*Department of Ecology and Evolution
Department of Biochemistry and Molecular Biology, University of Chicago
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Abstract |
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Introduction |
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The GH and the GHR also provide an opportunity to understand the coevolution of two proteins. Wallis (1994)
compiled the evolutionary rates of GHs from various mammalian taxa to show that, in general, GH evolves at a slow basal rate, but significant acceleration in evolutionary rate occurred in two lineages, one leading to the primates (human and Rhesus monkey) and the other to the artiodactyls (Wallis 1994
). The acceleration seen in the primate lineage is exceptionally high (Li 1997
). It has been proposed that positive selection may be a driving force for the observed acceleration (Wallis 1996
; Liu et al. 2001
; Wallis, Zhang, and Wallis 2001
). The acceleration was particularly conspicuous at "functionally important" sites (Liu et al. 2001
), which were inferred from functional epitope mapping of the hGH·(hGHR-ECD)2 complex (Cunningham and Wells 1989
, 1991
, 1993
), as well as the tertiary structure of the hGH and human prolactin receptor complex (Somers et al. 1994
).
Among these functionally important sites, one pair of residues is of particular interest with regard to the origin of species specificity of the hGHR. In general, protein hormones from nonprimates are active in humans. However, human and Rhesus monkey GHRs are unresponsive to GHs from nonprimate mammals (Carr and Friesen 1976
; Peterson and Brooks 2000
). In contrast, the hGH is fully functional when administered to other mammalian species. This pattern of high specificity and cross-reactivity is not well understood at either the biochemical or the evolutionary level. On the basis of the sequence comparisons, Souza et al. (1995)
proposed that the interaction between residue 171 of the GH (GH171Asp in human, which is equivalent to GH170His in nonprimates) and residue 43 of the GHR (GHR43Arg in human and GHR43Leu in nonprimates) is the major determinant of the hGHR species specificity. At the hGH:hGHR site 1 interface, GH171Asp and GHR43Arg are known to form a strong salt bridge and are, therefore, assumed to be essential for the formation of the hormone-receptor complex (De Vos, Ultsch, and Kossiakoff 1992
; see fig. 1
). Among the residues that are known to form favorable salt bridges and hydrogen bonds at the site 1 interface, these two are the only pair of structurally complementary residues that differ at both positions between primates and nonprimates (see table 1
).
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In this study we investigated the range of binding affinities of squirrel monkey growth hormone receptor (smGHR) by conducting in vitro binding assays between the squirrel monkey growth hormone receptor ECD (smGHR-ECD) and the GHs from human, squirrel monkey, and rat (a nonprimate outgroup). The surface plasmon resonance (SPR) measurement technique was used to measure the kinetic rate constants and the corresponding binding affinities. Our study suggests that the smGHR-ECD does represent an intermediary form of species specificity. To further test whether this is caused by the compatible interaction between GH171 (GH170 in nonprimates) and GHR43, we engineered and produced human and squirrel monkey GHR-ECDs with converse amino acids at residue 43. By this, we were able to alter smGHR-ECD to acquire species specificity, whereas hGHR-ECD with Leu at position 43 gained an affinity toward rat GH, thereby losing its species specificity. In contrast to these significant effects of mutations at residue 43 of primate GHRs on the interactions with the nonprimate GH, the same mutations have very little effect on the binding affinities toward primate GHs. This demonstrates an example of a single mutation in a protein hormone that has very different functional consequences on its interactions with receptors from distinct phylogenetic groups.
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Materials and Methods |
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Expression and Purification of Periplasmic Proteins
BL21 cells were transformed with each expression plasmid. From the resulting plates, a single colony was picked and inoculated in the Luria-Bertani media containing 60 µg/ml ampicillin in 1/100th volume of the final culture. The cells were grown for 4 h at 37°C, then washed with and transferred into a larger volume of low-phosphate AP-5 media to induce the alkaline phosphatase promoterdirected transcription of the recombinant proteins (Fuh et al. 1990
). The optimized growth conditions for all the GHR-ECD variants are at 20°C for 28 h, with shaking at 225 rpm. Expression plasmid for smGH was grown at 32°C for 11 h. Cells were then harvested to be frozen at -80°C. Periplasmic proteins were extracted through repeated freezing-thawing steps in 1/100 volume of the original culture of 10 mM Tris-Cl (pH 8.0). Whether the protein of expected size was present or not in the periplasmic protein fraction was checked from this stage by SDS polyacrylamide gel electrophoresis (PAGE) or by Western blotting (or both), using the anti-hGH (catalog number E45600M, BioDesign International, Kennebunk, Me.) for GHs and MAb against hGHR (product number 7005B, American Diagnostica Inc., Greenwich, Conn.). The supernatant was then ammonium sulfate precipitated to be resuspended into 3 ml of 10 mM Tris-HCl, pH 8.0, buffer and then further processed according to the different purification procedures discussed subsequently.
Fast Protein Liquid Chromatography Purification of hGH
The ammonium sulfateprecipitated periplasmic protein fraction containing hGH was desalted on a PD-10 column (Amersham Pharmacia Biotechnology, Piscataway, NJ) and applied onto a Resource Q column (Amersham Pharmacia Biotechnology) equilibrated with 10 mM Tris-Cl (pH 8.0). The hormone was then eluted by a 0- to 300-mM NaCl gradient using a Fast Protein Liquid Chromatography apparatus (Varian Prostar model 210). The resulting fractions were concentrated on a Centricon 10k filtering unit (Amicon) and analyzed on SDS-PAGE, reverse-phase HPLC (Shimadzu LC10AD VP system with a Vydac Protein C4 column), and mass spectrometry (PE Sciex API 150Ex). The concentrations of all samples were determined by absorbance at 280 nm. Extinction coefficients were derived from the tryptophan and tyrosine content (for example, 280 hGH = 16,200/[M cm],
280 hGHR-ECD =55,000/[M cm]). The recombinant rat GH used in this study was obtained from the National Hormone and Peptide Program (by courtesy of Dr. A. F. Parlow, http://www.humc.edu/hormones).
Affinity Chromatography for the Isolations of GHR-ECDs
Each ammonium sulfateprecipitated periplasmic protein fraction containing GHR-ECD was applied to a hGH affinity column (Fuh et al. 1990
), which was washed and equilibrated in 20 mM Tris-HCl, pH 8.0. The 2-ml column was then washed with the same buffer containing 10 mM MgCl2 and finally with 1.0 M NaCl. The specifically bound proteins were then eluted with 2.5 ml 4.5 M MgCl2. The eluted proteins were immediately desalted on a PD-10 column (Amersham Pharmacia Biotechnology) in 10 mM sodium phosphate buffer, pH 7.4, and then further analyzed as described above.
Affinity Chromatography for smGH Isolation
To purify smGH, an smGHR-ECD affinity column was constructed. First, 5 mg of smGHR-ECD237Cys from a 10-liter culture was purified using an hGH affinity column. Because this GHR-ECD contained 237Cys mutation, it was glutathione modified on the unpaired cysteine by E. coli. After removing the glutathione modification to free the thiol residue (see Biosensor Surface Preparation for the detailed protocol), the purified smGHR-ECD was biotinylated using the EZ-LinkTM PEO-Maleimideactivated biotin (Pierce, Rockford, Ill.) and then coupled to immobilized streptavidincontaining beads (UltraLinkTM immobilized streptavidin plus, Pierce). We were able to obtain >95% pure smGH using this column. Preliminary binding experiments with hGHR-ECD using the pure smGH obtained from this column led to an observation that the smGH binds to hGHR-ECD with high affinity. After this, we constructed a higher-capacity hGHR-ECD affinity column for the purification of a larger amount of smGH, using >15 mg of hGHR-ECD immobilized through random amine coupling using CNBr-activated Sepharose beads (Amersham Pharmacia Biotechnology). The smGH was then purified following the same affinity column purification steps described earlier and desalted in PBS (pH 7.4).
Preparation of GHR-ECDs for Immobilization
As mentioned earlier, a point mutation Ser237Cys was introduced in the receptor. The Ser237Cys mutation does not affect hormone binding and allowed us to immobilize the GHR-ECDs in a homogeneously oriented manner, using a thiol-disulfide exchange coupling method. In all cases, the extra unpaired cysteine residue on the receptor introduced by this mutation became covalently modified in E. coli by glutathione. This modification was removed by a 30-min treatment in 1 mM dithiothreitol (DTT) on ice for the Ser237Cys mutants of hGHR-ECD and hGHR-ECD43Leu. The Ser237Cys-containing smGHR-ECD and smGHR-ECD43Arg were treated in a milder reducing reagent, 7.5 mM ß-mercapthoethanolamine for 15 min at 37°C in the presence of 50 mM EDTA.
Biosensor Surface Preparation
BIAcore Pioneer C-1 sensor chips were used for the analyses. Reagents for this, N-ethyl-N'-(s-dimethyl-aminopropyl)-carbodiimide hydrochloride (EDC), N-hydrosuccinimide (NHS), and 2-(2-pyridinyldithio) ethaneamine hydrochloride (PDEA), were purchased from Biacore (Uppsala, Sweden). First, the surface of the Pioneer C-1 chip was treated with 15 µl of 100 mM Na-glycinate and 0.3% Triton-X solution (pH 12.0) and was then equilibrated with degassed HBS buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.005% Tween 20, pH 7.4). Then, the surface carboxyl groups were activated with a 0.05 M NHS0.2 M EDC solution. Activated thiol groups were introduced by a 40-µl injection of 80 mM PDEA (in 0.1 M sodium carbonate buffer pH 8.5). This step was followed by manual injection of the Ser237Cys-containing GHR-ECDs of 0.1-mg/ml concentration in 10 mM sodium acetate pH 4.5 buffer. The excess reactive thiol compound was quenched by a 30-µl injection of 50 mM glutathione. The control surface was activated and blocked as mentioned before but had no protein immobilized. The same HBS buffer, as mentioned before, was used for all the measurements with the flow rate of 50 µl/min.
SPR Measurements and Analyses of Binding Kinetics Data
A BIAcore2000 measures the refractive index near the sensor surface by SPR (see Fisher et al. 1994
for an application of this technology). We injected twofold serial dilutions of the GH solutions into cuvettes containing the analysis chip. Buffer-only injection representing 0 nM analyte was also included and subtracted from all the sensorgrams in the same set to minimize the noise introduced by the experimental procedure. All measurements were repeated at least four times to ensure reproducibility. On average, two independent measurements from two chips were performed to assess the interactions.
Because of the short and rigid linker of the C-1 chip, the immobilized receptor molecules cannot interact with each other. Thus, GH binding specifically through site 1 can be analyzed without the complication of subsequent interactions through site 2 (Bernat et al., unpublished data). The kinetics of the site 1 GH:GHR-ECD interaction can, therefore, be accurately described by a simple bimolecular or Langmuir interaction:
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Using the sensorgrams from analytes of different concentrations, a global fit of both association and dissociation rate constants for each interaction was generated. The data were analyzed using the BIA evaluation 3.0 software (Biacore), applying the simple 1:1 (Langmuir) binding model for the fitting.
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Results |
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All Ser237Cys variants were modified by glutathione at the unpaired cysteine residue during the expression. This modification was successfully removed by DTT without affecting the native disulfides of the protein for 237Cys mutants of hGHR-ECD and hGHR-ECD43Leu. This was not the case for smGHR-ECD and its variant. The same condition turned out to be too harsh for the 237Cys smGHR-ECD variant, leading to reduction of the native disulfide bonds and ultimate denaturation of the protein. Despite the small number of amino acid sequence differences between human and squirrel monkey GHRs, the stability and reactivity of their disulfides are measurably different, suggesting subtle tertiary structure differences. A milder reducing reagent ß-mercapthoethanolamine was used to empirically determine an optimal treatment condition. A 20-min treatment with 7.5 mM ß-mercapthoethanolamine solution at 37°C with the presence of 50 mM EDTA was effective and safe for the removal of the glutathione modification.
Determination of Binding Kinetics
SPR is a method capable of measuring the binding interactions between molecules by using the changes in refractive index to measure the mass changes of molecules bound to the analysis chip. These changes were reported as "response units (RUs)." The RU values are linearly proportional to the amount (mass) of the analytes binding to the ligands. A typical sensorgram is composed of two characteristic phases. In the first (association) phase, the GH is injected above the GHR-ECDcontaining surface, allowing the association of the GH to the immobilized GHR-ECD. However, simultaneous dissociation of the GH from the resulting GH:GHR-ECD complex also occurs in this phase. A sufficiently long injection time leads to a steady-state response level characterized by equal rates of association of the GH to GHR-ECD and of dissociation of the GH from the GH:GHR-ECD complex. In the second (dissociation) phase, instead of the GH, only the running buffer is injected. In this phase only the dissociation of the GH from the previously formed GH:GHR-ECD complexes occurs because there is no further supply of GH injected over the surface.
Different Species Specificities of Human and Squirrel Monkey GHR-ECDs
Figure 2A
shows the sensorgrams generated from the responses of hGHR-ECD237Cys to the three different GHs: hGH, smGH, and rat GH. The interaction between hGH and hGHR-ECD has a Kd = 3.5 nM (table 3
). Interestingly, the smGH binds to the hGHR-ECD with approximately the same affinity as hGH does (Kd = 3.3 nM). As expected, the rat GH does not bind to the hGHR-ECD.
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GHR-ECD Residue 43 is a Major Determinant of Species Specificity
As described above, the wild-type hGHR-ECD shows no binding affinity toward the rat GH in accord with the well-established species specificity of Old World primate GHRs. However, introduction of a single mutation Arg43Leu provided binding toward the rat hormone that was only slightly weaker than the wild-type hGHR-ECD:hGH interaction (fig. 3A
and table 3
), whereas it conserved strong binding to the hGH and the smGH. Moreover, we introduced a converse mutation to smGHR-ECD. The resulting variant smGHR-ECD43Arg (with a human residue at position 43) completely lost its affinity toward rat GH (fig. 3B
and table 3
), while maintaining its affinity toward hGH and smGH. In other words, it acquired a species specificity to respond only to primate GHs. This strongly suggests that the amino acid at position 43 is likely to be the primary determinant for the so-called species specificity: Arg confers, whereas Leu disables, the species specificity.
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The purified smGH binds tightly to both its own receptor and hGHR, with 2.3- and 2.0-nM dissociation constants, respectively (table 3 ). The binding between smGH and smGHR-ECD with the human residue Arg at position 43 was weaker than the wild-type interactions (5.7- nM compared with 2.3-nM dissociation constant). Binding of smGH toward the mutant hGHR-ECD, encoding residue Leu at positon 43, is about twofold weaker than the binding toward the wild-type hGHR-ECD (6.0- vs. 3.3-nM dissociation constants).
Overall, there is no directional effect of residue 43 on the within-species binding affinities observed. Therefore, in contrast to the dramatic effect of residue 43 on the binding toward the rat GH, within human and squirrel monkey GHRs, whether residue 43 is Leu or Arg does not affect the binding affinities in the same direction. For both species, the binding affinities were greater for the wild-type interactions. At the same time the wild-type interactions were not the strongest; we observed the strongest binding interactions between hGH and smGHR-ECD. Although hGH showed similar binding affinity across the various GHR-ECDs used, smGH showed more variation in terms of binding affinity toward different GHR-ECDs.
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Discussion |
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Inferring the selective advantage or disadvantage of a specific amino acid substitution by means of functional data is not straightforward. For example, the effects of mutations may not be additive, but synergistic. Indeed, Clackson et al. (1998)
reported that some of the mutations outside the structural epitopes of hGHR could indirectly influence the binding affinity. Therefore, inferring the significance of individual residues serves as only the first step in understanding the phenotypic consequences of specific substitutions. Another limitation of the measurement of choice presented in this report lies in the fact that the present study focused only on the interaction between GHR-ECDs and GHs through site 1 binding. It should be noted that the species specificity was originally described in a biological system (Carr and Friesen 1976
) in which both site 1 and site 2 interactions occur. The site 1 binding affinity accounts for the first step of the cascade of the biological signal transduction pathway (Kossiakoff 1995
). It is possible that the kinetics of site 1 and site 2 binding may behave differently in response to a single mutation. Unfortunately, this aspect of GH:GHR interactions has not been investigated in detail yet because of the fact that it is technically challenging to measure the site 1 and site 2 binding affinities separately. A new SPR technique for this purpose, however, has been developed recently (Bernat et al., unpublished data). Determining whether the effect of a mutation that is critical in site 1 binding affinity, such as the GH171 or GHR43 residue, has the same degree of significance at the site 2 binding affinity will be important to elucidate the underlying biological consequences and to facilitate the understanding of the trajectories of functional evolution.
Origin and the Evolution of the Major Determinant of Old World Primate GHR Specificity
The species specificity of the Old World primate GHR is an example of the dramatic functional differences of the same proteins from different phylogenetic units. Although most of the hormones produced in one mammalian species are physiologically effective when injected into another species, the incompatibility of human and Rhesus monkey GHRs toward nonprimate GHs stands out as a notable exception (Carr and Friesen 1976
).
Efforts to elucidate the molecular basis of species specificity became successful only after detailed structural and biochemical knowledge of the GH and GHR interaction became available. A high-resolution X-ray structure of the ternary hGH·(hGHR-ECD)2 complex (De Vos, Ultsch, and Kossiakoff 1992
) revealed the amino acid residues that are directly involved in the formation of the ternary complex (table 1
). Complementary binding studies using systematic mutagenesis strategies both on the hormone (Cunningham and Wells 1989
; Cunningham et al. 1989
) and on the receptor (Clackson and Wells 1995
; Clackson et al. 1998
) identified the most important residues for the site 1 interaction (table 1
). On the basis of this information, it was speculated that the structurally complementary residues Asp171 of the hormone and Arg43 of the receptor were the principal determinants of species specificity (Souza et al. 1995
).
Recently, Liu et al. (2001)
and Wallis, Zhang, and Wallis (2001)
studied three New World monkey species, i.e., squirrel monkey, spider monkey, and marmoset, and found that position 171 of the GH is an Asp in all three species, suggesting that the His
Asp change occurred before the radiation of New World monkeys. Liu et al. (2001)
further found that the GHR of the squirrel monkey is encoded by Leu at position 43, the so-called nonprimate residue. Therefore, the states of the GH and the GHR in the New World monkeys would appear to be the "missing link" of the emergence of species specificity. Whether the situation of the squirrel monkey really represents a functional intermediary state was the motivating question of this study. Our functional analysis supported this hypothesis. Thus, the species specificity is an Old World primatespecific trait; smGHR can bind to the nonprimate GH as well as to the primate GH, in contrast to the hGHR, which can only bind to the higher primate GH. We further demonstrated that this functional characteristic could be altered by a single amino acid change at residue 43. In other words, we were able to reconstruct the evolutionary trajectory of the emergence of species specificity; it can occur through a single nucleotide change (CTA
CGA) in New World monkey GHR, where the GH171His
GH171Asp substitution has already occurred (Liu et al. 2001
).
Biochemical Basis of the Major Determinant of Old World Primate GHR Specificity
There are stereochemical reasons why the His Asp mutation in the hormone must have preceded the Leu
Arg mutation in the receptor. The GHR43Arg residue is known to be the center of an extensive network of structural interactions necessary for the stable hormone-receptor complex (De Vos, Ultsch, and Kossiakoff 1992
; Behncken et al. 1997
; Clackson et al. 1998
). The hGH:hGHR interaction is stabilized by a salt bridge between hGHR43Arg and hGHAsp171 and by a hydrogen bond between hGH175Thr and hGHR43Arg (fig. 1
). In addition, the alkyl portion of the side chain of Arg43 of the hGHR is known to form extensive van der Waals interactions with the side chain of Trp169 and also contacts Ile103 and Trp104 (fig. 1
). Because of the extensive array of interactions involving GHR43Arg, it is to be expected that any change in this residue will result in a substantial decrease in affinity.
However, when changed to Leu, we did not observe a significant reduction in site 1 binding affinity (table 3
; see also Clackson et al. 1998
). This leads to a suggestion that the Arg43 guanidium group interactions are not as important as the hydrophobic interaction with the receptor Trp169, which presumably still occurs with Leu residue at GHR43 (Behncken et al. 1997
; Clackson et al. 1998
). Leu can maintain this intramolecular packing, even though it lacks the electrostatic charge caused by the deletion of the guanidinium group (see fig. 3
of Behncken et al. 1997
for suggested structural interface for this interaction).
Substitution of Asp by His at GH171 will cause a steric hindrance and repulsive interactions with Arg43 (see fig. 3
of Behncken et al. 1997
) and, therefore, result in the observed incompatibility. However, with GHR43Leu this hindrance is removed; hence, no change in site 1 affinity is observed. Therefore, although the GHR43 change could not have preceded the GH171 change (because it will lose affinity to its own GH), once the GH171 change occurred, the Leu
Arg substitution became possible without a dramatic change in the binding affinity.
Was the Emergence of the Species Specificity of GHR the Result of Positive Selection?
What triggered the evolution of the GH171Asp:GHR43Arg pair as seen in Old World primates? A selective hypothesis postulates that these two residues coevolved toward increased affinity and specificity; in other words, the species specificity evolved as a result of positive selection. From our binding analysis, we found no direct evidence to support this hypothesis. For the smGHR, the pairs of molecules engineered to have the GH171Asp:GHR43Arg interaction exhibited a lower binding affinity than the wild-type interaction, which is intermediary in the evolution of functional specificity (GH171Asp:GHR43Leu). In the case of the hGHR, the intermediary interaction had essentially the same binding affinity as the wild-type interactions (GH171Asp:GHR43Arg). Therefore, although the incompatibility between GH171His and GHR43Arg was the major determinant of the Old World primate GHR specificity, the interaction between GH171Asp and GHR43Arg does not suggest selective advantage, at least starting from the sequence of smGHR. It should also be noted that in these measurements the hGH had a high affinity toward smGHR-ECD, which has Leu at residue 43. Therefore, for the interaction between GH and GHRs in Old World primates, the GH171Asp:GHR43Arg interaction does not appear to be a significant improvement over the intermediate status of GH171Asp:GHR43Leu.
Additionally, Liu et al. (2001)
reported that the intermediary state of the GH171Asp:GHR43Leu pair is found in both squirrel monkey and spider monkey. These two species represent the two most divergent families of the Haplorhine Superfamily Ceboidea, which has an estimated age of 25 Myr (Goodman et al. 1998
). This indicates that the intermediate state has persisted for a very long period of time within Platyrrhines. If the new mutation of GHR43Leu
GHR43Arg has a significant selective advantage, then the intermediary state is more likely to be short-lived. The observation that there is no increase in binding affinity for the evolved interaction compared with the ancestral interaction and the phylogenetic distribution of this substitution both indicate that the evolution of the GHR species specificity of Old World primates may not be a direct consequence of selectively driven coevolutionary substitutions in the GH and the GHR.
An alternative explanation is that the new mutation (T G) was selectively neutral and became fixed in the population by chance, and the evolution of the species specificity is a byproduct of this substitution. The lack of direct evidence for a selective advantage of the new mutation in the smGHR favors this explanation. In the light of this conclusion, it will be of great interest to investigate whether the GH171His
GH171Asp change had any significant selective advantage. This substitution provided the biochemical basis for the emergence of the species specificity of Old World primate GHRs. Interestingly, the timing of this substitution coincides with the acceleration in the evolutionary rate observed in the ancestral lineage leading to higher primates (Liu et al. 2001
).
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Acknowledgements |
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Footnotes |
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Present address: Array BioPharma, Boulder, Colorado
Keywords: growth hormone
growth hormone receptor
species specificity
surface plasmon resonance
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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References |
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