The Interaction of Heparin/Polyanions with Bovine, Porcine, and Human Growth Hormone

SANGEETA B. JOSHI, TIM J. KAMERZELL, CHRIS McNOWN, C. RUSSELL MIDDAUGH Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, Kansas 66047

Received 11 February 2007; revised 22 April 2007; accepted 1 May 2007 Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.21056

ABSTRACT: The interaction of polyanions with proteins is of potential pharmaceutical and cellular signicance. A partial thermodynamic description of the interaction of four representative polyanions with human, bovine, and porcine growth hormone is described. A heparin bead-binding assay conrms all growth hormones bind to heparin but to varying extents. Moderate-binding constants and high ratios of bound protein to the more extended polyanions, heparin, and dextran sulfate were measured by isothermal titration calorimetry and dynamic light scattering. The binding constants and ratio of protein bound to ligand were signicantly smaller for the low molecular weight polyanions phytic acid and sucrose octasulfate (SOS). The effect of polyanion binding on the bovine, porcine, and human growth hormones (hGH) structural and colloidal stability was also explored. Heparin and dextran sulfate inhibit porcine somatotropin (pST) and bovine somatotropin (bST) aggregation to the greatest extent, as compared to phytic acid and SOS, while decreasing secondary and tertiary structural stability as measured by the temperature dependence of their circular dichroism and intrinsic uorescence. Somewhat surprisingly, the polyanions do not appear to affect the structure or stability of hGH. The potential biological signicance of growth hormone polyanion interactions is discussed. 2007 Wiley-Liss, Inc. and the American Pharmacists
Association J Pharm Sci

Keywords: thermodynamics; polyanion; heparin; uorescence; circular dichroism; isothermal titration calorimetry; stability; growth hormone

INTRODUCTION
Growth hormones are globular proteins of $191 amino acids.1 Also known as the pituitary somatotropins, they are involved in a wide variety of biological functions including growth and metabolism. The structure of bovine (bST, bovine
Sangeeta B. Joshi and Tim J. Kamerzell contributed equally to this work. Abbreviations: pST, porcine somatotropin; bST, bovine somatotropin; hGH, human growth hormone; DS, dextran sulfate; PA, phytic acid; REES, red edge excitation spectroscopy; ITC, isothermal titration calorimetry; CD, circular dichroism; DLS, dynamic light scattering. Correspondence to: C. Russell Middaugh (Telephone: 785864-5813; Fax: 785-864-5814; E-mail: Middaugh@ku.edu)
Journal of Pharmaceutical Sciences 2007 Wiley-Liss, Inc. and the American Pharmacists Association

somatotropin), porcine (pST, porcine somatotropin), and human growth hormone (hGH) are similar with greater than 90% sequence homology between bST and pST.2 The crystal structure has been solved for a modied Met-pST analogue,3 and for afnity matured,4 and apo wild-type hGH at 2.5 A resolution.5 The structure shows that the growth hormones consist of a four alpha helix bundle motif with a left-handed super-helical twist. All three growth hormones contain lysine, histidine, and arginine rich clusters suggestive of sites for potential electrostatic interactions with negatively charged ligands. In fact, hGH has been shown to interact with the polyanion heparin leading to its stabilization against interfacial unfolding.6 Electrostatic interactions between
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positively charged protein residues and various polyanionic compounds have been shown to signicantly enhance the physical stability and activity of some protein molecules,714 many of them hormones. For example, human FGF1 is unstable at physiological temperatures but its physical stability is markedly increased upon addition of a wide variety of polyanions.13,14 Furthermore, RNA and heparin both bind to overlapping sites of epidermal growth factor like domains of Factor VII activating protease and increase activity15 in similar fashion as the activation of Factor XII by heparin and chondroiton sulfate E.16 It is now well established that in the case of several growth factors, the interaction of the proteins with cell surface proteoglycans is of physiological signicance. It has been proposed and experimental data has been obtained in support of the idea that some growth factors may be presented bound to molecules such as heparan and chondroitan sulfate to their proteinaceous cell surface receptors. This may facilitate the oligomerization of target receptors initiating further signaling events. It has also been postulated that the proteoglycan may serve as storage sites for such hormones with their release by heparinase and related enzymes regulating their interaction with receptors. There is little evidence, however, that the growth hormones are involved in any such activities. In this work, a variety of methods are employed to explore the potential interaction of polyanions with human, bovine, and porcine growth hormones. The polyanions, dextran sulfate ($50000 amu), heparin ($1216000 amu), phytic acid ($923 amu), and sucrose octasulfate (SOS; $1287 amu) were used. Heparin was chosen for its similarity to the cell surface proteoglycans, while the other polyanions were investigated because of their ranges of size and charge density which can be used to probe the effect of varying polyanionicity. The effect of polyanion binding on all three growth hormones, including a partial thermodynamic description, a determination of the number of proteins bound per ligand, associated secondary and tertiary structural changes, and the effect on inhibition of protein aggregation are described. Surprisingly, we nd that bovine and porcine growth hormone bind a variety of polyanions with moderate binding constants but with a reduction of their thermal stabilization, while hGH exhibits much less interaction with all of the polyanions studied.
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EXPERIMENTAL PROCEDURES (MATERIALS AND METHODS)

Materials Bovine growth hormone (bST) was obtained from Monsanto (St. Louis, MO). Porcine growth hormone (pST) from Monsanto and Pzer, Inc., and hGH from Pharmacia, Inc. (Piscataway, NJ). All three proteins produce a single band upon SDS PAGE. Dextran sulfate was purchased from Spectrum (New Brunswick, NJ), heparin and phytic acid from Sigma (St. Louis, MO) and SOS from TRC, Inc. (Ontario, Canada). All other chemicals were of reagent grade and were obtained from Sigma and Fisher Scientic (Pittsburgh, PA).

Methods Protein Sample Preparation Bovine and porcine growth hormones were obtained as lyophilized solids and were stored at 208C. hGH was obtained as a concentrated protein solution in Hepes buffer and was stored at 208C. bST protein solutions were prepared by dissolving bST (1 mg/mL) in bicarbonate buffer (35 mM, pH 9.5) followed by overnight dialysis against Hepes buffer (20 mM Hepes, pH 8.0). The dialyzed protein solution was centrifuged for 5 min at 14000 rpm to remove aggregated protein before concentration determination. Protein concentration was determined at room temperature by absorbance measurement at 280 nm (A280 nm 0.65 at 1 cm, 0.1%) using an Agilent 8453 UVVisible spectrophotometer (Palo Alto, CA) tted with a Peltier temperature controller. The sample solutions of pST and hGH were prepared by diluting the concentrated stock solutions of proteins in 20 mM Hepes buffer, pH 8.0. Heparin Bead-Binding Isotherms Samples were prepared in 20 mM Hepes, pH 8.0 by adding increasing amounts of protein (bST, pST, hGH) to a solution containing 100 uL of heparinagarose beads (Sigma, St. Louis, MO) in 2 mL polypropylene microcentrifuge tubes. The total sample volume was 200 mL. The samples were placed on a rotating mixer for 1 h at 48C and then centrifuged at 14000 rpm for 2 min in a microcentrifuge. The unbound protein in the supernatant was measured by the UV absorbance at 280 nm and was subtracted from the total protein to calculate the amount of heparin bound
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protein. Protein concentration was determined by employing the extinction coefcients of 0.65, 0.66, and 0.85 (A280 nm at 1 mg/mL) for bST, pST, and hGH, respectively. The samples of pST in the presence of heparin beads were also prepared in Hepes buffer containing either 0.15 or 1.0 M NaCl. Isothermal Titration Calorimetry (ITC) All calorimetric titrations were performed using a CSC Model 4200 isothermal titration calorimeter (Calorimetry Sciences, Lindon, UT) at 258C and were performed with full sample (1300 mL) and reference cells, with the sample cell stirrer set at 297 rpm. The reference cell contained puried water and all solutions were degassed before use. ITCRun Software (Calorimetry Science) was used for instrument operation and collection of data. The differential heats of binding were integrated and analyzed utilizing Bindworks 3.0 software. Data collected from all experiments were t to an independent set of multiple binding sites model with the total heat determined by: Q VDH " p# 1 MnK 1 MnK LK 2 4KL L 2K (1)

solution, stirred, and equilibrated for $1200 s before measurements were taken. Five runs at each ratio of macromolecule to ligand were performed, and repeated three times. Particle size distributions were analyzed by multiple methods. Titrations were initially analyzed by the method of cumulants using a gaussian analysis, least squares t. For more complex multimodal distributions, data were analyzed employing a nonlinear, least squares calculation (with a nonnegative constraint) of the inverse Laplace transform of the autocorrelation function. Furthermore, all calculations were performed using both a number and intensity average of the population. The resulting diffusion coefcients were converted to hydrodynamic radii using the Stokes/Einstein equation. Red Edge Excitation Spectroscopy (REES) Red edge excitation uorescence (REE) measurements were conducted with a JASCO FP-6500 spectrouorometer (Tokyo, Japan). Fluorescence emission spectra were collected after varying the excitation wavelength every 1 nm from 285 310 nm. Collection of emission spectral data began 7 nm past the excitation wavelength with the temperature held at 108C. A bandwidth of 3 nm was used for both emission and excitation. The ligands, dextran sulfate and heparin were combined with each growth hormone in a 1:1 molar ratio, while SOS and phytic acid were used at a 1:1 weight ratio ($17:1 and 24:1 molar ratio SOS:GH and PA:GH). Emission maxima were determined utilizing 1st and 2nd derivative analysis. A center of mass determination was also used to nd the emission lmax. For spectral center of mass determination, Eq. (2) was used P lem Fi C:M: P (2) Fi where lem is the emission wavelength, and Fi is the uorescence intensity at the respective emission wavelength. Turbidity Studies The kinetics of protein aggregation was monitored by increases in the turbidity at 360 nm using a Fluostar Galaxy microplate reader (BMG Labtechnologies, Offenberg, Germany). Preliminary studies established solution conditions under which the growth hormones exhibit conveniently measurable aggregation behavior. The aggregation of proteins (bST and pST, 0.02 mM) in Hepes
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where DH is the enthalpy of binding, K is the binding constant, n is the number of binding sites, V is the volume of the cell, [L] the total ligand concentration, and [M] the protein concentration.17 Titrations consisted of 3001200-s equilibration times with 5-min intervals between injections. Titrations of polyanion into macromolecule utilized a titration regimen of 25 10 mL or 40 5 mL. Heats of dilution were accounted for by subtracting the integrated heats of dilution from the binding heats. Dynamic Light Scattering (DLS) Dynamic light scattering measurements were performed with a Brookhaven Model BI200SM instrument (Brookhaven, NY). Light scattering was detected at 908 using a continuous wave 532 nm diode pumped solid-state laser. All samples were ltered prior to titration. DLS measurements were conducted under conditions which simulated the ITC experiments. Ten microliters of ligand were directly injected into the macromolecular
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buffer (20 mM Hepes, pH 8.0) was monitored continuously at 378C in the presence of DTT (nal concentration 30 mM) for 2 h. Aggregation studies of protein samples were performed both with and without polyanions with the aggregation seen with protein alone used as a control. The maximum OD observed in control samples at 2 h was used as the maximum extent of aggregation since aggregation was complete by this time. Inhibition of aggregation by polyanions was then characterized by their ability to lower the maximum OD observed after 2 h. Circular Dichroism Studies CD studies were performed with a Jasco 720 spectrophotometer (Tokyo, Japan) equipped with a Peltier temperature controller. Thermal unfolding experiments were performed at 0.18C intervals using a 158C/h temperature ramp rate to monitor the change in ellipticity at 222 nm as a function of temperature. A protein concentration of 7.0 mM was used in all the studies. Samples of GH containing various polyanions, a 1:1 molar ratio of protein to dextran sulfate and heparin and a 1:1 weight ratio of protein to phytic acid and SOS ($17:1 and 24:1 molar ratio SOS:GH and PA:GH) were contained in a 0.1-cm path length cell sealed with a Teon stopper. A resolution of 0.1 nm and a scanning speed of 20 nm/min with a 2-s response time were employed. The CD signals were converted to molar ellipticity using the Jasco Spectra Manager software. Fluorescence Spectroscopy Fluorescence studies were performed using a PTI Quanta Master Spectrophotometer (Lawrenceville, NJ) equipped with a thermostated cuvette holder. A protein concentration of 9.0 mM was used in all studies and the amount of polyanion was the same as described above. The intrinsic uorescence spectrum of tryptophan was monitored using an excitation wavelength of 295 nm (>95% Trp emission). Emission spectra were collected over a range of 305435 nm. Excitation and emission slits were set at 4 nm and a 1-cm path length quartz cuvette was used in all experiments. The spectra were collected from 108C to 858C at 2.58C intervals with a 5-min equilibration time at each temperature. Buffer baselines were subtracted from each spectrum prior to data analysis. Data analysis was performed using FelixTM (PTI) software. Emission peak positions were determined by rst derivative
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analysis. Reproducibility of the thermal unfolding temperature (Tm, the midpoint of the transition) was 28C.

RESULTS
Binding to Heparin-Conjugated Agarose Beads Initially, the binding of human, bovine, and porcine growth hormones to heparinized beads was conrmed by a simple binding assay. Each growth hormone in increasing amounts was incubated with heparin-derivatized agarose beads and the extent of adsorption was determined by the measurement of protein remaining in solution employing UV absorbance at 280 nm. All growth hormones bound heparin beads but to varying extents (Fig. 1). The amount of protein bound relative to the amount added was greatest for bST, with pST having intermediate values, and hGH binding the least. The binding assay was also conducted in the presence of various concentrations of sodium chloride. The extent of binding of protein with heparin beads decreased with increasing salt concentration (Fig. 1), suggesting the expected involvement of electrostatic interactions in the binding process. ITC Titrations A thermodynamic description of the growth hormones interactions with various polyanions is shown in Table 1. A representative thermogram

Figure 1. Binding isotherms of bovine, porcine, and human growth hormones to heparin beads in 20 mM hepes buffer, pH 8.0 at various ionic strengths. (&) bST without salt, (~) hGH without salt, (&) pST without salt, (~) pST with 0.15 M NaCl, (*) and pST with 1.0 M NaCl. Each point represents an average of three experiments.
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DLS Prot/Sacch

DS (J/mol K)

Table 1. Thermodynamic Analysis of Growth Hormone/Polyanion Interactions

DG (kJ/mol)

Adj.DH (kJ/Charge)

1080 20 436 21 82 20 7 3 1600 100 990 176 12 0.2 NA NA

DH (kJ/mol)

3.4 7.4 10.3 1.2 5 17 1.5 NA NA

37 33 25 35 39 37 31 NA NA

3500 1350 190 94 5200 3200 64 NA NA

2.8E6 2.7E5 5.1E5 2.5E4 2.3E4 4.4E3 4.3E5 2.5E5 6.4E6 1.6E6 3.6E6 1.2E6 2.7E5 9E4 NA NA

K (M1)

of pST (3.71 mg/mL) titrated with dextran sulfate (2.2 mg/mL) at 258C using a program of 25 injections of 10 mL is shown in Figure 2. The heats of dilution were calculated by a blank titration of ligand into dialysis buffer and subtracted in the nal analysis. The integrated peak areas were t to an independent set of multiple binding sites model from which the parameters DH (enthalpy of binding in kJ/mol ligand), K (binding constant), and n (binding stoichiometry) were obtained. Utilizing the equation DG RTlnK, the Gibbs free energy was calculated and subsequently the entropy from DG DHTDS. Because of the large number of binding sites demonstrated for dextran sulfate and heparin, the apparent enthalpy of interaction was divided by the total

ITC Prot/Sacch n

0.02 0.03 0.1 0.3 0.02 0.02 0.4 NA NA

Prot/sacch, proteins bound per saccharide.

Dextran sulfate Heparin Sucrose octasulfate Phytic acid Dextran sulfate Heparin Sucrose octasulfate Phytic acid All

Ligand

1/3 1/1.3 NA NA 1/3 1/0.7 NA NA NA

$1/3 $1/1.5 NA NA $1/3 $1/1 NA NA NA

Figure 2. Representative isothermal titration calorimetry binding isotherm (A) and raw data (B) for pST (3.71 mg/mL) titrated with dextran sulfate (2.2 mg/mL) at 258C using a program of 25 injections of 10 mL.
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Protein

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pST pST pST pST bST bST bST bST hGH

JOSHI ET AL.

number of () charges per mole of ligand. This number should then represent the heat of binding per () charge of ligand. The number of proteins bound per ligand was calculated from the inverse of the stoichiometry. The number of proteins bound per saccaharide was determined by dividing the number of protein molecules bound per ligand by the average number of disaccharides per mole of ligand. Measured enthalpies of interaction for the titration of polyanions into both pST and bST were large and negative. The greatest enthalpic effect was produced by the interaction of protein with the much larger polyanions dextran sulfate and heparin. Dextran sulfate and heparin possess a relatively greater number of negative charges per molecule than the smaller polyanions phytic acid and SOS. This large number of charges should result in more electrostatic binding sites and a greater enthalpic contribution to binding. Even when normalized on a single charge basis, the heats of binding of the larger polysaccharides are greater than the smaller ones (Tab. 1). Upon interaction, the two components of the biomolecular complex may change conformation resulting in a more negative or positive DS. The complex itself could become more tightly folded, thereby reducing internal degrees of freedom resulting in a less favorable DS. In contrast, translational and rotational motion may increase, resulting in a more positive entropy contribution. Thermodynamic parameters are state functions, and the measured enthalpy and calculated entropy are the sum of all processes that occur in the reaction process. Thus, it is inherently difcult to assign individual contributions of binding phenomenon to the individual thermodynamic parameters. Stoichiometric measurements of the biomolecular interaction are also possible through the use of ITC. Varying degrees of binding stoichiometry were measured for complexation of bST and pST with the polyanions (Tab. 1). The much larger polyanions dextran sulfate and heparin, bind large numbers of protein molecules. A similar result was observed for aFGF bound to heparin.10 Approximately 1 molecule of pST was bound for every 3 monosaccharides of dextran sulfate, and 1.25 monosaccharides of heparin, 1 bST for every 3 monosaccharides of dextran sulfate and 1 of heparin. Similarly, converting to bound proteins per ligand results in approximately 40 proteins per ligand for pST DS, 25 proteins per ligand for
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pST Heparin, 40 bST molecules per dextran sulfate, and 40 bST molecules per heparin chain. Measurements of the association constants, (Ka), were also compared. As indicated above, an ITC experiment is based on measuring the total enthalpy change. Therefore, K values are only apparent binding constants. Moderate to strong binding constants were observed for all growth hormone polyanion interactions ranging from 103106 M1. The strongest afnity was observed for the interaction of dextran sulfate and heparin with protein (Tab. 1). Undetectable heats of binding were observed for hGH as well as phytic acid binding to bST, no doubt reecting the weak interaction in these cases. The major binding event in all of the polyanion protein interactions of this study is probably electrostatic in nature. In some situations, macromolecules that bind charged compounds are entropically favored with minor DH values. This can be explained by the release of water molecules solvating the free-charged compounds. There are, however, examples of electrostatic interactions between macromolecules and ions that are enthalpically driven.1820

Dynamic Light Scattering DLS measurements were performed to determine the polyanion/protein complex size and conrm the stoichiometry estimated by the ITC experiments. Light scattering experiments were designed to simulate the conditions of the ITC titrations employing the same concentrations of macromolecule and ligand. Binding stoichiometry was determined based on the saturation point or plateau where no further increase in diameter of the polyanion\protein complex was observed. The stoichiometry based on DLS experiments was of the same magnitude as that determined by ITC, $40 proteins per ligand for pST DS, $25 proteins per ligand for pST Heparin, $40 bST molecules per dextran sulfate, and 40 bST molecules per heparin chain. This value was calculated by determining the molar ratio of proteins bound per ligand based on the injection point where saturation is reached (approximately the 7th injection for pST DS) (Fig. 3). The effective diameter versus injection number is presented in Figure 3. DLS measurements of growth hormones in the presence of SOS and phytic acid were inconclusive as to the nature of the stoichiometry. Consistent
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with low energy quanta (red edge) a small distribution of uorophores will be selectively excited assuming equilibrium is not attained and solvent relaxation is not complete. The emission energies of the uorophores will necessarily be lower than the mean of the distribution and the emission spectra will be shifted toward longer wavelengths.22 All three growth hormones show a signicant red shift upon increasing excitation wavelength (results not shown). Thus, the tryptophan residue(s) appear to be positioned in a conformationally restricted environment. When polyanions are added, however, no change in the extent of the peak position shift is observed, arguing that ligand binding has little or no effect on the proteins conformational dynamics, at least in the vicinity of the single indole side chain.
Figure 3. Hydrodynamic diameter observed versus injection number of polyanions for pST employing dynamic light scattering. Each data point represents the mean () standard error of three samples of protein ligand complex formed by sequential titration of 10 mL of 1.5 mg/mL heparin (~), 2.5 mg/mL dextran sulfate (&), 1 mg/mL phytic acid (), or 1 mg/mL sucrose octasulfate (^), into 4 mg/mL pST.

Aggregation Studies To further examine the interactions between the three growth hormones and polyanions, an aggregation-based turbidity assay was developed to produce conditions that could achieve signicant aggregation. This technique monitors the physical stability of proteins by following the kinetics of aggregation of structurally altered protein by measurement of the degree of turbidity (light scattering) of the solution at 360 nm. It was found that elevated temperatures (378C) and the presence of DTT induced signicant aggregation of two of the proteins. All polyanions produced some degree of inhibition of bST and pST aggregation with almost 100% inhibition observed in the presence of dextran sulfate and heparin. Various ratios of protein to polyanions were tested (Tab. 2) with an example of the aggregation behavior (pST in the presence of polyanions at a 1:1 molar or weight ratios of protein to polyanion) shown in Figure 4. The larger polyanions were potent inhibitors of aggregation even at very low molar ratios (Tab. 2). The inhibitory effect of phytic acid appeared to decrease with an increasing weight/molar ratio of phytic acid to protein. An inhibition assay was not developed for hGH due to the inherent stability of the protein except under extreme conditions of temperature.

with other methods of analysis, DLS measurements of hGH upon titration with polyanions showed no signicant increase in Stokes radius upon addition of any ligand over the range of concentration employed.

Red Edge Excitation Shifts (REES) Red edge excitation spectroscopy was employed to probe the conformational dynamics of the protein before and after binding of polyanions. Fluorescence emission spectra are usually thought to be independent of excitation wavelength. Excitation dependent spectra, however, have been observed for a large number of proteins.2124 Monitoring the peak position at excitation wavelengths above 290 nm will provide information concerning the microenvironment of these tryptophan residues as well as their dynamics. Upon excitation, the uorophores dipole moment changes and solvent dipoles can reorient in response. Solvent relaxation may occur faster, slower, or simultaneously with emission decay. When relaxation occurs slower than or on the same time scale as emission, a complex excitation-dependent emission spectrum may be observed. When excitation occurs
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Intrinsic Fluorescence The effect of polyanions on the thermal stability of bST, pST, and hGH was monitored using the
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Table 2. Percent Inhibition of Protein Aggregation by Polyanions (Polyanion:Protein) Molar Ratio 0.1 1.0 2.5 0.1 1.0 2.5 0.04 0.2 0.4 0.06 0.3 0.59 (Polyanion:Protein) Weight Ratio 0.23 2.3 5.7 0.06 0.64 1.6 1.0 5.0 10.0 1.0 5.0 10.0 % Inhibition (bST) 96 97 92 94 99 99 69 57 35 55 64 64 % Inhibition (pST) 108 100 98 95 103 95 91 76 35 57 59 47

Polyanion Dextran sulfate Dextran sulfate Dextran sulfate Heparin Heparin Heparin Phytic acid Phytic acid Phytic acid Sucrose octasulfate Sucrose octasulfate Sucrose octasulfate
Errors less than 1%.

temperature dependence of the proteins intrinsic tryptophan uorescence. A stabilizer or a destabilizer is identied when an increase or a decrease, respectively, in the transition temperature (Tm) of the protein is observed in the presence of that compound. In addition, the effect of polyanions on the aggregation of these proteins was also examined by simultaneously measuring the scattered light at the wavelength of excitation at 1808 from the uorescence signal. The emission wavelength maximum of bST at room temperature is 321 nm. The change in uorescence intensity of bST as a function of temperature was monitored at 321 nm in the presence of various polyanions (Fig. 5B). The uorescence intensity of bST increases around

Figure 4. Time course of pST turbidity formation in the presence of various polyanions on the aggregation of pST at 378C. Samples contained 400 mg/mL protein (20 mM) in hepes buffer, pH 8.0 with (^) no polyanion; (&) dextran sulfate; (~) heparin; (o) pST phytic acid, and () pST sucrose octasulfate.
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$608C with a transition occurring between 608C and 708C. The thermal stability of bST is signicantly reduced in the presence of various polyanions, as indicated by a shift in Tm values toward lower temperatures (Tab. 3). The change in uorescence emission maxima (peak position) of bST as a function of temperature in the presence of polyanions is shown in Figure 5C. A red shift in the peak positions from 321 to 329 nm with increases in temperature is observed. This corresponds to an increase in solvent exposure of the tryptophan side chains at elevated temperatures. This small shift suggests that bST is not extensively unfolded but a more subtle change in tertiary structure is occurring. In contrast to the uorescence intensity results, however, the peak shift versus temperature plots looked quite similar in the presence and absence of polyanions with a transition occurring at the much lower temperature of approximately 408C. The light scattering at right angles exhibits a sharp increase above 558C for bST alone and in the presence of SOS and phytic acid, indicative of thermally induced aggregation (Fig. 5D). The scattering intensity did not, however, change signicantly with temperature in the presence of dextran sulfate and heparin (Fig. 5D) suggesting a marked inhibition of bST aggregation upon addition of these polyanions. Plots of uorescence intensity as a function of temperature suggest a transition around 708C for unliganded pST. As seen with bST, the Tm of pST determined by this method was decreased in the presence of polyanions with the much larger polyanions decreasing the Tm more than PA and SOS (Fig. 6B and Tab. 3). A temperature-induced
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Figure 5. Effect of various polyanions on the thermal unfolding of bST as measured by circular dichroism and uorescence spectroscopy. (A) Molar ellipticity at 222 nm of (a) bST, (b) bST dextran sulfate, and (c) bST heparin. (B) Fluorescence intensity, (C) wavelength of emission maximum, and (D) light scattering intensity of (^) bST, (&) bST dextran sulfate, (~) bST heparin, (o) bST phytic acid, and () bST sucrose octasulfate.

red shift occurs with pST alone and in presence of all polyanions from $324 nm to 333 nm at approximately 368C when emission peak position is monitored but no stabilization by polyanions is seen (Fig. 6C). The relative light scattering intensity of pST exhibits a marked increase above 658C when measured alone or in the presence of phytic acid and SOS (Fig. 6D). The scattering was dramatically reduced with the addition of the larger ligands, again indicating stabilization against aggregation.
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hGH was affected the least of the growth hormones by addition of the various polyions. hGH is very stable in solution and shows little to no aggregation upon increasing the temperature up to 858C. The expected decrease with temperature in the uorescence intensity of hGH was observed both in the presence and absence of polyanions (Fig. 7B), although suggestions of a small transition are evident above 808C. A small red shift was observed ($3 nm) for hGH in the presence and absence of polyions (Fig. 7C)
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Table 3. Transition Midpoint (Tm) of Growth Hormones Alone and in the Presence of Polyanions Method Circular dichroism Polyanion Protein alone Dextran sulfate Heparin Phytic acid SOS Protein alone Dextran sulfate Heparin Phytic acid SOS Protein alone Dextran sulfate Heparin Phytic acid SOS Transition Midpoint (8C) bST 66 53 59 65 65 43 45 42 43 41 63 51 53 58 60 Transition Midpoint (8C) pST 74 60 71 74 72 35 38 37 36 37 70 55 53 66 65 Transition Midpoint (8C) hGH 85 85 85 85 85 NDa ND ND ND ND ND ND ND ND ND ND

Fluorescence lmax shift

Fluorescence intensity

Errors are approximately 12%. a Not determined.

indicative of a structural transition or change in Trp environment due to a direct effect of the ligand. The relative light scattering intensity of hGH in the presence or absence of polyanions did not change signicantly with temperature (Fig. 7D). Overall, the addition of polyanions does not appear to signicantly perturb the secondary or tertiary structure of hGH.

transition temperature upon addition to pST (Tab. 3). As now expected, none of the polyanions appeared to have a measurable effect on the thermal behavior of hGH (Fig. 7A, Table 3).

DISCUSSION
The interaction of proteins with polyanions is a phenomenon of increasingly wide interest.9 Despite the importance of the mammalian somatotropins and their correspondingly extensive biophysical characterization, with the exception of a single report,6 there is little recognition that these proteins fall into this category. The biological signicance of such polyanion/protein interactions are still unclear although there is growing recognition that they are important, playing a role in a number of activities such as storage, transport, protein folding, and delivery. Unfortunately, a direct test of the biological relevance of the interactions seen in this article is difcult since the relatively moderate binding constants of the polyanions for the GHs results in rapid dissociation when they are introduced into a biological milieu or cell culture. This would not necessarily be the case on the surface or inside cells, however. The potential use of polyanions in therapeutic protein dosage forms is wide ranging. As previously
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Circular Dichroism The effect of temperature on the secondary structure of bST, pST, and hGH in the presence of various polyanions was analyzed by changes in CD ellipticity at 222 nm. CD measurements show a thermal transition near 608C for bST alone. In the presence of the larger polyanions, dextran sulfate and heparin, the transition begins at the lower temperatures of 498C and 568C, respectively (Fig. 5A), suggesting destabilization of bST by these polyanions. In contrast, the low molecular weight polyanions phytic acid and SOS did not appear to have a signicant effect on the Tm value and hence on the thermal stability of bST (Tab. 3). Dextran sulfate appeared to have a major destabilizing effect on pST, as demonstrated by a large downward shift in the Tm value (approximately 148C, Fig. 6A). The other polyanions did not produce a major effect on the
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11

Figure 6. Effect of various polyanions on the thermal unfolding of pST as measured by circular dichroism and uorescence spectroscopy. (A) Molar ellipticity at 222 nm of (a) pST, (b) pST dextran sulfate, and (c) pST heparin. (B) Fluorescence intensity, (C) wavelength of emission maximum, and (D) light scattering intensity of (^) pST, (&) pST dextran sulfate, (~) pST heparin, (o) pST phytic acid, and () pST sucrose octasulfate.

mentioned, the thermal stability of many proteins is increased upon polyanion addition. The addition of polyanions to growth hormones, however, results in decreased Tm values. Decreased transition midpoints do not in themselves necessarily indicate an inferior formulation. For example, in this work and others, polyanions have been shown to signicantly inhibit protein aggregation. Protein self-association is one of, if not the major pathway of physical degradation of protein pharmaDOI 10.1002/jps

ceuticals. In addition, polyanions may protect many proteins from mechanical stresses encountered during manufacturing such as shipping, lling, etc. (e.g., agitation shear-stress). The polyanion/protein interaction seen for bovine and porcine growth hormones results in signicant exothermic (enthalpic) contributions to the binding equilibrium. The greatest enthalpic effect was produced by the interaction of protein with the larger polyanions dextran sulfate and
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Figure 7. Effect of various polyanions on the thermal unfolding of hGH as measured by circular dichroism and uorescence spectroscopy. (A) Molar ellipticity at 222 nm of (a) hGH, (b) hGH dextran sulfate, and (c) hGH heparin. (B) Fluorescence intensity, (C) wavelength of emission maximum, and (D) light scattering intensity of (^) hGH alone, (&) hGH dextran sulfate (~), hGH heparin, (o) hGH phytic acid, and () hGH sucrose octasulfate.

heparin. Dextran sulfate and heparin possess a relatively greater number of negative charges than the smaller polyanions phytic acid and SOS. Therefore, a greater number of charges would result in potentially more electrostatic binding contacts and greater enthalpic contribution to binding despite the well-known entropic contribution to electrostatically driven binding interJOURNAL OF PHARMACEUTICAL SCIENCES

actions. There are examples of electrostatic interactions between macromolecules and ions that are enthalpically driven,1820 including binding of a target peptide to the calmodulin binding site of rabbit smooth muscle myosin light chain kinase.25 Moreover, the degree of the entropy change may be sensitively dependent on the number of binding sites on the macromolecule or
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ligand. The enthalpic contribution to the binding event increases with increasing number of () charged protein residues (bST polyanion > pST polyanion ) hGH polyanion). Even on a per charge basis, binding of the larger polyanions is greater than the smaller one, perhaps suggesting

positively cooperative binding. A large number of growth hormones appear to bind to the larger polyanions. This may seem surprising at rst, however, a number of studies indicate that multiple proteins can bind to single polysaccharide chains.10,26,27 Under such conditions,

Table 4. Multiple Sequence Alignments of Human, Bovine, and Porcine Somatotropins Using all Default Clustal W Parameters

Positively charged residues Histidine, Lysine, and Arginine are highlighted in pink.

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micro-aggregated states are probably formed in which multiple proteins bind to extended polyanion chains which become crosslinked. Dynamic light scattering experiments demonstrate a heterogeneous distribution of particle sizes supporting the existence of such micro-aggregated states and prohibiting a more rigorous analysis of the thermodynamic data. In the presence of dextran sulfate and heparin, bST and pST showed a marked reduction in the thermal transition temperature as measured by uorescence. Interestingly, plots of the uorescence intensity (Figs. 5B and 6B) as a function of temperature indicate different midpoints of thermal unfolding as compared to both uorescence peak position (Figs. 5C and 6C) and molar ellipticity (Figs. 5A and 6A) as a function of temperature. Fluorescence and CD measurements support the idea that the secondary and tertiary structure of both bST and pST is altered upon interaction with polyanions. Dextran sulfate and heparin, however, inhibit aggregation of bST and pST as indicated by a decrease in light scattering intensity (Figs. 5D and 6D). Furthermore, a signicant red shift of the emission

maximum is observed with bST and pST indicating increased solvent exposure of the tryptophan residues. Surprisingly, however, the polyanions do not appear to signicantly affect the physical stability or secondary and tertiary structure of hGH. The uorescence emission maximum as a function of temperature for hGH alone and in the presence of polyanions shifts approximately 3 nm indicating some alteration of the tryptophan environments (Fig. 7C), although polyanions do not appear to alter the magnitude of the shift. It is clear, however, that hGH binds heparin, albeit signicantly more weakly than the other two proteins. The mechanism of growth hormone aggregation and inhibition of aggregation by polyanions is not directly investigated in this work. It may be hypothesized that polyanions bind to a more native-like conformation of the growth hormone reducing the number and diversity of partially unfolded protein states. These partially unfolded ensembles may be prone to self-association. Another possible explanation for the inhibition of growth hormone aggregation upon polyanion binding involves charge repulsion. The high

Figure 8. Calculated electrostatic potential maps using the online server protein continuum electrostatics (http://bioserv.rpbs.jussieu.fr/PCE) of bovine somatotropin with positive potentials shown in blue and negative potentials shown in red. Calculation parameters include 4 as the protein internal dielectric, 80 as the solvent dielectric constant, and 0.1 as the ionic strength.
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Figure 9. Calculated electrostatic potential maps using the online server protein continuum electrostatics (http://bioserv.rpbs.jussieu.fr/PCE) of human somatotropin with positive potentials shown in blue and negative potentials shown in red. Calculation parameters include 4 as the protein internal dielectric, 80 as the solvent dielectric constant, and 0.1 as the ionic strength.
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negative charge density of polyanions may prevent aggregation because of unfavorable energetics upon self-association. Steric hindrance may also prevent protein aggregation. These specic polyanions are highly exible and proteinprotein interactions could be blocked by bulky chains. The temperature dependence of the uorescence emission maximum (lmax) and uorescence intensity ( Fi) are two different physical properties that may be dissimilar depending on the specic changes in protein structure and uorophore environment. Therefore, it is not surprising that we observe different results from these two measurements. The temperature dependence

of lmax and Fi both reect changes in the Trp environment, but they may not necessarily directly correlate. For example, Lys residues may inuence the uorescence intensity by quenching with relatively little effect on the emission maximum. Furthermore, changes in the local dielectric constant and permitivity surrounding indole side chains may inuence the lmax to a much greater extent compared to uorescence intensity. A number of cellular polyanions are thought to mediate protein/receptor interactions. For example, the interaction of the FGFs with the FGFRs requires heparan sulfate glycosaminoglycans.

Figure 10. Molecular surface representations of (A, B) bovine somatotropin, (C) human somatotropin bound to two receptors, and (D) human somatotropin alone with the residues histidine, lysine, and arginine shown in blue and labeled. Visual molecular dynamics (VMD) was used to produce the surface representations. Protein Data Bank structures 1bst, 1hgu, and 3hhr were used for bovine somatotropin, human somatotropin, and the human growth hormone receptor complex, respectively.
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Heparan sulfate binds both the receptor and FGF facilitating the formation of a large FGF/FGFR/ HSPG complex essential for signaling.28,29 Many examples exist of this phenomenon.8,30,31 The sites of polyanion binding have in many cases been determined from X-ray crystallography and NMR studies and the general conclusion is that polyanions bind to clusters of basic amino acids on the surfaces of such proteins. In some instances, receptor interaction with the target protein results in the alignment of a contiguous patch of basic amino acid residues facilitating the docking of polyanionic proteoglycans. In addition to mediating cell signaling, cellular polyanions can interact with proteins in a wide range of biologically functional situations. Heparan sulfate proteoglycans regulate the formation of concentration gradients, and localize the response and strength of signaling of many proteins including growth factors and chemokines.8,30,32 It is possible that growth hormones interact with cellular polyanions in a functionally similar context to that of the broblast growth factors, many chemokines and other proteins, but this remains to be directly demonstrated. A multiple sequence alignment of bovine, porcine, and human somatotropin is shown in Table 4 with the basic amino acid residues highlighted. The number of lysine, arginine, and histidine residues is greatest for bST,28 followed by pST27 and hGH.22 This decrease in the polycationic nature of hGH compared to the other two proteins can also be seen on the surface of hGH as observed by electrostatic surface representations (Figs. 8 and 9). Two patches of basic residues in the surface of pST and bST involving Lys30 and Arg34, as well as Arg108 and Lys112 are absent in hGH. These observations may help explain the reduced interaction of hGH with the polyanions studied. An integrated bioinformatics software program, the Sting Millenium Suite,3335 was used to further probe the nature of GH basic residues by determining accessible surface areas and internal residue contacts. The solvent accessible surface area is quite high for the basic residues missing in hGH with values of 124.5, 157.2, 160.5, and 174.7 for K30, R108, R34, and K112, respectively. Potential polyanion binding hot spots are shown in Figures 810 and it appears that bST and hGH may share a common potential polyanion binding surface. Common clusters of basic residues include R17, 166, 177,183 and K167, 171 of bST and R16, 167,178,183 and K168, 172 of hGH. The
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hGH receptor complex, as revealed by crystallographic studies,36 suggests a potential polyanion binding site across the face of the complex involving a patch of basic amino acid residues (Lys121,167 and Arg 71,126,183,213) (Fig. 10CD). In vivo, one growth hormone molecule binds and dimerizes two receptor molecules in a sequential fashion,3742 initially via a high afnity event then through a relatively low afnity allosterically coupled interaction.43 Although the mechanism of GHR activation is still debated, it has been shown that alignment of the receptor molecules may be important in signaling and that dimerization alone is insufcient to activate GHR.44,45 Thus, polyanions might facilitate the formation and alignment of the hGH receptor complex in a similar fashion to the FGF/FGFR/HSPG complex by binding simultaneously to both proteins.

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