Episodic Evolution of Growth Hormone in Primates and Emergence of the Species Specificity of Human Growth Hormone Receptor

Jaw-Ching Liu, Kateryna D. Makova, Ronald M. Adkins, Susan Gibson and Wen-Hsiung Li

Department of Integrative Biology, Pharmacology and Physiology, University of Texas–Houston Medical School
Department of Ecology and Evolution, University of Chicago
Biology Department, University of Massachusetts–Amherst
Department of Comparative Medicine, University of South Alabama


    Abstract
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 literature cited
 
Growth hormone (GH) evolution is very conservative among mammals, except for primates and ruminant artiodactyls. In fact, most known mammalian GH sequences differ from the inferred ancestral mammalian sequence by only a few amino acids. In contrast, the human GH sequence differs from the inferred ancestral sequence by 59 amino acids. However, it is not known when this rapid evolution of GH occurred during primate evolution or whether it was due to positive selection. Also, human growth hormone receptor (GHR) displays species specificity; i.e., it can interact only with human (or rhesus monkey) GH, not with nonprimate GHs. The species specificity of human GHR is largely due to the Leu->Arg change at position 43, and it has been hypothesized that this change must have been preceded by the His->Asp change at position 171 of GH. Is this hypothesis true? And when did these changes occur? To address the above issues, we sequenced GH and GHR genes in prosimians and simians. Our data supported the above hypothesis and revealed that the species specificity of human GHR actually emerged in the common ancestor of Old World primates, but the transitional phase still persists in New World monkeys. Our data showed that the rapid evolution of primate GH occurred during a relatively short period (in the common ancestor of higher primates) and that the rate of change was especially high at functionally important sites, suggesting positive selection. However, the nonsynonymous rate/synonymous rate ratio at these sites was <1, so relaxation of purifying selection might have played a role in the rapid evolution of the GH gene in simians, possibly as a result of multiple gene duplications. Similar to GH, GHR displayed an accelerated rate of evolution in primates. Our data revealed proportionally more amino acid replacements at the functionally important sites in both GH and GHR in simians but, surprisingly, showed few coincidental replacements of amino acids forming the same intermolecular contacts between the two proteins.


    Introduction
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 literature cited
 
Human and rhesus monkey growth hormone (GH) and growth hormone receptor (GHR) differ from their nonprimate homologs in several significant ways. First, most nonprimate GHs differ from each other by only 0–4 amino acids in the mature peptide (190 amino acids long), but incredibly, human (or rhesus monkey) and nonprimate GHs differ by 59–63 amino acids (i.e., a difference of ~33%). This high rate of evolution in primate GH is rarely observed in mammalian proteins (Ohta 1993Citation ; Li 1997Citation ). However, the rate of GH evolution in the bushbaby (a prosimian) was similar to those in nonprimates (Adkins, Nekrutenko, and Li 2001). These observations raise the questions of (1) whether the rate increase was a persistent phenomenon during the evolution from prosimians to the common ancestor of the Old World primates or whether most of the replacements occurred during a short period of time and (2) whether the rapid evolution was due to positive selection (Wallis 1994Citation ). Interestingly, the proximal promoter region of the GH gene also evolved rapidly in Old World primates (Krawczak, Chuzhanova, and Cooper 1999Citation ); it has not been studied in other primates.

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. 1989Citation ; Golos et al. 1993Citation ). 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. 1995Citation )! Correspondingly, nonprimate GHs fail to stimulate GHR, and consequently, growth, in the rhesus monkey (Carr and Friesen 1976Citation ). 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. 1995Citation ; Behncken et al. 1997Citation ). 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. 1995Citation ).

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, 1996Citation ) 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 1996Citation ). GH binds to two GHR molecules sequentially, and this dimerization is necessary to activate signal transduction within the cell (Wells 1996Citation ). X-ray crystallography (de Vos, Ultsch, and Kossiakoff 1992Citation ) and alanine-scanning mutagenesis (Cunningham and Wells 1989Citation ; Bass, Mulkerrin, and Wells 1991Citation ; Clackson and Wells 1995Citation ) demonstrate that only a relatively small subset of residues account for the majority of GH-GHR binding affinity (Wells 1996Citation ). This information was used as an empirical basis for our evolutionary inferences of GH-GHR interaction.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 literature cited
 
Sequencing of the GH and GHR Genes
Squirrel monkey (Saimiri boliviensis) mRNA was isolated from pituitary gland and from liver; the GH gene is expressed only in the pituitary gland, while the GHR gene is expressed in the liver and many other tissues. The mRNA was purified using the MessengerMaker mRNA isolation system (Life Technologies). The mRNA was used for cDNA synthesis with Superscript Reverse Trancriptase (Life Technologies). The resulting cDNAs served as templates for polymerase chain reaction (PCR) with a GH-specific (5'-A T G G C T A C A G G C T C C C G G A C G T C C C T G C T C C -3') or GHR-specific (5'-A T G G A T C T C T G G C A G C T G C T G T T G A C C T T G G-3') primer and poly-dT. The PCR products were cloned and sequenced from several clones.

The sequence of the tarsier GH gene (except for the last 20 codons) was obtained by "touch-down PCR" (Don et al. 1991Citation ) for the two species Philippine tarsier (Tarsius syrichta) and Western tarsier (Tarsius bancanus). Three primer sets (first—5'-CCCAKCRCCTGCACCARCT-3' and 5'-AGKAGAGCAGCCCGTAGTTCTT-3', second—5'-GCCCTGCTCTGCCTGCCC-3' and 5'-GCCACACCTTCGCTTGTGC-3', third—5'-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)Citation and Goodman et al. (1998)Citation .

The hypotheses of the heterogeneity of the nonsynonymous/synonymous ({omega} = dN/dS) rate ratio in GH and GHR among primate lineages were tested using the codonml program of the PAML software package (Yang 1997Citation ). 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 ({omega}1) different from the background ratio {omega}0. The two-ratio model with a constraint assumes that the dN/dS ratio is 1 ({omega}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 {omega}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{Delta}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 {chi}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. 2000Citation ). 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 {chi}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.


    Results
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 literature cited
 
GH and GHR Sequences
The nucleotide sequence data generated in this study are summarized in table 1 . We obtained the complete coding sequences of squirrel monkey GH and GHR genes, the genomic sequences of the GH gene of the two tarsier species (except for the last 20 codons), the sequences of GH exon 5 from three prosimians and five simians, and the sequences of GHR exon 4 from five prosimian and five simian species.


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Table 1 GH and GHR Genes Sequenced

 
Emergence of Human GHR Species Specificity
Our sequence data of GH genes from three lemurs and two tarsiers revealed His at position 171 of GH, the same as in nonprimate and bushbaby GHs (fig. 1 ). In contrast, our sequence data of GH genes from two New World (NW) monkeys, two Old World (OW) monkeys, and two apes revealed Asp at position 171 of GH, the same as in rhesus monkey and human GHs (fig. 1 ). These data clearly indicated that the His171Asp replacement in GH occurred in the common ancestor of simians (fig. 1 ). Our sequence data from GHR genes showed that the amino acid at position 43 of GHR was Leu in the six prosimians and two NW monkeys studied, the same as in nonprimate GHRs, but it was Arg in the three OW monkeys and two apes studied, the same as in human GHR (fig. 1 ). Thus, the Leu43Arg replacement in GHR occurred in the common ancestor of OW monkeys, apes, and humans (fig. 1 ). As this common ancestor is about 15 Myr younger than the common ancestor of simians (Goodman et al. 1998Citation ), the His171Asp change in GH obviously preceded the Leu43Arg change in GHR.



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Fig. 1.—Amino acid replacements at position 171 of GH and position 43 of GHR. The schematic phylogeny of primates is according to Goodman et al. (1998)Citation and Yoder et al. (1996)Citation and is not drawn according to scale. The "human-like" replacements are shown in bold. NWM = New World monkeys; OWM = Old World monkeys

 
Periods of Rapid GH Evolution in Primates
We first applied likelihood tests to examine whether the rate of evolution of the GH gene was uniform among primate lineages (human, rhesus monkey, squirrel monkey, Philippine tarsier, Western tarsier, and bushbaby). As pig GH has been shown to be virtually the same as the ancestral sequence of eutherian GHs (Wallis 1994Citation ), we used it as an outgroup. The free-ratio model, which assumed an independent dN/dS ratio for each branch, fitted the data significantly better than the one-ratio model, which assumes the same dN/dS ratio ({omega}0) for all branches (table 2 ). This indicated that the dN/dS ratio was indeed different among primate lineages, so we used the maximum-likelihood estimates of parameters under the free-ratio model to calculate the rates of synonymous and nonsynonymous substitutions for each branch (fig. 2 ). Figure 2a shows that few nonsynonymous changes (in fact, only 1 amino acid change) have occurred between the pig and the common ancestor of primates. Bushbaby GH has evolved slowly, although faster than pig GH. Rapid evolution started only after the divergence of the tarsier and simian lineages. The highest rate occurred in branch s, which connects the common ancestor of the simian and tarsier lineages and the common ancestor of simians (fig. 2a ). The average nonsynonymous rate in the two tarsier lineages was significantly (sixfold) faster than that in the bushbaby lineage, and the average nonsynonymous rate in the simian lineages was, in turn, significantly (2.8-fold) faster than that in the two tarsier lineages (fig. 2a ). The synonymous rate also showed a similar pattern of acceleration, but to a lesser extent (fig. 2b ). This finding supports a correlation between nonsynonymous and synonymous rates (Graur 1985Citation ).


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Table 2 Maximum-Likelihood Ratio Statistics for the GH Data

 


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Fig. 2.—Rates of nucleotide substitutions in the GH gene estimated under the free-ratio model. a, The number of nonsynonymous substitutions per site (dN) is shown above each branch. The dN/dS rate ratios along each branch are given in parentheses. b, The number of synonymous substitutions per site (dS) is shown above each branch. s indicates the branch of the simian common ancestor. There were, in total, 94 synonymous and 425 nonsynonymous sites

 
The absolute substitution rates in the branches with rapid evolution can be calculated from divergence times. The tarsiers and simians diverged about 58 MYA, and the NW and OW monkeys split about 18 Myr later (Goodman et al. 1998Citation ). Dividing the numbers of nonsynonymous and synonymous substitutions by divergence times leads to 1.0 x 10-9 and 9.0 x 10-9 substitutions per nonsynonymous site per year in the tarsier lineage and in branch s (fig. 2 ), respectively, and to the corresponding synonymous rates of 12.1 x 10-9 and 25.1 x 10-9. These rates are high in comparison with the average synonymous (3.5 x 10-9) and nonsynonymous (0.74 x 10-9) rates in mammals (Li 1997Citation ). In particular, the synonymous and nonsynonymous rates in branch s are 7 and 12 times as high as the respective average rates in mammals!

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 ({omega}S = 0.354) is significantly elevated compared with the rates in the other lineages under study ({omega}0 = 0.081), but it is still <1, providing no evidence for positive selection. The dN/dS ratio for the tarsier lineage ({omega} = 0.081) estimated under the free-ratio model (fig. 2 ) is lower than that for the entire tree ({omega}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 1992Citation ), 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, 1993Citation ; 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 1961Citation ). Among the 19 GH amino acids that are involved in GH-PRLR binding (Somers et al. 1994Citation ) or important for GH-PRLR interaction as shown by alanine-scanning mutagenesis (Cunningham and Wells 1991Citation ), 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 ({chi}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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Table 3 Divergence of GH at Functionally Important Sites

 
Evolution of GHR
Next, we examine (1) whether the synonymous and nonsynonymous substitution rates were uniform in the primate GHR, by comparing human, rhesus monkey, baboon, and squirrel monkey sequences (pig and rabbit GHR sequences were used as outgroups), and (2) whether GHR evolved under positive selection in the common ancestor of higher primates. Similar to GH, for GHR the free-ratio model fitted the data significantly better than the one-ratio model (table 4 ). This suggests different dN/dS rate ratios among branches, so we calculated the synonymous and nonsynonymous rates using the parameters estimated under the free-ratio model (fig. 3 ). The ancestral branch of higher primates (branch p) for the GHR data exhibited an accelerated nonsynonymous substitution rate (fig. 3a ). This was similar to an accelerated nonsynonymous rate in branch s in GH (fig. 2a ). However, while the difference in the GH nonsynonymous rates between the ancestral simian and the tarsier or the bushbaby lineages was several-fold, the GHR nonsynonymous rate in branch p was only 68% and 20% higher than those in the pig and rabbit lineages, respectively. The dN/dS ratios estimated under the free-ratio model were elevated for most of the primate branches in figure 3 (except for the baboon lineage) and were especially high for the ancestral primate branch ({omega}P = 0.912) and for the human branch (branch h, {omega}H = 1.268), as compared with the average dN/dS ratio among branches estimated under the one-ratio model ({omega}0 = 0.341; table 4 ). In fact, the comparison of each of the two two-ratio models (table 4 , models C and D) and the one-ratio model (model A) indicated that the dN/dS ratios were significantly higher for the human and ancestral primate branches than for other branches. Under the free-ratio model, the dN/dS ratio in the human branch was >1; however, the difference between the two-ratio model (model C) and the two-ratio model with the constraint of {omega}H = 1 (model E) was not significant (table 4 ). This suggests that the evolution of the GHR gene in the human branch is not significantly different from the neutral expectation. It should be noted that our conclusions from the analysis of the GHR data held even when we employed Bonferroni's correction for multiple tests. However, the null hypotheses about the human branch were not formulated prior to the analysis, and thus, strictly speaking, this might have increased the probability of rejecting the null hypothesis in the comparison between models C and A (table 4 ).


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Table 4 Maximum-Likelihood Ratio Statistics for the GHR Data

 


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Fig. 3.—Rates of nucleotide substitutions in the GHR gene estimated under the free-ratio model. a, The number of nonsynonymous substitutions per site (dN) is shown above each branch. The dN/dS rate ratios along each branch are given in parentheses. b, The number of synonymous substitutions per site (dS) is shown above each branch. p and h indicate the branches of the primate common ancestor and of the human lineage, respectively. There were, in total, 523 synonymous and 1,337 nonsynonymous sites

 
Similar to GH, a maximum-likelihood selection model with heterogeneous dN/dS ratios among sites did not identify any sites with dN/dS > 1 in GHR. Interestingly, however, a high rate of evolution was found at functionally important sites in GHR in simians. By comparing GHR sequences from the squirrel monkey, the rhesus monkey, the baboon, and the human with the nonprimate consensus sequence at 24 functionally important sites (Bass, Mulkerrin, and Wells 1991Citation ; de Vos, Ultsch, and Kossiakoff 1992Citation ; Clackson and Wells 1995Citation ; Wells 1996Citation ), we identified 10 amino acid changes in simians (table 5 ). This is in contrast to 50 amino acid changes at 222 other sites of the extracellular domain of GHR. Thus, similar to GH, simian GHR has undergone proportionally more changes at functionally important sites than at other sites; the difference was again statistically significant ({chi}2 = 4.30, P < 0.05). In contrast, there were no changes at these functionally important sites in the outgroup pig and rabbit sequences.


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Table 5 Divergence at GHR Functional Sites

 
Let us examine in more detail contact sites between GH and GHR. How often were both a GH site and a corresponding GHR site replaced in simians compared with the nonprimate consensus (table 3 )? Of 13 intermolecular contacts (de Vos, Ultsch, and Kossiakoff 1992Citation ), only one has been changed at both GH and GHR in the human and the macaque. This is an interaction between sites 171 (GH) and 43 (GHR) that has been discussed before. In the squirrel monkey, two intermolecular contacts have been simultaneously changed in both GH and GHR: 41 (GH)–127 (GHR) and 167 (GH)–127 (GHR); amino acid 127 in GHR interacts with two GH amino acids (at sites 41 and 167). Thus, the majority of the changes at functionally important sites in simian GH are not at the sites that interact with the changed sites in simian GHR.


    Discussion
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 literature cited
 
Our data from prosimians and simians support the hypothesis that the His171Asp change in GH preceded the Leu43Arg change in GHR, which confers species specificity on human GHR. It is tempting to think that the evolution of the species specificity of human GHR, i.e., the Leu43Arg change, was adaptive. However, this change has not occurred in NW monkeys, although the His171Asp change in GH had already occurred in the common ancestor of simians (fig. 1 ). That is, the transitional phase still persists in NW monkeys, despite the divergence of ~40 Myr between the NW and OW monkey lineages. This finding suggests that there is no advantage for Leu43 to change to Arg in NW monkey GHR, although we cannot exclude the possibility that the change in the common ancestor of the OW primates was adaptive. It will be interesting to investigate the binding affinities between GH and GHR of NW monkeys.

Although it was known that human and rhesus monkey GHs evolved rapidly (Wallis 1994, 1996Citation ), 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. 1998Citation ), the rates of GH evolution in the common ancestor of simians are still several-fold higher than the average rates for mammalian proteins (Li 1997Citation ).

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 1997Citation ). 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)Citation 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 rarely—only 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. 2000Citation ) 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. 2000Citation ). 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.


    Acknowledgements
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 literature cited
 
This study was supported by NIH grants HD38287, GM55759, and P40 RR01254. We thank two anonymous reviewers for suggestions.


    Footnotes
 
Naruya Saitou, Reviewing Editor

1 These authors contributed equally to this work. Back

1 Abbreviations: GH, growth hormone; GHR, growth hormone receptor. Back

2 Keywords: growth hormone growth hormone receptor rapid evolution coevolution species specificity Back

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 . Back


    literature cited
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 literature cited
 

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Accepted for publication January 30, 2001.