Crystal structure and site 1 binding energetics of human placental lactogen

In primates, placental lactogen (PL) is a pituitary hormone with fundamental roles during pregnancy involving fetal growth, metabolism, and stimulating lactation in the mother. Human placental lactogen (hPL) is highly conserved with human growth hormone (hGH) and both hormones bind to the hPRLR extracellular domain (ECD), the first step in receptor homodimerization, in a Zn2+-dependent manner. A modified surface plasmon resonance method was developed to measure the kinetics for hPL and hGH binding to the hPRLR ECD, with and without Zn2+ and showed that hPL has about a tenfold higher affinity for the hPRLR ECD1 than hGH. The crystal structure of the free state of hPL has been determined to 2.0 A resolution showing the molecule possesses an overall structure similar to other long chain four-helix bundle cytokines. Comparison of the free hPL structure with the 1:1 complex structure of hGH bound to the hPRLR ECD1 suggests that two surface loops undergo conformational changes >10 A upon binding. An 18 residue Ala-scan was used to characterize the binding energy epitope for the site 1 interface of hPL. Individual alanine substitutions at five positions reduced binding affinity by a DeltaDeltaG > or = 3 kcal mol(-1). A comparison of the hPL site 1 epitope with that previously determined for hGH indicates contributions of individual residues track reasonably well between hPL and hGH. In particular, residues involved in the zinc-binding site and Lys172 constitute the principal binding determinants for both hormones. However, several residues that are identical between hPL and hGH contribute quite differently to the binding of the hPRLR ECD1. Additionally, the overall magnitudes of the DeltaDeltaG changes observed from the Ala-scan of hPL were markedly larger than those determined in the comparative scan of hGH to the hPRLR ECD1. The structural and biophysical data presented here show that subtle changes in the structural context of an interaction can lead to significantly different effects at the individual residue level.     

Structure of a phage display-derived variant of human growth hormone complexed to two copies of the extracellular domain of its receptor: evidence for strong structural coupling between receptor binding sites

The structure of the ternary complex between the phage display- optimized, high-affinity Site 1 variant of human growth hormone (hGH) and two copies of the extracellular domain (ECD) of the hGH receptor (hGHR) has been determined at 2.6 A resolution. There are widespread and significant structural differences compared to the wild-type ternary hGH hGHR complex. The hGH variant (hGH(v)) contains 15 Site 1 mutations and binds>10(2) tighter to the hGHR ECD (hGH(R1)) at Site 1. It is biologically active and specific to hGHR. The hGH(v) Site 1 interface is somewhat smaller and 20% more hydrophobic compared to the wild-type (wt) counterpart. Of the ten hormone-receptor H-bonds in the site, only one is the same as in the wt complex. Additionally, several regions of hGH(v) structure move up to 9A in forming the interface. The contacts between the C-terminal domains of two receptor ECDs (hGH(R1)- hGH(R2)) are conserved; however, the large changes in Site 1 appear to cause global changes in the domains of hGH(R1) that affect the hGH(v)-hGH(R2) interface indirectly. This coupling is manifested by large changes in the conformation of groups participating in the Site 2 interaction and results in a structure for the site that is reorganized extensively. The hGH(v)- hGH(R2) interface contains seven H-bonds, only one of which is found in the wt complex. Several groups on hGH(v) and hGH(R2) undergo conformational changes of up to 8 A. Asp116 of hGH(v) plays a central role in the reorganization of Site 2 by forming two new H-bonds to the side-chains of Trp104(R2) and Trp169(R2), which are the key binding determinants of the receptor. The fact that a different binding solution is possible for Site 2, where there were no mutations or binding selection pressures, indicates that the structural elements found in these molecules possess an inherent functional plasticity that enables them to bind to a wide variety of binding surfaces.     

Shotgun alanine scanning shows that growth hormone can bind productively to its receptor through a drastically minimized interface

The high affinity binding site (Site1) of the human growth hormone (hGH) binds to its cognate receptor (hGHR) via a concave surface patch containing about 35 residues. Using 167 sequences from a shotgun alanine scanning analysis of Site1, we have determined that over half of these residues can be simultaneously changed to an alanine or a non-isosteric amino acid while still retaining a high affinity interaction. Among these hGH variants the distribution of the mutation is highly variable throughout the interface, although helix 4 is more conserved than the other binding elements. Kinetic and thermodynamic analyses were performed on 11 representative hGH Site1 variants that contained 14-20 mutations. Generally, the tightest binding variants showed similar associated rate constants (k(on)) as the wild-type (wt) hormone, indicating that their binding proceeds through a similar transition state intermediate. However, calorimetric analyses indicate very different thermodynamic partitioning: wt-hGH binding exhibits favorable enthalpy and entropy contributions, whereas the variants display highly favorable enthalpy and highly unfavorable entropy contributions. The heat capacities (DeltaCp) on binding measured for wt-hGH and its variants are significantly larger than normally seen for typical protein-protein interactions, suggesting large conformational or solvation effects. The multiple Site1 mutations are shown to indirectly affect binding of the second receptor at Site2 through an allosteric mechanism. We show that the stability of the ternary hormone-receptor complex reflects the affinity of the Site2 binding and is surprisingly exempt from changes in Site1 affinity, directly demonstrating that dissociation of the active signaling complex is a stepwise process.     

Growth hormone binding and stimulation of amino acid uptake in epithelial cells of rat ventral prostate

The binding of [125I]-human growth hormone (hGH) was studied in epithelial cells isolated from rat ventral prostate. Binding and degradation were dependent on time and temperature. The effect of a lysosomotropic agent suggested internalization and lysosomal degradation of the hormone. Dissociation and stoichiometric studies indicated the existence of a single class of GH receptors with a Kd of 0.7 nM and a binding capacity of 46 fmol hGH bound mg-1 cell protein. The receptor appeared to possess a somatotrophic nature since lactogenic hormones such as human placental lactogen and rat prolactin exhibited a very low degree of competition (whereas a variety of unrelated hormones and neuropeptides showed no effect). GH-stimulated leucine uptake by the cells in a time- and dose-dependent manner, half maximal effect being observed at 0.32 nM GH thus suggesting a direct relationship with the binding step.     

Increased site 1 affinity improves biopotency of porcine growth hormone. Evidence against diffusion dependent receptor dimerization

Based on phage display optimization studies with human growth hormone (GH), it is thought that the biopotency of GH cannot be increased. This is proposed to be a result of the affinity of the first receptor for hormone far exceeding that which is required to trap the hormone long enough to allow diffusion of the second receptor to form the ternary complex, which initiates signaling. We report here that despite similar site 1 kinetics to the hGH/hGH receptor interaction, the potency of porcine GH for its receptor can be increased up to 5-fold by substituting hGH residues involved in site 1 binding into pGH. Based on extensive mutations and BIAcore studies, we show that the higher potency and site 1 affinity of hGH for the pGHR is primarily a result of a decreased off-rate associated with residues in the extended loop between helices 1 and 2 that interact with the two key tryptophans Trp104 and Trp169 in the receptor binding hot spot. Our mutagenic analysis has also identified a second determinant (Lys165), which in addition to His169, restricts the ability of non-primate hormones to activate hGH receptor. The increased biopotency of GH that we observe can be explained by a model for GH receptor activation where subunit alignment is critical for effective signaling.     

Dissecting the energetics of an antibody-antigen interface by alanine shaving and molecular grafting.

Alanine-scanning mutagenesis on human growth hormone (hGH) identified 5 primary determinants (Arg 8, Asn 12, Arg 16, Asp 112, and Asp 116) for binding to a monoclonal antibody (MAb 3) (Jin L, Fendly BM, Wells JA, 1992, J Mol Biol 226:851-865). To further analyze the energetic importance of residues surrounding these five, we mutated all neighboring residues to alanine in groups of 7-16 (a procedure we call alanine shaving). Even the most extremely mutated variant, with 16 alanine substitutions, caused less than a 10-fold reduction in binding affinity to MAb3. By comparison, mutating any 1 of the 5 primary determinants to alanine caused a 6- to > 500-fold reduction in affinity. Replacing any of the 4 charged residues (Arg 8, Arg 16, Asp 112, and Asp 116) with a homologous residue (i.e., Arg to Lys or Asp to Glu) caused nearly as large a reduction in affinity as the corresponding alanine replacement. It was possible to graft the 5 primary binding determinants onto a nonbinding homologue of hGH, human placental lactogen (hPL), which has 86% sequence identity to hGH. The grafted hPL mutant bound 10-fold less tightly than hGH to MAb3 but bound as well as hGH when 2 additional framework mutations were introduced. Attempts to recover binding affinity by grafting the MAb3 epitope onto more distantly related scaffolds having a similar 4-helix bundle motif, such as human prolactin (23% sequence identity) or granulocyte colony-stimulating factor, were unsuccessful.(ABSTRACT TRUNCATED AT 250 WORDS)     

The functional binding epitope of a high affinity variant of human growth hormone mapped by shotgun alanine-scanning mutagenesis: insights into the mechanisms responsible for improved affinity

A high-affinity variant of human growth hormone (hGH(v)) contains 15 mutations within site 1 and binds to the hGH receptor (hGHR) approximately 400-fold tighter than does wild-type (wt) hGH (hGH(wt)). We used shotgun scanning combinatorial mutagenesis to dissect the energetic contributions of individual residues within the hGH(v) binding epitope and placed them in context with previously determined structural information. In all, the effects of alanine substitutions were determined for 35 hGH(v) residues that are directly contained in or closely border the binding interface. We found that the distribution of binding energy in the functional epitope of hGH(v) differs significantly from that of hGH(wt). The residues that contributed the majority of the binding energy in the wt interaction (the so-called binding "hot spot") remain important, but their contributions are attenuated in the hGH(v) interaction, and additional binding energy is acquired from residues on the periphery of the original hotspot. Many interactions that inhibited the binding of hGH(wt) are replaced by interactions that make positive contributions to the binding of hGH(v). These changes produce an expanded and diffused hot spot in which improved affinity results from numerous small contributions distributed broadly over the interface. The mutagenesis results are consistent with previous structural studies, which revealed widespread structural differences between the wt and variant hormone-receptor interfaces. Thus, it appears that the improved binding affinity of hGH(v) site 1 was not achieved through minor adjustments to the wt interface, but rather, results from a wholesale reconfiguration of many of the original binding elements.     

Allosteric effects of monoclonal antibodies on human growth hormone

We have previously shown that a monoclonal antibody (MAb) recognizing the human growth hormone (hGH) antigenic domain left exposed after binding to lactogenic receptors enhanced hGH binding probably through allosteric effects on the hormone binding site. Since receptors displaying different specificities would not recognize exactly the same hGH region, we explored whether some of our MAb could affect hGH binding to somatogenic receptors from rabbit liver and to human liver hGH-specific receptors.The effect of MAbAE5, AC8 and F11 on hGH binding was measured by determining the formation of¹²⁵I-MAb:hGH:receptor complexes using two different experimental approaches. Results from procedure A, which involved the previous binding of the hormone to microsomes before adding¹²⁵I-MAb, indicated that the hGH domain defined by epitopes AE5, AC8 and F11 is uncovered in the various hormone:receptor complexes.Procedure B was devised to reveal any alteration in the hGH molecule induced by the MAb. In this case preformed¹²⁵I-MAb:hGH complexes were added to microsomes. Data showed that¹²⁵I-MAb AE5:hGH complexes bound better to the various receptors than¹²⁵I-MAb AE5 to hGH:receptor complexes. On the contrary, hGH previously bound to¹²⁵I-MAb AC8 or¹²⁵I-MAb F11 was less recognized by the receptors than the free hormone. Furthermore, binding of MAb AE5 or MAb F11 to hGH 20 K (a natural hGH variant lacking residues 32–46) also enhanced its affinity to the various receptors whereas MAb AC8 did not inhibit hGH 20 K binding.Results indicated that MAb recognizing the hGH antigenic area that remains unmasked after binding to different membrane-bound receptors are able to affect hormone binding site. MAb would induce either positive or negative allosteric changes in the hormone region involved in its binding to lactogenic, somatogenic and hGH-specific receptors.     

Evidence for a direct effect of growth hormone on osteoblasts

In order to determine whether growth hormone (GH) exerts a direct effect on osteoblasts, in vitro and in vivo immunocytological studies were carried out on newborn rat calvaria and a clonal osteoblast-like cell line (MC3T3-E1) isolated from newborn mouse calvaria. After exposure to human growth hormone (hGH) or 1,25 dihydroxyvitamin D3 (1,25(OH)₂D₃), a significant increase in alkaline phosphatase activity was observed in MC3T3-E1 cells. Simultaneous exposure of MC3T3-E1 cells to hGH and 10 nM 1,25(OH)₂D₃ showed a synergistic effect of the two hormones on this activity. The optimal dose of hGH was 0.1 nM. An immunocytological procedure was performed on ultrathin frozen sections from 7-day-old rat calvaria and MC3T3-E1 cells cultured with hGH. GH-like immunoreactivity was observed in both cases. In calvaria, endogenous GH-like immunoreactivity was localized at the same ultrastructural level (plasma membrane, cytoplasmic and nuclear matrices) as exogenous GH-like immunoreactivity in MC3T3-E1 cells. Following the initial step of binding to the plasma membrane, GH may be internalized in the cytoplasmic matrix and nucleus. In situ hybridization revealed the presence of mRNA coding for GH receptor in calvaria cells. The density of these receptors seemed to be lower in osteoblasts than in hepatocytes. In MC3T3-E1 cells, hGH induced a dose-dependent secretion of insulin-like growth factor 1. In conclusion, these results indicate that GH may act directly on osteoblasts.     

Homology modeling of rabbit prolactin hormone complexed with its receptor

The pituitary hormone prolactin (prl) is implicated in a number of biological functions, especially lactation, which is mediated through specific lactogenic receptors (PrlR). Human growth hormone (hGH) is also a pituitary hormone responsible for linear growth. While the growth hormone receptor (hGHR) binds only hGH, hPrlR can interact with both hGH and hPrl. Using structural information from the human growth hormone (hGH)/receptor (hGHR) complex, we modeled by homology a complex between rabbit prolactin hormone (rbPrl) and its receptor (rbPrlR). While the somatogenic hormone/somatogenic receptor (hGH/hGHR) and somatogenic hormone/lactogenic receptor (hGH/hPrlR) interactions are now known and well studied, here we propose a model for the interaction of the lactogenic hormone with its receptor (rbPrl/rbPrlR), and compare these three kinds of ligand/receptor interaction. We identified residues contributing to the active site and tested the potential dimerization of the receptor. Biochemical studies and information deduced from the modeled complex do not exclude a homodimeric form but point to a functional heterodimeric complex. Proteins 27: 459–468, 1997. © 1997 Wiley-Liss, Inc.     

Intramolecular cooperativity in a protein binding site assessed by combinatorial shotgun scanning mutagenesis

Combinatorial shotgun alanine-scanning was used to assess intramolecular cooperativity in the high affinity site (site 1) of human growth hormone (hGH) for binding to its receptor. A total of 19 side-chains were analyzed and statistically significant data were obtained for 145 of the 171 side-chain pairs. The analysis revealed that 90% of the side-chain pairs exhibited no statistically significant pair interactions, and the remaining 10% of side-chain pairs exhibited only small interactions corresponding to cooperative interaction energies with magnitudes less than 0.4 kcal/mol. The statistical predictions were tested by measuring affinities for purified mutant proteins and were found to be accurate for five of six side-chain pairs tested. The results reveal that hGH site 1 behaves in a highly additive manner and suggest that shotgun scanning should be useful for assessing cooperative effects in other protein-protein interactions.     

Binding and signalling properties of a growth hormone enhancing monoclonal antibody

We have used a sequential, qualitative biosensor based assay to demonstrate that OA15, a monoclonal antibody which enhances in vivo the activity of bovine growth hormone (bGH) does not disrupt the interaction between bGH and its cognate receptor (as represented by recombinant bovine GH binding protein -rbGHBP). We have confirmed this using a classical cell-based radio-receptor assay with the GH-responsive mouse pre-adipocyte cell line 3T3-F442A. The fact that OA15 binding to bGH still allows hormone to interact with its receptor, allows us to test the hypothesis that there is any amplification of signalling events following hormone-MAb treatment of 3T3-F442A cells. We have used as a reporter of GH activity the rapid stimulation of JAK-2 tyrosine phosphorylation which is a critical first step in GH signalling events. We demonstrate that binding of rbGH by OA15 attenuates hormone stimulation of JAK-2 tyrosine phosphorylation. We conclude that although OA15 does not disrupt GH-GH receptor (GHR) interactions it does interfere with subsequent GH activity at the molecular and cellular level. We further speculate therefore that the biological enhancing activity of this antibody is most likely due to an in vivo effect as presentation of antibody-hormone complexes to a GH-target cell inhibits hormone activity.     

Alternative views of functional protein binding epitopes obtained by combinatorial shotgun scanning mutagenesis

Combinatorial shotgun scanning mutagenesis was used to analyze two large, related protein binding sites to assess the specificity and importance of individual side chain contributions to binding affinity. The strategy allowed for cost-effective generation of a plethora of functional data. The ease of the technology promoted comprehensive investigations, in which the classic alanine-scanning approach was expanded with two additional strategies, serine- and homolog-scanning. Binding of human growth hormone (hGH) to the hGH receptor served as the model system. The entire high affinity receptor-binding sites (site 1) of wild-type hGH (hGH_wt) and of an affinity-improved variant (hGH_v) were investigated and the results were compared. The contributions that 35 residue positions make to binding were assessed on each hormone molecule by both serine- and homolog-scanning. The hormone molecules were displayed on the surfaces of bacteriophage, and the 35 positions were randomized simultaneously to allow equal starting frequencies of the wild-type residue and either serine or a homologous mutation in separate libraries. Functional selections for binding to the hGH receptor shifted the relative wild-type/mutant frequencies at each position to an extent characteristic of the functional importance of the side chain. Functional epitope maps were created and compared to previous maps obtained by alanine-scanning. Comparisons between the different scans provide insights into the affinity maturation process that produced hGH_v. The serine and homolog-scanning results expand upon and complement the alanine-scanning results and provide additional data on the robustness of the high affinity receptor-binding site of hGH.     

Turning the growth hormone receptor on: Evidence that hormone binding induces subunit rotation

Atomistic molecular dynamics simulations have been used to investigate the conformational changes associated with the binding of human growth hormone (hGH) to the extracellular domains (ECD) of the human growth hormone receptor (hGHR), thereby shedding light on the mechanism of activation. It is shown that the removal of hGH from the hormone‐bound receptor complex results in a counter‐clockwise rotation of the twosubunits relative to each other by 30°–64° (average 45° ± 14°), in close agreement in terms of both the magnitude and direction of the rotation with that proposed based on mutagenesis experiments. In addition to providing evidence to support a rotational activation mechanism, the simulations have enabled the nature of the interaction interfaces in both the cytokine‐bound and unliganded hGHR states to be analyzed in detail. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Comparison of the intermediate complexes of human growth hormone bound to the human growth hormone and prolactin receptors.

The crystal structures of complexes of human growth hormone (hGH) with the growth hormone and prolactin receptors (hGHR and hPRLR, respectively), together with the mutational data available for these systems, suggest that an extraordinary combination of conformational adaptability, together with finely tuned specificity, governs the molecular recognition processes operative in these systems. On the one hand, in the active 1:2 ligand-receptor complexes, 2 copies of the same receptor use the identical set of binding determinants to recognize topographically different surfaces on the hormone. On the other hand, comparing the 1:1 hGH-hGHR and hGH-hPRLR complexes, 2 distinct receptors use this same set of binding determinants to interact with the identical binding site on the ligand, even though few residues among the binding determinants are conserved. The structural evidence demonstrates that this versatility is accomplished by local conformational flexibility of the binding loops, allowing adaptation to different binding environments, together with rigid-body movements of the receptor domains, necessary for the creation of specific interactions with the same binding site.     

Cloning and expression of ovine placental lactogen

Ovine placental lactogen (oPL) is active in a wide range of GH and PRL assays, a property that it shares with human GH (hGH). In addition, oPL is one of a small number of hormones that bind the human GH receptor with high affinity. In order to compare the sequence of oPL to the sequences of other members of the GH family, full-length cDNA clones have been isolated. These clones predict that the full sequence of oPL contains 198 amino acids preceded by a 38 amino acid signal sequence. The mature oPL sequence includes six cysteine and two tryptophan residues and shows substantially more identity to bovine PL (67%) and oPL (49%) than to mouse (31%) or human (25%) PL or to oGH (28%) or (26%) hGH. Like the natural hormone, oPL expressed in mammalian tissue cells binds with high affinity to a soluble form of the recombinant hGH receptor. Thus, oPL binds to the human receptor in spite of having a sequence that is considerably divergent from hGH. Interestingly, the sequence of oPL differs from hGH at most of the amino acids recently found by mutagenesis studies to be important residues in the binding of hGH to the human receptor.     

Evidence that the thyroid-stimulating hormone (TSH) receptor transmembrane domain influences kinetics of TSH binding to the receptor ectodomain

Thyroid-stimulating hormone (TSH)-induced reduction in ligand binding affinity (negative cooperativity) requires TSH receptor (TSHR) homodimerization, the latter involving primarily the transmembrane domain (TMD) but with the extracellular domain (ECD) also contributing to this association. To test the role of the TMD in negative cooperativity, we studied the TSHR ECD tethered to the cell surface by a glycosylphosphatidylinositol (GPI) anchor that multimerizes despite the absence of the TMD. Using the infinite ligand dilution approach, we confirmed that TSH increased the rate of dissociation (k(off)) of prebound (125)I-TSH from CHO cells expressing the TSH holoreceptor. Such negative cooperativity did not occur with TSHR ECD-GPI-expressing cells. However, even in the absence of added TSH, (125)I-TSH dissociated much more rapidly from the TSHR ECD-GPI than from the TSH holoreceptor. This phenomenon, suggesting a lower TSH affinity for the former, was surprising because both the TSHR ECD and TSH holoreceptor contain the entire TSH-binding site, and the TSH binding affinities for both receptor forms should, theoretically, be identical. In ligand competition studies, we observed that the TSH binding affinity for the TSHR ECD-GPI was significantly lower than that for the TSH holoreceptor. Further evidence for a difference in ligand binding kinetics for the TSH holoreceptor and TSHR ECD-GPI was obtained upon comparison of the TSH K(d) values for these two receptor forms at 4 °C versus room temperature. Our data provide the first evidence that the wild-type TSHR TMD influences ligand binding affinity for the ECD, possibly by altering the conformation of the closely associated hinge region that contributes to the TSH-binding site.     

Solubilization and characterization of a lactogenic receptor from human placental chorion membranes

Prolactin has a wide range of actions, including osmoregulation and the control of mammary gland development and lactation. These effects are mediated through a high-affinity cell surface receptor, which has been well characterized in a number of animal tissues. The molecular characteristics of the human receptor are unknown, however. The present studies were initiated, therefore, to determine the binding and molecular characteristics of the lactogenic receptor of human placental chorion membranes. Subcellular fractionation studies showed that the bulk of the receptor sedimented in the microsomal fraction at 45,000gav. Endogenous ligand was dissociated from the receptor with 3.5 M MgCl2 or 0.05 M acetate buffer (pH 4.8) with preservation of binding activity. The microsomal receptor bound human growth hormone (hGH), human prolactin (hPRL), ovine prolactin (oPRL), and human placental lactogen (hPL) but not non-primate growth hormones, indicating a narrow specificity for lactogenic hormones. The binding was only partially reversible in agreement with the known binding kinetics of animal lactogenic receptors. The receptor was solubilized with 45% yield from the microsomes using 16 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulphonate (CHAPS) detergent-250 mM NaCl, and the binding activity was fully restored by a two-fold dilution in the binding reaction to reveal a KD of 0.8 nM for hGH and a binding capacity of 200 fmol of specifically bound hGH per mg of microsomal protein. Gel filtration chromatography indicated the minimum molecular weight of the ligand-receptor complex was approximately 60,000 daltons, and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) of covalently cross-linked 125I-hGH-receptor complexes revealed a molecular size of 58,000 daltons. When account was taken of the contribution of the ligand, a molecular weight of 36,000 for the receptor's binding domain was obtained. These data indicate that the chorion lactogenic receptor has very similar binding and molecular characteristics to the lactogenic receptors from other mammalian species. Chorion membranes are thus a convenient source of material for the further purification and characterization of the human lactogenic receptor.     

Contribution of individual histidines to the global stability of human prolactin

A member of the family of hematopoietic cytokines human prolactin (hPRL) is a 23k kDa polypeptide hormone, which displays pH dependence in its structural and functional properties. The binding affinity of hPRL for the extracellular domain of its receptor decreases 500‐fold over the relatively narrow, physiologic pH range from 8 to 6; whereas, the affinity of human growth hormone (hGH), its closest evolutionary cousin, does not. Similarly, the structural stability of hPRL decreases from 7.6 to 5.6 kcal/mol from pH 8 to 6, respectively, whereas the stability of hGH is slightly increased over this same pH range. hPRL contains nine histidines, compared with hGH's three, and they are likely responsible for hPRL's pH‐dependent behavior. We have systematically mutated each of hPRL's histidines to alanine and measured the effect on pH‐dependent global stability. Surprisingly, a vast majority of these mutations stabilize the native protein, by as much as 2–3 kcal/mol. Changes in the overall pH dependence to hPRL global stability can be rationalized according to the predominant structural interactions of individual histidines in the hPRL tertiary structure. Using double mutant cycles, we detect large interaction free energies within a cluster of nearby histidines, which are both stabilizing and destabilizing to the native state. Finally, by comparing the structural locations of hPRL's nine histidines with their homologous residues in hGH, we speculate on the evolutionary role of replacing structurally stabilizing residues with histidine to introduce pH dependence to cytokine function.     

Somatotropic and lactotropic receptors in transgenic mice expressing human or bovine growth hormone genes

The somatotropic and lactotropic receptors were studied in liver microsomal preparations from transgenic mice carrying the human growth hormone (hGH) or bovine growth hormone (bGH) gene fused to mouse metallothionein-I (MT) or phosphoenolpyruvate carboxykinase promoter/regulator (PEPCK). Specificity studies indicated that, similarly to normal mice, liver microsomes from the transgenic animals possess a mixed population of somatotropic and lactotropic binding sites. In transgenic animals of both sexes, the binding capacity of somatotropic receptors was significantly increased without corresponding changes in affinity. Expression of the MT-hGH hybrid gene was associated with the induction of somatotropic receptors which was approximately twice as great as that measured in animals expressing the MT-bGH hybrid gene. The binding capacity of lactotropic receptors in liver microsomes (quantitated, by the use, of labelled ovine prolactin) was increased 2–3 fold in transgenic females and approximately 10-fold in transgenic males as compared to the respective normal controls. We conclude that lifelong excess of GH up-regulates hepatic GH and prolactin receptors, and that lactogenic activity of GH is not essential for induction of prolactin receptors in the liver of transgenic mice.