2001a,Citation 2001bCitation ). It therefore seems that concerns of amplifying pseudogenes or paralogs by using an intronless gene from a large gene family are unfounded in the case of A2AB. Several nodes in the A2AB tree, however, are poorly resolved and supported. At the ordinal level, medium support is seen for Afrosoricida (average 73% in unrooted and 54% in rooted analyses), Rodentia (75%–72%), and Chiroptera (69%–68%), and weak support is seen for Primates (38%–38%) and Eulipotyphla (48%–49%). However, all analyses support rodent monophyly, varying from 54% in rooted unweighted parsimony to 1.00 in Bayesian probability analyses. This consistent support for monophyly is in line with all recent analyses of other nuclear genes (Huchon, Catzeflis, and Douzery 1999Citation ; Adkins et al. 2001Citation ; DeBry and Sagel 2001Citation ) and of combined nuclear and mitochondrial data sets (Madsen et al. 2001Citation ; Murphy et al. 2001a,Citation 2001bCitation ) but contradicts analyses on total mitochondrial proteins that still indicate rodent para- or polyphyly (Reyes, Pesole, and Saccone 2000Citation ; Janke et al. 2002Citation ).

The division of lipotyphlan insectivores into two orders, Eulipotyphla and Afrosoricida, is supported by A2AB. Eulipotyphla is found in 17 of the 20 analyses but with low confidence values (fig. 3 ). However, we never observed a basal position for Erinaceidae (hedgehog and gymnure), as is found for whole mitochondrial DNA analyses (Krettek, Gullberg, and Arnason 1995Citation ; Mouchaty et al. 2000Citation ); Erinaceidae always groups inside Laurasiatheria, far from the eutherian root (see below). Support for Afrosoricida (golden moles and tenrecs) is mixed (branch m, fig. 3 , table 2 ). Notably, there is an extreme difference in support between rooted and unrooted Bayesian probability analyses (0.01 and 0.99, respectively). This is caused by the placement of the root between the Malagasy tenrecs and continental African otter shrew in rooted Bayesian probability analyses (see below). Also, analyses which include unweighted third codon positions give low support for Afrosoricida. In most of these analyses, golden mole groups with aardvark due to third position transitions, whereas weighting as transversions-only restores Afrosoricida (data not shown). When compared with our previous analyses of A2AB (Stanhope et al. 1998Citation ), the increased taxon sampling of Afrosoricida has a positive influence on the support for this group. Within Afrosoricida, we observe a well-supported affiliation of otter shrew with the two Malagasy tenrecs (node n, mean support 94% and 80%). This confirms our prediction from sequence signatures that the otter shrew is a member of Afrotheria (van Dijk et al. 2001Citation ).

There is compelling molecular evidence, from nuclear and mitochondrial genes and from SINE elements, that whales are nested within Artiodactyla as sister to hippo's, and that ruminants are the sister group of the hippo-whale clade (e.g., Ursing and Arnason 1998Citation ; Gatesy et al. 1999Citation ; Nikaido, Rooney, and Okada 1999Citation ). For A2AB, the mean support for the hippo-whale clade is 71% and 71% (unrooted-rooted), whereas ruminants (bovine) cluster with hippo-whale, with average support of 64% and 61% (nodes b and c; table 2 ). The position of pigs (Suiformes) and alpacas (Tylopoda) within Cetartiodactyla is unresolved by A2AB.

Recent analyses in which A2AB was combined with other genes strongly supported the division of Eutheria into four major groups (Madsen et al. 2001Citation ; Murphy et al. 2001a,Citation 2001bCitation ). These four groups are also supported, albeit not conclusively, by A2AB itself: Afrotheria (branch k, mean support 94% and 50%), Laurasiatheria (branch g, mean 58% and 58%), Euarchontoglires (branch h, mean 55% and 57%), and Xenarthra (branch to sloth). In contrast to Laurasiatheria and Euarchontoglires, Afrotheria is strongly affected by rooting the tree, lowering the average support from 94% to 50%. Moreover, in the rooted analyses, the support ranges from 1% to 87%, caused by a tendency of the root to nest inside Afrotheria in some analyses (see table 2 ). The low support in rooted analyses, and the occasional rooting inside Afrotheria, indicates a basal position of Afrotheria in the A2AB tree, in accordance with the concatenated sequence evidence (Murphy et al. 2001bCitation ). It also illustrates the difficulty in establishing a stable root for the Eutherian tree in single gene analyses, an often neglected problem.

Joining of Laurasiatheria and Euarchontoglires in the Boreoeutheria (Springer and de Jong 2001Citation ) is reasonably well supported (branch j). Within the three superordinal clades, not much further resolution of orders is observed. The only relationship that is consistently supported with high bootstrap and probability values is Paenungulata within Afrotheria (node l, mean support 87% and 84%). Furthermore, it can be noted that most analyses support an aardvark-elephant shrew-Afrosoricida clade and rabbit as sister to tree shrew. The latter, however, is in contrast with the increasing molecular evidence for the grouping of rodents and lagomoprhs in Glires (e.g., Murphy et al. 2001aCitation ).

The inability of A2AB and other individual nuclear genes to robustly resolve various regions of the eutherian tree strongly advocates their concatenation in ever larger data sets. This may accumulate enough signal to ultimately resolve the remaining weakly supported and ambiguous relationships in the tree. Alternatively, one may hope that qualitative, rare genomic changes (Rokas and Holland 2000Citation ), like transposons and indels, may be helpful in this respect.

In conclusion, the comparison of A2AB sequences from a broad range of well-selected mammalian species has served a dual purpose. It has contributed—in combination with other genes—to the resolution of mammalian phylogeny and has additionally revealed the importance of sequence variation in the protein with respect to function and structure. Residues and regions involved in ligand binding and G-protein coupling are highly conserved, as are the major parts of the TM domains. Highest variation is found in the very long, third intracellular loop IL3, where only a few residues and a polyglutamyl domain are conserved. The presence of this polyglutamyl domain in all A2ABs, albeit with varying lengths, confirms its proposed role in agonist-dependent phosphorylation of IL3. Thus, comparing orthologous protein sequences from more mammalian species other than only humans, rat, and mouse is likely to give valuable additional information about important parts of such a protein.


    Acknowledgements
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Acknowledgements
 References
 
We thank Dr. Gert Vriend for critical reading of the manuscript, and Drs. François Catzeflis and Mark Springer for tissue and DNA samples. This work was supported by a TMR grant from the European Commission.


    Footnotes
 
Peer Bork, Reviewing Editor

1 Present address: Center for Genomics and Bioinformatics, Karolinska Institutet, Stockholm Back

Keywords: protein evolution GPCR A2AB mammalian phylogeny Back

Address for correspondence and reprints: Wilfried W. de Jong, Department of Biochemistry 161, University of Nijmegen, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands. E-mail: w.dejong{at}ncmls.kun.nl . Back


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Accepted for publication August 7, 2002.