Department of Integrative Biology, Pharmacology and Physiology, University of TexasHouston Medical School
Department of Ecology and Evolution, University of Chicago
Biology Department, University of MassachusettsAmherst
Department of Comparative Medicine, University of South Alabama
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Abstract |
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Introduction |
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Second, most nonprimate mammals and the bushbaby have a single-copy GH gene (Adkins, Nekrutenko, and Li 2001), whereas the human and the rhesus monkey possess five GH-like genes (Chen et al. 1989
; Golos et al. 1993
). Was the high rate of GH evolution in primates due to relaxation of purifying selection as a consequence of gene duplication?
Third, human GH has a high affinity for nonprimate GHRs, but nonprimate GHs have about 3,000-fold lower affinity for human GHR than does human GH (Souza et al. 1995
)! Correspondingly, nonprimate GHs fail to stimulate GHR, and consequently, growth, in the rhesus monkey (Carr and Friesen 1976
). This phenomenon is called "species-specificity" of human GHR; the term is imprecise because the specificity extends beyond the human species to apes and Old World monkeys. The species specificity of human GHR is primarily due to the Leu
Arg change at position 43 of GHR, leading to an unfavorable charge repulsion/steric hindrance between Arg43 of human GHR and His170 of nonprimate GHs (Souza et al. 1995
; Behncken et al. 1997
). In contrast, human GHR has a high affinity for human GH because the His
Asp change at position 171 (position 170 in nonprimate GHs) forms a favorable salt bridge with Arg43. For this reason, it has been hypothesized that the His171Asp change in GH must have preceded the Leu43Arg change in GHR (Souza et al. 1995
).
To test the above hypothesis, delineate the periods of rapid GH evolution in primates, and study the coevolution between GH and GHR, we sequenced GH and GHR genes from several prosimian and simian species. The GH and GHR sequences of the human (GenBank accession numbers J03071 and NM_000163) and the rhesus monkey (L16556 and U84589) had previously been obtained, while the bushbaby GH had been sequenced recently (Adkins, Nekrutenko, and Li 2001). Using the new and published data, we also investigated whether the rapid evolution of primate GH was due to positive selection (Wallis 1994, 1996
) and whether the emergence of human GHR species specificity was connected with periods of rapid evolution of GH.
The binding of GH to GHR has been very well characterized (Wells 1996
). GH binds to two GHR molecules sequentially, and this dimerization is necessary to activate signal transduction within the cell (Wells 1996
). X-ray crystallography (de Vos, Ultsch, and Kossiakoff 1992
) and alanine-scanning mutagenesis (Cunningham and Wells 1989
; Bass, Mulkerrin, and Wells 1991
; Clackson and Wells 1995
) demonstrate that only a relatively small subset of residues account for the majority of GH-GHR binding affinity (Wells 1996
). This information was used as an empirical basis for our evolutionary inferences of GH-GHR interaction.
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Materials and Methods |
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The sequence of the tarsier GH gene (except for the last 20 codons) was obtained by "touch-down PCR" (Don et al. 1991
) for the two species Philippine tarsier (Tarsius syrichta) and Western tarsier (Tarsius bancanus). Three primer sets (first5'-CCCAKCRCCTGCACCARCT-3' and 5'-AGKAGAGCAGCCCGTAGTTCTT-3', second5'-GCCCTGCTCTGCCTGCCC-3' and 5'-GCCACACCTTCGCTTGTGC-3', third5'-CCTCAGGCAGACCTACGACA-3' and 5'-ATGAYCCGCAG-GWACGTCTC-3') were used to amplify the GH gene in three overlapping parts. The PCR products were cloned and sequenced in both directions.
In addition to the above GH and GHR sequences, we amplified and sequenced exon 5 of the GH gene for the crowned lemur (Lemur mongoz coronatus), the red ruffed lemur (Varecia variegata rubra), the greater dwarf lemur (Cheirogaleus major), the spider monkey (Ateles chamek), the baboon (Papio cynocephalus), the Angolan colobus (Colobus angolensis), the orangutan (Pongo pygmaeus), and the chimpanzee (Pan troglodytes) using primers 5'-GCTGGAAGATGGCAGCCCCCGGACTG-3' and 5'-CTAGAAGCCACAGCTGCCCTCCACA-3'. This exon was selected because it contained the critical amino acid site 171 of GH. We also amplified and sequenced exon 4 of the GHR gene for the taxa mentioned above, as well as for the bushbaby (Galago moholi) and the Philippine tarsier, using primers 5'-TTCTTCTAAGGAGCCTAAATTCACCAAGTG-3' and 5'-ATAGAACAGCTGTATGGGTCCTAGGTTC-3'. This exon was selected because it contained critical amino acid residue 43. Polymerase chain reaction conditions used in this study are available on request.
Analysis of GH and GHR Gene Sequences
To test whether the His171Asp change in GH preceded the Leu43Arg change in GHR, we superimposed amino acids at position 171 of GH and amino acids at position 43 of GHR on the primate phylogeny inferred by Yoder et al. (1996)
and Goodman et al. (1998)
.
The hypotheses of the heterogeneity of the nonsynonymous/synonymous ( = dN/dS) rate ratio in GH and GHR among primate lineages were tested using the codonml program of the PAML software package (Yang 1997
). Several models were considered. All models incorporated the transition/transversion and the codon usage biases (F3x4, the nucleotide frequencies at the three codon positions were used to calculate codon frequencies). The one-ratio model assumes the same dN/dS ratio in the entire tree, and the free-ratio model assumes an independent dN/dS ratio for each branch. The two-ratio model assumes that a particular branch (or branches) has a dN/dS ratio (
1) different from the background ratio
0. The two-ratio model with a constraint assumes that the dN/dS ratio is 1 (
1 = 1) for a particular branch. The three hypotheses were tested by comparing different models. First, the likelihood values under the one-ratio and free-ratio models were compared to test whether the dN/dS ratio was heterogeneous among lineages. If the rate ratio was heterogeneous, then the maximum-likelihood estimates of parameters under the free-ratio model were used to calculate dN and dS along each branch. Second, the one-ratio and two-ratio models were compared to test whether a dN/dS ratio for a particular branch was significantly different from that for other branches. And third, the two-ratio models with and without the constraint
1 = 1 were compared to test whether a dN/dS ratio for a particular branch was significantly >1. When this was true, the evolution in this branch was considered to be under positive selection. The tree was user-defined. The significance of the difference between the two models was tested by computing 2
l = 2 (l1 - l0), where a more general model involves p parameters and has log likelihood l1, and a simpler model has q parameters with log likelihood l0, and by comparison with a
2 distribution with df = p - q. Additionally, a selection model with heterogeneous dN/dS ratios among sites was employed to identify sites under positive selection (model M2 from Yang et al. 2000
). For this case, the dN/dS ratio was assumed to be equal for all branches. Similar analyses were performed for GH and GHR. For the GH gene, 519 nucleotides (nt) encoding the 5' portion of the mature peptide (without the last 60 nt, as they were not available for tarsiers) were analyzed from the human (XM_008250), the rhesus monkey (L16556), the squirrel monkey (this study), the Philippine tarsier (this study), the Western tarsier (this study), the bushbaby (Adkins, Nekrutenko, and Li 2001), and the pig (X53325). The pig sequence was used as an outgroup. For the GHR gene, 1,860 nt encoding the mature peptide were analyzed for the human (X06562), the rhesus monkey (U84589), the baboon (AF150751), the squirrel monkey (this study), the pig (X54429), and the rabbit (AF015252). The pig and rabbit sequences were used as outgroups.
To test whether there were proportionally more amino acid replacements at functionally important sites than at the other sites in simian GHs and GHRs, a 2 x 2 contingency table with the numbers of changed sites and unchanged sites as rows and the numbers of functionally important sites and the numbers of other sites as columns was analyzed with the 2 test. A site was considered "changed" if there was an amino acid replacement in at least one of the simian sequences in comparison with the nonprimate consensus sequence. If the amino acid replacements occurred several times along the primate lineage at a particular site, they were all counted. Sites with indels were not taken into consideration.
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Results |
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Did GH Evolve by Positive Selection?
We now examine whether GH evolved under positive selection, using the nonsynonymous rate/synonymous rate ratio (dN/dS). By comparing the one-ratio and two-ratio models (table 2
, models A and C), we conclude that in the ancestral simian lineage (branch s), the dN/dS ratio (S = 0.354) is significantly elevated compared with the rates in the other lineages under study (
0 = 0.081), but it is still <1, providing no evidence for positive selection. The dN/dS ratio for the tarsier lineage (
= 0.081) estimated under the free-ratio model (fig. 2
) is lower than that for the entire tree (
0 = 0.112) estimated under the one-ratio model (table 2
).
A maximum-likelihood selection model with heterogeneous dN/dS ratios among sites did not identify any sites with dN/dS > 1 in GH. However, let us examine only amino acid changes at functionally important sites (table 3
). Among the 11 GH amino acids that form salt bridges and hydrogen bonds with GHR (de Vos, Ultsch, and Kossiakoff 1992
), none changed in the bushbaby, and only one (Gln46Arg) changed in the tarsier, but four (His19Arg, Arg41Lys, Lys167Arg, and His171Asp) changed in the simian lineages. Among the 24 GH amino acid sites that were shown to be important in GH-GHR interaction by alanine-scanning mutagenesis (Cunningham and Wells 1989, 1993
; table 3
), only 1 changed in the bushbaby and in the tarsier (site 48), but 11 changed once in the simian lineages (table 3
). In addition to binding to GHR, human GH binds to the prolactin receptor (PRLR) and is unique in its lactogenic activity (Chadwick, Folley, and Gemzell 1961
). Among the 19 GH amino acids that are involved in GH-PRLR binding (Somers et al. 1994
) or important for GH-PRLR interaction as shown by alanine-scanning mutagenesis (Cunningham and Wells 1991
), none changed in the bushbaby lineage, and only 1 changed in the tarsier lineages, but 10 changed in the simian lineages (table 3
). Altogether, among the functionally important sites (table 3
), 1 change occurred in the bushbaby, and only 2 out of the 26 sites studied in tarsier GH changed, whereas 20 changes among the 34 sites (59%) studied have occurred in squirrel monkey, human, or rhesus monkey GH. In contrast, among the 155 other sites in simian GHs, only 58 changes have occurred (37%). Thus, there were proportionally more amino acid changes at functionally important sites than at the other sites in simian GHs, suggesting a role of positive selection in the evolution of these sites. The difference is statistically significant (
2 = 5.27, P < 0.05). In branch s at these sites, dN is 0.348 (data not shown), twice as high as dN for the GH mature peptide (0.162; fig. 2a
), but still lower than dS for the mature peptide (0.451; fig. 2b
), providing no evidence of positive selection at these sites. The large number of amino acid changes at the functionally important sites of GH in the simian lineage is particularly striking when compared with the apparent lack of changes at these sites in the other mammals.
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Discussion |
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Although it was known that human and rhesus monkey GHs evolved rapidly (Wallis 1994, 1996
), our data indicate that the rapid evolution occurred before the NW-OW monkey split. As the rapid evolution predates the emergence of the species specificity of human (OW monkey) GHR, it is not a consequence of the latter event. Nor is it the cause of the latter, because most of the amino acid replacements in primate GH occurred before the NW-OW monkey split, which was at least 10 Myr before the evolution of the species specificity.
Our data enabled us to show that the majority of the amino acid substitutions found previously between human GH and nonprimate GHs occurred in a short period, i.e., before the radiation of higher primates but after the separation of the higher primate lineage from the tarsier lineage (branch s in fig. 2a
). The period in which the majority of amino acid replacements occurred might have been even shorter than the time span of branch s, but unfortunately, there are no extant primate species that allow such an analysis to be made. However, it is clear that the evolution of GH in primates was episodic, revealing a low rate in most prosimians and an extremely high rate in the common ancestor of higher primates, but subsequently a much lower rate in extant higher primates. Even if 58 Myr is an underestimate for the tarsier-simian divergence, and the time between the tarsier-simian split and the NW-OW monkey split was actually longer than 18 Myr (as suggested by fig. 4 in Goodman et al. 1998
), the rates of GH evolution in the common ancestor of simians are still several-fold higher than the average rates for mammalian proteins (Li 1997
).
The fact that the rate of evolution is especially high in functionally important sites suggests that some of the amino acid changes during the period of GH rapid evolution might have been due to positive Darwinian selection. However, the cause or biological basis for the rapid evolution is unclear. One speculation is that primate GH might have once played a dual role; i.e., in addition to growth promotion, it might have played a role in maintaining the nutritional balance between mother and dependent young through its lactogenic action (Wallis 1997
). When the second role was assumed by duplicate GH genes that are expressed in the placenta, the evolution of the pituitary GH went back to a conservative mode, as seen in extant higher primates. How well this alleged dual role can explain the rapid evolution remains to be seen.
Other factors instead of or in addition to positive selection may have played a role in the overall rapid evolution of GH in simians. One possibility is relaxation of purifying selection as a consequence of multiple duplications of the GH locus. Our preliminary data showed multiple copies of the GH gene in the tarsier and the squirrel monkey. This suggests that the GH gene duplication occurred before the OW-NW monkey split, probably beginning in the common ancestor of tarsiers and simians, and this might have led to relaxation of purifying selection. Wallis (1996)
has argued against the possibility of relaxation of purifying selection because similar amino acid changes were found in duplicate genes in the human GH gene cluster. However, the similarity in amino acids in duplicate genes could be the result of gene conversion. Nucleotide sequences of GH genes from tarsiers and New World monkeys may clarify this issue.
The presence of multiple copies of the GH gene may have potentially complicated our analysis. However, the presented GH sequences for the bushbaby and the squirrel monkey should be the authentic growth hormone for the following reasons. First, our previous experiment showed that there was only one copy of GH in the bushbaby (Adkins, Nekrutenko, and Li 2001). Second, squirrel monkey GH sequence was obtained from mRNAs isolated from the pituitary gland, where no other GH-like genes are expressed. We are less certain if we are dealing with the authentic growth hormone in the case of tarsiers. As we did not have fresh tarsier pituitary tissue to isolate RNA, amplification from genomic DNA was our method of choice. PCR with the primers based on the primate consensus sequence yielded only one product. We are currently screening the tarsier genomic library to investigate potential GH gene duplications.
An accelerated rate of GH evolution in the common ancestor of the NW and OW monkeys (branch s) corresponds to an accelerated rate of GHR evolution (branch p), although GHR sequences of prosimians are not available. This suggests the coevolution between GH and GHR, but the complete GHR sequences of the bushbaby and the tarsier are needed to directly correlate the evolution of GH and GHR in the common ancestor of simians. In addition, simian GHR, similar to simian GH, has proportionally more amino acid changes at the functionally important sites than at the other sites compared with the nonprimate consensus. This and the fact that in GHR the dN/dS ratio is approaching 1 in the common ancestor of primates and is >1 (although not significantly) in the human lineage suggest that some of the sites in GHR might have been under positive selection. However, changes of both GH and GHR amino acids at the same intermolecular contacts happened very rarelyonly at one contact in the human and the macaque (or the baboon) and at two contacts in the squirrel monkey. For both GH and GHR, a selection model with heterogeneous dN/dS rate ratios (Yang et al. 2000
) failed to identify sites under positive selection. This is because models that assume heterogeneous rates among sites but the same dN/dS ratio for all branches have a low power when sites are under positive selection only along particular lineages (Yang et al. 2000
). Models that allow the dN/dS rate ratio to vary among sites and lineages are currently unavailable.
In conclusion, the dN/dS ratio was found to be <1 for the GH gene and for the functionally important sites of the gene, not giving support for positive selection. However, some of the amino acid replacements at functionally important sites in simian GH might have been adaptive, as suggested by the high rate of evolution at these sites and a correlation in the overall evolutionary tempo of GH and GHR. Other factors, such as relaxation of purifying selection, also might have been responsible for the rapid GH evolution, because there have been multiple GH gene duplications in primates and there was no correlation between GH and GHR evolution at contact sites.
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Acknowledgements |
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Footnotes |
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1 These authors contributed equally to this work.
1 Abbreviations: GH, growth hormone; GHR, growth hormone receptor.
2 Keywords: growth hormone
growth hormone receptor
rapid evolution
coevolution
species specificity
3 Address for correspondence and reprints: Wen-Hsiung Li, Department of Ecology and Evolution, University of Chicago, 1101 East 57th Street, Chicago, Illinois 60637. whli{at}uchicago.edu
.
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