©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Rational Design of a Mouse Granulocyte Macrophage- Colony-stimulating Factor Receptor Antagonist (*)

(Received for publication, June 6, 1994; and in revised form, November 4, 1994)

Scott W. Altmann Robert A. Kastelein (§)

From the Department of Molecular Biology, DNAX Research Institute of Molecular and Cellular Biology, Palo Alto, California 94304-1104

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Mouse granulocyte-macrophage colony-stimulating factor (mGM-CSF) proteins with substitutions at residues in the first alpha-helix were examined for biological activity and receptor binding properties. Substitution at the buried residue His affected both bioactivity and receptor binding. Of the four surface-exposed positions examined (Arg, Lys^14, Lys, and Glu) only substitutions at Glu impaired bioactivity. Proteins with charge reversal substitutions at this position were partial agonists and weak antagonists of native mGM-CSF action. All substitutions at Glu abrogated high affinity binding. Lys^14 and Lys substitution proteins showed various receptor binding defects. Qualitative and quantitative measurement of these binding defects identified Lys^14 as a residue that interacts specifically with the beta subunit of the mGM-CSF receptor, whereas Lys appeared to exist at the GM-R alpha-subunit/GM-R beta-subunit interface as substitutions at this position produce both high and low affinity binding losses. These determinations permitted the design of a more potent mGM-CSF antagonist.


INTRODUCTION

Granulocyte-macrophage colony-stimulating factor (GM-CSF) (^1)is a pleiotropic cytokine made by various cell types, such as T cells and monocytes, and is likely to play an important role in the regulation of hemeatopoiesis(1, 2) . GM-CSF interacts with specific high affinity cell surface receptors (GM-Ralphabeta, K 5 times 10M) that are complexes containing at least two subunits, both of which are members of the cytokine receptor superfamily(3) . The receptor alpha chain (GM-Ralpha) binds GM-CSF specifically with low affinity (K 3 times 10M), whereas the receptor beta chain (GM-Rbeta), common to the receptors for GM-CSF, IL-3, and IL-5, does not bind this protein detectably by itself, but confers high affinity binding when co-expressed with GM-Ralpha(4, 5, 6) . Formation of this complex with GM-CSF is required for receptor activation and cellular signaling(7) .

Structurally, GM-CSF has been characterized as a four anti-parallel alpha-helical bundle protein(8) . This topological fold is shared by many of the known cytokine structures, including interleukins-2(9) , -4 (10) , and -5(11) , macrophage colony-stimulating factor(12) , and growth hormone(13) .

Molecular genetic studies and studies with neutralizing anti-GM-CSF antibodies have identified regions and residues in both mouse and human GM-CSF that interact with specific subunits of GM-Ralphabeta(14, 15, 16, 17, 18, 19, 20, 21) . These studies suggest the functional importance of the first and fourth helix of GM-CSF; in particular, the N-terminal helix of mGM-CSF interacts directly with mGM-Rbeta in the context of mGM-Ralphabeta to form the high affinity ligand-receptor complex(15) . Alanine scanning mutagenesis of this region identified Glu as essential for this interaction in both mouse and human GM-CSF(16, 17, 18) . Although substitution of Glu with alanine in mGM-CSF results in a loss of high affinity binding, biological activity remains essentially intact. The biological activity of hGM-CSF Glu substitution proteins is reduced, and a basic side chain at this position causes the most severe defect(18) .

In this report we examine further the biological and receptor binding properties of mGM-CSF mutant proteins with substitutions at residues in the first helix of mGM-CSF. We show that all Glu substitution proteins display only low affinity binding, but that the biological activity depends on the nature of the substituted side chain. Substitutions at several other examined residues lead to a loss in either high or low affinity binding. We also show that this knowledge can be used to design mGM-CSF derivatives with antagonistic properties.


MATERIALS AND METHODS

Bacterial Host Strains, Cell Lines, and Vectors

The Escherichia coli K12 strains XL-1 (Stratagene) and JM101 (22) were used as host strains for the propagation and maintenance of plasmid and M13 DNA. Strain CJ236 (23) was used to prepare uracil-DNA for use in site-directed mutagenesis procedures. M13mp19 containing the ompA leader sequence and the entire mGM-CSF gene was used as the template for site-directed mutagenesis(24) . Strain AB1899 (25) was used as the host for expression of mutant GM-CSF proteins. The plasmid pINIIIompA2 (26) was used as the expression vector for mutant GM-CSF proteins. The use of this secretory E. coli system to express biologically active, mature GM-CSF has been described elsewhere (27) . A mouse GM-CSF-dependent myeloid leukemia cell line, NFS60, was maintained in RPMI 1640 medium supplemented with 5% (v/v) fetal bovine serum and 50 µM 2-mercaptoethanol in the presence of mGM-CSF (1 ng/ml) (Schering-Plough).

Construction of mGM-Ralpha Expression Plasmid and Transfection to Mouse L-cells

The mouse GM-Ralpha clone 71 cDNA (6) (kindly provided by L. Park, Immunex) was inserted downstream of the SRalpha promoter in the expression vector pME18-Neo, a neomycin-resistant derivative of the vector pCEV4 (28) (kindly provided by A. Miyajima, DNAX). Mouse L-cells washed with 20 mM HEPES-buffered saline (pH 7.1) were resuspended at 10^7 cells/ml, and 50 µg of linearized plasmid DNA was added to 0.8 ml of cell suspension in a 0.4-cm electroporation cuvette (Bio-Rad). Electroporation was performed using a Gene Pulser (Bio-Rad) at 960 microfarads and 400 V. Transfectants were selected with G418 (1.5 mg/ml) (Schering-Plough) and cells expressing mGM-Ralpha were confirmed by their ability to bind I-mGM-CSF.

Mutagenesis, Recombinant DNA, and Sequencing Protocols

Site-directed mutagenesis was performed using the method described by Kunkel et al.(23) . Oligonucleotides, 21 nucleotides in length, corresponding to mGM-CSF sequences incorporating the desired amino acid substitution were used as primers in the mutagenesis reactions. Individual clones were sequenced using the method and modifications described in the Sequenase (United States Biochemical Corp., Cleveland, OH) protocol(29) . M13 (replicative form) DNA containing confirmed mutations was digested with XbaI and BamHI and subcloned into pINIIIompA2.

Preparation and Quantitation of Protein Samples

Expression and quantitation of mutant proteins has been described previously(24) . Briefly, mutant proteins were expressed in E. coli AB1899 and periplasmic extracts were generated by osmotic shock. The concentration of mutant protein in the periplasmic extracts was determined by quantitative immunoblot analysis. Extracts were titrated along with a standard curve of purified E. coli derived recombinant GM-CSF. Either hybridoma mg 1.8.2 (30) or 22E9 (31) were used as primary antibody, and the secondary antibody was I-labeled sheep anti-rat IgG (Amersham Corp.). The amount of immunoreactive protein was quantified by integrating the area times density from an exposed screen using a PhosphorImager (Molecular Dynamics). The error in the calculated concentrations of GM-CSF mutant proteins by this method did not exceed 2-fold based on repetitive analysis of individual samples.

Biological Assays for GM-CSF Activity

Protein extracts were assayed for their ability to stimulate the proliferation of the mouse GM-CSF- dependent cell line NFS60. Samples were titrated in quadruplicate using serial 3-fold dilutions. The 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) (Sigma) assay (32) was used to measure the extent of proliferation, and the absorbance value difference of 570-650 nm was measured with a V(max) kinetic microplate reader (Molecular Devices). The concentration of each mutant and wild-type mGM-CSF protein that gave 50% maximal response was determined using the Softmax program (Molecular Devices). The correlation coefficient of the 4-parameter curve fit to the measured values exceeded 0.990 for all curves presented. Protein extracts were assayed for their ability to antagonize the GM-CSF proliferative response of NFS60 cells. Samples were titrated in quadruplicate using serial 3-fold dilutions in the presence of 3.5 times 10M mGM-CSF using the MTT assay format.

Competitive Receptor Binding Assays of Mutant GM-CSF Proteins

Mouse GM-CSF was labeled with I using the Bolton and Hunter reagent (Amersham; specific activity 400 mCi/mmol) to a specific activity of 20-40 µCi/µg as described previously(16) . Competition binding assays were performed as follows: NFS60 cells maintained in media containing mGM-CSF were harvested, washed, and incubated with 1 ml of ice-cold 10 mM NaPO(4), 150 mM NaCl (pH 3.0) for 2 min then diluted to 50 ml with 10 mM NaPO(4), 150 mM NaCl (pH 7.0). Following centrifugation the cells were resuspended in 1 times Hanks' balanced salts solution (Life Technologies, Inc.) containing 0.1% bovine serum albumin, 0.02% NaN(3), and 10 mM HEPES (pH 7.5) (HBAH buffer). 2.5 times 10^6 cells in 200 µl of HBAH buffer were incubated with decreasing concentrations of unlabeled competitor in the presence of 3.0 times 10MI-mGM-CSF at 4 °C with continuous agitation for 4 h. Samples were titered in triplicate using serial 3-fold dilutions. Nonspecific binding was determined from the data collected for the competition of unlabeled wild-type mGM-CSF. Cell-bound radioactivity was separated from free ligand by centrifugation at 4 °C (2 min, 12,000 times g) through dioctylphthalate/dibutylphthalate (2:3). Bound and total radioactivity was measured with a Cobra 5010 -counter (Packard). Low affinity receptor competition binding assays were performed in a similar manner except 7.5 times 10^5 mouse L-cells expressing mGM-Ralpha were incubated with unlabeled competitors in the presence of 2.0 times 10MI-mGM-CSF for 2 h. The equilibrium binding data were analyzed using the Ligand program (33) . Curve fitting for both one- and two-site models were performed on each sample. Curves were either fit using a fixed high and low affinity equilibrium binding constants (K; 5.0 times 10M, K; 3.0 times 10M) or by fitting the control wild-type mGM-CSF and the mutant protein competition curve simultaneously. Binding curves are presented as the concentration of competitor versus the specific counts/minute bound as a percentage of total bound. Analysis of the binding data for Lys substitutions exposed a limitation of the Ligand software. Substitutions for Lys showed a range of defects in high affinity binding. For substitutions resulting in small changes a two site model with reliable high and low affinity K values was statistically significant. For substitutions with reduced high affinity binding approaching that of the low affinity, the Ligand program was unable to discriminate a two-site model. This is reflected in the unreliable binding constants with small F and large p (>0.05) values as well as high affinity K values with values higher than would be expected from examination of the binding curves (Table 1). We presume that this is the result of combining both high and low affinity values into a single binding constant. Only by measuring the affinity of Lys substitution proteins for the low affinity receptor alone (mGM-Ralpha) were we able to asign binding defects of Lys mutant proteins to the mGM-Rbeta (Table 4).





Mouse GM-CSF Molecular Modeling

The coordinates for hGM-CSF were kindly provided by Dr. A. Karplus (Cornell University, Ithaca, NY). The mouse GM-CSF Calpha backbone conformation was modeled using SegMod (34) followed by refinement of side chain conformations using the program CARA (35) within the LOOK suite of programs (Molecular Applications Group, Palo Alto, CA). The structure was displayed using Insight II software (Biosym Technologies, San Diego, CA).


RESULTS

Biological Response of mGM-CSF Mutant Proteins

A panel of amino acid substitutions was generated for selected residues in the first alpha-helix of mGM-CSF. Based on previous studies (16) the following residues were targeted: Arg (R11), Lys^14 (K14), His (H15), Lys (K20), and Glu (E21). Position Glu, although surface exposed and located between Glu^14 and Glu, was not included in this analysis, since all available evidence points to at best a minor involvement of this residue(15, 16) . Mutant protein was produced in E. coli and quantitated as described previously(24) . Biological activity was assessed by measuring the proliferative response of the mGM-CSF-dependent myeloid cell line NFS60. Of the four surface-exposed residues (Arg, Lys^14, Lys, and Glu), only substitution of Glu significantly altered the biological activity of mGM-CSF (Fig. 1, panel 5A, Table 3). Charge reversal substitutions at this position (E21R and E21K) reduced the biological activity to <1% and reduced the magnitude of the plateau. Several other Glu substitution proteins (Ala, Gly, Ser, and Leu) had lower biological activity (10-30%), although the magnitude of the response was not affected. All other substitutions at these four positions had only a marginal effect on the biological activity (>30%) with the exception of proline substitutions of Lys and Glu. The introduction of proline residues at these positions in the first alpha-helix reduced the activity of the resulting proteins to less than 1% (Fig. 1, panels 4A and 5A, Table 2and Table 3). The magnitude of the plateau for E21P but not K20P was reduced. Proline substitutions of Arg and Lys^14, located just outside and just within the first alpha-helix, respectively, did not significantly alter the biological activity (Fig. 1, panels 1A and 2A, Table 1).


Figure 1: Biological activity and competition binding of wild-type and mutant proteins. A, proliferation of NFS60 cells was measured as a function of protein concentration using the MTT assay(32) . Assays were performed in quadruplicate with symbol error bars indicating standard deviation. Curves were generated using a 4-parameter logistical fit and all correlation coefficients exceeded 0.990. B, competition of I-mGM-CSF on NFS60 cells were performed with a constant concentration of 3.0 times 10MI-mGM-CSF in triplicate with symbol error bars indicating standard deviation. Under these conditions both high and low affinity sites were detected using the Ligand program (33) . Each pair of A and B panels represents multiple amino acid substitutions for a specific residue as indicated Arg (1), Lys^14 (2), His (3), Lys (4); and Glu (5). Substitutions are represented by each symbol as follows: bullet, wild-type; , Gly; , Met; Pro; circle, Ala; box, Leu; , Val; &cjs0513;, His; , Phe; down triangle., Trp; ., Tyr; up triangle, Gln; down triangle, Ser; &cjs1730;., Arg; , Glu; &cjs1730;, Lys.







Based on homology to hGM-CSF, His of mGM-CSF appears to occupy a buried position in the first alpha-helix. This buried position is less tolerant to substitution than the surface exposed residues (with the exception of Glu). Substitution at His with charged or polar residues resulted in a reduced biological activity, whereas hydrophobic or aromatic substitutions are well tolerated (Fig. 1, panel 3A). All His mutants reached full plateau.

Competitive Receptor Binding of mGM-CSF Mutant Proteins

The effects of amino acid substitution on the ability of mGM-CSF to bind to the high affinity mGM-CSF receptor (mGM-Ralphabeta) on NFS60 cells was measured using conditions designed to detect both high and low affinity binding (see ``Materials and Methods''). Some proteins had no loss in high affinity binding, and such mutant proteins were not investigated further. Some proteins had decreased high affinity binding, and the mutant protein was then tested for its ability to bind to the low affinity mGM-CSF receptor (mGM-Ralpha) on stably transfected L-cells (see ``Materials and Methods''). This step was necessary so that the observed reduction in affinity could be assigned to either a loss of binding to the mGM-Rbeta component in the mGM-Ralphabeta complex, to a loss of binding to the mGM-Ralpha component, or to a loss of binding to both.

For most substitution mutants at Arg, Lys^14, His, and Lys, a two-site fit of the high affinity receptor binding data was statistically significant (data shown for Lys^14 and Lys; Table 1and Table 2). Substitution at Arg with either a negatively charged (R11E) or positively charged (R11K) residue, neutral (R11G), hydrophobic (R11L), or aromatic (R11W) residue had no effect on either high or low affinity binding constants (data not shown). This residue was not studied further.

Mutant proteins with charged and polar substitutions at position His showed a reduced ability to compete for I-mGM-CSF (Fig. 1, panel 3B), in agreement with the observed loss in biological activity (Fig. 1, panel 3A). Since this is a buried position in the protein, losses in activity and binding are probably due to structural perturbations of the protein core. Proteins mutated at this position were also not studied further.

Mutations at position Lys^14 did not alter noticeably the biological activity of the resulting mutant protein. However, significant binding defects were observed (Fig. 1, panels 2A and 2B, Table 1). For some Lys^14 mutants, only one affinity site is detected (Glu, Gly, Ser, Val, Trp, and Tyr). For others, two sites were still detected but both were reduced in affinity (e.g. Phe and Gln). In general, reduction of the high affinity binding constant was less than 10-fold, while values for the low affinity K(d) showed wider variation with a concomitant increase in the %CV, indicating that the calculated value for the low affinity binding constant was less reliable under these conditions. Several Lys^14 mutants displaying a range of high affinity defects were selected and analyzed for their low affinity binding to mGM-Ralpha expressed alone on stably transfected L-cells (Fig. 2A). Of the five mutant proteins tested, four had a low affinity K(d) similar to mGM-CSF (K(d) = 7.2 times 10M; Table 4). From this analysis we conclude that the reduction seen in the high affinity binding constant for Lys^14 substitution proteins results from losses in binding to the mGM-Rbeta in the mGM-Ralphabeta complex. Protein K14P is the exception. A proline substitution at this position affected the low as well as the high affinity binding constant (Table 1).


Figure 2: Competition binding of mutant and wild-type mGM-CSF proteins. A, competition of I-mGM-CSF on L cells transfected with mGM-Ralpha by bullet, mGM-CSF; circle, K14A; , K14E; , K14G; , K14P; and down triangle, K14S. B, competition of I-mGM-CSF on L cells transfected with mGM-R-alpha by bullet, mGM-CSF; , K20G; , K20M; , K20P; circle, K20A; box, K20L; , K20V; box+, K20F; up triangle+, K20H; down triangle+, K20W; +, K20Y; up triangle, K20Q; down triangle, K20S; &cjs1730;+, K20R; , K20E. Assays were performed in triplicate using a constant concentration of 2.0 times 10MI-mGM-CSF with symbol error bars indicating standard deviation.



Mutant proteins with Lys substitutions also showed losses in both high and low affinity binding as measured on NSF60 cells (Fig. 1, panel 4B, Fig. 2B, Table 2and Table 4). To distinguish between losses in binding to the mGM-Rbeta and/or mGM-Ralpha, Lys substitution proteins were analyzed on mGM-Ralpha-expressing L-cells. Whereas most substitutions at Lys did not affect the low affinity binding constant as measured on L-cells, small amino acid substitutions (Ala, Gly, and Ser) at this position led to a 10-fold reduction in mGM-Ralpha binding (Fig. 2B, Table 4). The introduction of proline at this position profoundly disturbed the binding of the resulting protein to both high and low affinity receptor.

In contrast to the other mutant proteins examined, all Glu substitution proteins competed I-mGM-CSF for only a single class of binding sites on NSF60 cells (Fig. 1, panel 5B, Table 3). The affinity of Glu substitution proteins (K(d) = 1-4 times 10M) for these sites suggested that binding is to mGM-Ralpha. Scatchard analysis of one of the Glu substitution proteins, E21A, has shown that both affinity and number of binding sites were consistent with binding of this mutant protein to mGMRalpha only(16) . Again, the exception was E21P. This protein had a low affinity binding constant that was at least three orders of magnitude higher than the other Glu proteins (Fig. 5B, Table 3), probably caused by severe structural changes(36) .


Figure 5: Two views of the mGM-CSF Calpha backbone indicating important structural features and identifying specific amino acid side chains. Helix A (red) and helix D (blue) form an anti-parallel helix pair and comprise the receptor binding site(38) . Disulfide bonds are indicated in yellow. Amino acid residues significant to this study are colored according to the properties of their side chains, negatively charged (E), red; positively charged (K and R), blue; and aromatic (H), green.



Design of mGM-CSF Antagonists

Mutant proteins E21R and E21K were unable to trigger a maximal biological response. Such partial agonists should be antagonists of mGM-CSF at concentrations equal to or greater than those at which they bind the receptor. E21K was a partial agonist with about 40% residual activation on NFS60 cells (Fig. 3). At concentrations which elicited the partial agonist activity, E21K was an antagonist of mGM-CSF activity (Fig. 4). However, its efficacy was limited by its relatively high residual activation level. We reasoned that the residual ability of E21K to activate mGM-Ralphabeta might result from contacts with mGM-Rbeta which were still intact in E21K. Identification of Lys^14 as a residue that exclusively interacts with mGM-Rbeta suggested the possibility that a further reduction of the submaximal response of E21K could be achieved by combining mutations at these two positions. Fig. 3shows that the K14E, E21K double mutant protein had, in fact, lost almost (>90%) all ability to activate mGM-Ralphabeta on NFS60 cells. As expected, this protein showed much improved efficacy when used on NSF60 cells to antagonize mGM-CSF (Fig. 4).


Figure 3: Biological activity of wild-type and mutant mGM-CSF proteins. Proliferation of NFS60 cells in response to mGM-CSF (bullet), E21K (), and K14E/E21K () was measured as a function of protein concentration using the MTT assay(32) . Assays were performed in quadruplicate with symbol error bars indicating standard deviation.




Figure 4: Antagonism of the mGM-CSF response by mutant mGM-CSF proteins. Proliferation of NFS60 cells in response to E21K () and K14E/E21K () in the presence of 3.5 times 10M mGM-CSF using the MTT assay(32) . Assays were performed in quadriplicate with the symbol error bars indicating standard deviation.




DISCUSSION

The involvement of the amino-terminal region of mGM-CSF in receptor binding and biological activity is well documented(16, 17) . In particular, it is now clear that residues in the first alpha-helix of mGM-CSF directly contact the mGM-CSF receptors. The data presented here identify in detail the qualitative and quantitative contribution of several residues in this region to this interaction. This knowledge was essential to our designing a mGM-CSF receptor antagonist.

We have previously shown that Glu plays a pivital role in binding to the mGM-Rbeta in the high affinity complex. Substitution of Glu with Ala abrogates high affinity binding(16) . The data presented here show that none of the 14 amino acid substitutions are tolerated at this position. Previous work concludes that Glu can be replaced with Asp without serious consequences to bioactivity(36) . Analysis of the equivalent Glu position in hGM-CSF shows that Glu can be replaced with an Asp without noticeable loss in either bioactivity or binding(18) . Taken together, these data show the absolute requirement of an acidic residue at this position. All other substitutions we tested resulted in a complete loss of high affinity binding, while low affinity binding remained unaffected. These data identify Glu unequivocally as a residue that interacts with mGM-Rbeta only. Even though Glu mediates high affinity binding, only charge reversal substitutions cause a noticeable loss in biological activity. Maintaining biological activity in the absence of high affinity binding seemingly contradicts the observation that signaling occurs through a complex of the mGM-Ralpha and mGM-Rbeta subunits in the presence of ligand. However, we have previously shown for one of the Glu substitution proteins, E21A, that this protein still can be cross-linked to the mGM-Ralphabeta complex, even in the absence of any detectable high affinity binding(16) . Taken together these data indicate that the interaction of Glu with mGM-Rbeta, although pivital for the measured high affinity binding of mGM-CSF to its receptor, is not necessary for receptor activation. The absence of a Glu residue at position 21 apparently does not prevent mGM-Rbeta from participating in complex formation and signaling and suggests that other mGM-CSF residues contact mGM-Rbeta.

Studies on hGM-CSF and hIL-3, cytokines which share the beta receptor, show similar results. Substitution of the homologous acidic residues in these two cytokines also leads to a loss of high affinity binding(18, 37) . However, there is a closer correlation between loss of high affinity binding and loss of activity for both hGM-CSF and hIL-3. Taken together, these data suggest that Glu in GM-CSF and the homologous acidic residue in IL-3 and presumably also IL-5 are functionally identical; this residue determines high affinity binding. However, the extent to which its absence affects the biological activity varies and presumably depends on how many other residues contribute to the interaction with GM-Rbeta.

Our data revealed that at least 2 other residues in the first alpha-helix of mGM-CSF interact with mGM-Rbeta. Substitutions at Lys^14 and Lys both disrupted high affinity binding as indicated by decreased high affinity K(d) values. Lys^14 substitutions exclusively reduced high affinity binding, whereas some Lys substitutions affected high affinity binding and others low affinity binding. However, none of the substitutions at either position reduced the biological activity. The binding defects of Lys mutant proteins are generally somewhat smaller than Lys^14 defects and fell into two categories, those that were caused by defects in binding to mGM-Rbeta and those caused by defects in binding to mGM-Ralpha. Substitutions that reduced the binding to mGM-Ralpha had small side chains (Ala, Ser, and Gly), suggesting that size plays a critical role at this position. Homology modeling based on the high resolution crystal structure of hGM-CSF (8) indicates the most likely location of these residues on the alpha-helix 1 mGM-CSF backbone (Fig. 5). Both Lys^14 and Glu are located on the exterior of alpha-helix 1 toward the groove formed by the alpha-helix 1 and alpha-helix 3 and well situated for possible direct interaction with mGM-Rbeta. It has been suggested that the groove between the first and third helix provides most of the GM-Rbeta contact(38, 39) . Our data on Lys^14 and Glu support this hypothesis. On the other hand, Lys is at the interface of alpha-helix 1 and alpha-helix 4, which is the region thought to provide mGM-Ralpha specific contacts. Our data support a dual role for Lys in binding mGM-Ralpha and mGM-Rbeta. It is somewhat surprising that Glu does not play a more important role in mGM-Rbeta binding considering its location between Lys^14 and Glu(15, 16, 17) . Recent mutagenesis of the hGM-CSF equivalent position, Asn, has confirmed that this residue contributes little to binding and bioactivity(38) . Fig. 5shows that the Glu side chain is somewhat removed from the groove between helix 1 and helix 3 and apparently does not contribute to binding of GM-Rbeta.

Based on functional and structural comparison of hGM-CSF and GH two potential receptor-binding sites on hGM-CSF have been suggested(39) . Analogous to site I of GH(40) , it was proposed that GM-CSF binds to its primary binding receptor, GM-Ralpha, through alpha-helix 4. The interaction of the first alpha-helix of GM-CSF with the GM-Rbeta in the high affinity complex appears to be similar to the site II interactions of GH and GH-R. Receptor antagonists of GH were made by mutations of site II residues that prevent the second GH-R from participating in the formation of the GH-R dimer(41) . We have extrapolated that finding to mGM-CSF and tested the partial agonist E21K as a receptor antagonist. E21K weakly antagonized mGM-CSF. By eliminating other mGM-Rbeta-specific contact residues in mGM-CSF, we anticipated improved antagonistic mGM-CSF derivatives. The K14E, E21K double mutant was indeed a better antagonist than E21K. These experiments show that detailed studies which not only identify key residues involved in protein-protein interactions, but also characterize the nature of that interaction, make it possible to rationally design cytokine derivatives with altered properties.

GM-CSF, IL-3, and IL-5 all bind specific Ralphas but share a common Rbeta. Identification of two Rbeta-specific contact residues in mGM-CSF led to derivatives with antagonistic properties. This finding may have direct implications for the design of IL-3 and IL-5 antagonists. A strategy to find such molecules should focus on elimination of Rbeta-specific contacts, in particular, the conserved acidic residue in the first alpha-helix of these cytokines and a second residue corresponding to mGM-CSF Lys^14.


FOOTNOTES

*
The DNAX Research Institute is supported by the Schering-Plough Corp. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Molecular Biology, DNAX Research Institute of Molecular and Cellular Biology, 901 California Ave., Palo Alto 94304-1104.

(^1)
The abbreviations used are: GM-CSF, granulocyte-macrophage colony-stimulating factor; mGM-CSF, mouse GM-CSF; hGM-CSF, human GM-CSF; GM-R, GM-CSF receptor; GM-Ralpha, GM-R alpha-subunit; GM-Rbeta, GM-R beta-subunit; IL-2, interleukin-2; IL-3, interleukin-3; IL-4, interleukin-4; GH, growth hormone; GH-R, GH receptor; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide.


ACKNOWLEDGEMENTS

We thank Dr. Fernando Bazan for modeling of mGM-CSF, Gerard Zurawski for valuable discussion and critical evaluation regarding the manuscript, and D. Liggett for synthesis of DNA.


REFERENCES

  1. Metcalf, D. (1985) Science 229, 16-22 [Medline] [Order article via Infotrieve]
  2. Metcalf, D. (1986) Blood 67, 257-267 [Abstract]
  3. Bazan J. F. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 6934-6938 [Abstract]
  4. Hayashida, K., Kitamura, T., Gorman, D. M., Arai, K., Yokota, T., and Miyajima, A. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 9655-9659 [Abstract]
  5. Kitamura, T., Sato, N., Arai, K., and Miyajima, A. (1991) Cell 66, 1165-1174 [Medline] [Order article via Infotrieve]
  6. Park L. S., Martin, U., Sorensen, R., Luhr, S., Morrissey, P. J., Cosman, D., and Larsen, A. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 4295-4299 [Abstract]
  7. Kitamura, T., Hayashida, K., Sakamaki, K., Yokota, T., Arai, K., and Miyajima, A. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 5082-5086 [Abstract]
  8. Diederichs, K., S., Boone, T., and Karplus, P. A. (1991) Science 254, 1779-1782 [Medline] [Order article via Infotrieve]
  9. McKay D. B. (1992) Science 257, 412-413
  10. Smith, L. J., Redfield, C., Boyd, J., Lawrence, G. M. P., Edwards, R. G., Smith, R. A. G., and Dobson, C. M. (1992) J. Mol. Biol. 224, 899-904 [Medline] [Order article via Infotrieve]
  11. Milburn, M. V., Hassell, A. M., Lambert, M. H., Jordon, S. R., Proudfoot, A. E. I., Graber, P., and Wells, T. N. C., (1993) Nature 363, 172-176 [CrossRef][Medline] [Order article via Infotrieve]
  12. Pandit, J., Bohm, A., Jancarik, J., Halenbeck, R., Koths, K., and Kim, S. (1992) Science 258, 1358-1362 [Medline] [Order article via Infotrieve]
  13. Abdel-Meguid, S. S., Shieh, H., Smith, W. W., Dayringer, H. E., Violand, B. N., and Bentle, L. A. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 6434-6437 [Abstract]
  14. Shanafelt, A. B., Johnson, K. E., and Kastelein, R. A. (1991) J. Biol. Chem. 266, 13804-13810 [Abstract/Free Full Text]
  15. Shanafelt, A. B., Miyajima, A., Kitamura, T., and Kastelein, R. A. (1991) EMBO J. 10, 4105-4112 [Abstract]
  16. Shanafelt, A. B. and Kastelein, R. A. (1992) J. Biol. Chem. 267, 25466-25472 [Abstract/Free Full Text]
  17. Meropol, N. J., Altmann, S. W., Shanafelt, A. B., Kastelein, R. A., Johnson, G. D., and Prystowsky, M. B. (1992) J. Biol. Chem. 267, 14266-14269 [Abstract/Free Full Text]
  18. Lopez, A. F., Shannon, M. F., Hercus, T., Nicola, N. A., Cambareri, B., Dottore, M., Layton, M. J., Eglinton, L., and Vadas, M. A. (1992) EMBO J. 11, 909-916 [Abstract]
  19. Brown, C. B., Hart, C. E., Curtis, D. M., Bailey M. C., and Kaushansky, K. (1990) J. Immunol. 144, 2184-2189 [Abstract/Free Full Text]
  20. Kanakura, Y., Cannistra, S. A., Brown, C. B., Nakamura, M., Seelig, G. F., Prosise, W. W., Hawkins, J. C., Kaushansky, K., and Griffin, J. D. (1991) Blood 77, 1033-1043 [Abstract]
  21. Seelig, G. F., Prosise, W. W., and Scheffler, J. E. (1994) J. Biol. Chem. 269, 5548-5553 [Abstract/Free Full Text]
  22. Messing, J. (1983) Methods Enzymol. 101, 20-78 [Medline] [Order article via Infotrieve]
  23. Kunkel, T. A., Roberts, J. D., and Zakour, R. A. (1987) Methods Enzymol. 154, 367-382 [Medline] [Order article via Infotrieve]
  24. Shanafelt, A. B., and Kastelein, R. A. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 4872-4876 [Abstract]
  25. Howard-Flanders, P., Simson, E., and Theriot, L. (1964) Genetics 49, 237-246 [Free Full Text]
  26. Lundell, D., Greenberg, R ., Alroy, Y., Condon, R., Fossetta, J. D., Gewain, K., Kastelein, R., Lunn, C. A., Reim, R., Shah, C., Van Kimmenade, A., and Narula, S. K. (1990) J. Indust. Microbiol. 5, 215-228 [Medline] [Order article via Infotrieve]
  27. Greenberg, R ., Lundell, D., Alroy, Y., Bonitz, S., Condon, R., Fossetta, J. D., Frommer, B., Gewain, K., Katz, M., Leibowitz, P. J., Narula, S. K., Kastelein, R., and Van Kimmenade, A. (1988) Curr. Microb. 17, 321-332
  28. Itoh, N., Yonehara, S., Schreurs, J., Gorman, D. M., Maruyama, K., Ishii, A., Yahara, I., Arai, K., and Miyajima, A. (1990) Science 247, 324-327 [Medline] [Order article via Infotrieve]
  29. Sanger, F., Nicklen, S., and Coulson A. R. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 5463-5467 [Abstract]
  30. Miyajima, A., Otsu, K., Schreurs, J., Bond, M. W., Abrams, J. S., and Arai, K. (1986) EMBO J. 5, 1193-1197 [Abstract]
  31. Abrams, J. S., Roncarolo, M-G., Yssel, H., Andersson, U., Gleich, G. J., and Silver, J. E. (1992) Immunol. Rev. 127, 5-44 [Medline] [Order article via Infotrieve]
  32. Mosmann, T. (1983) J. Immunol. Methods 65, 55-63 [CrossRef][Medline] [Order article via Infotrieve]
  33. Munson, P. J. (1983) Methods Enzymol. 92, 543-576 [Medline] [Order article via Infotrieve]
  34. Levitt, M. (1992) J. Mol. Biol. 226, 507-533 [Medline] [Order article via Infotrieve]
  35. Lee, C., and Subbiah, S. (1991) J. Mol. Biol. 217, 373-388 [Medline] [Order article via Infotrieve]
  36. Altmann, S. W., Johnson, G. D., and Prystowsky M. B. (1991) J. Biol. Chem. 266, 5333-5341 [Abstract/Free Full Text]
  37. Barry, S. C., Bagley, C. J., Phillips, J., Dottore, M., Cambareri, B., Moretti, P., D'Andrea, R., Goodall, G. J., Shannon, M. F., Vadas, M. A., and Lopez, A. F. (1994) J. Biol. Chem. 269, 8488-8492 [Abstract/Free Full Text]
  38. Hercus, T. R., Cambareri, B., Dottore, M., Woodcock, J., Bagley, C. J., Vadas, M. A., Shannon, M. F., and Lopez, A. F. (1994) Blood 83, 3500-3508 [Abstract/Free Full Text]
  39. Kastelein, R. A., and Shanafelt, A. B. (1993) Oncogene 8, 231-236 [Medline] [Order article via Infotrieve]
  40. de Vos, A. M., Ultsch, M., and Kossiakoff, A. A. (1991) Science 255, 306-312
  41. Fuh, G., Cunningham, B. C., Fukunaga, R., Nagata, S., Goeddel, D. V., and Wells, J. A. (1992) Science 256, 1677-1680 [Medline] [Order article via Infotrieve]

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