Proc. Natl. Acad. Sci. USAVol. 93, pp. 1-6, January 1996

Review

Binding in the growth hormone receptor complexJames A. WellsDepartment of Protein Engineering, Genentech, Inc., 460 Point San Bruno Boulevard, South San Francisco, CA 94080

ABSTRACT Binding reactions be-tween human growth hormone (hGH) andits receptor provide a detailed account ofhow a polypeptide hormone activates itsreceptor and more generally how proteinsinteract. Through high-resolution struc-tural and functional studies it is seen thathGH uses two different sites (site 1 andsite 2) to bind two identical receptor mol-ecules. This sequential dimerization reac-tion activates the receptor, presumably bybringing the intracellular domains intoclose proximity so they may activate cy-tosolic components. As a consequence ofthis mechanism it is possible to buildantagonists to the receptor by introducingmutations in hGH that block binding atsite 2 and to build even more potentantagonists by combining these with mu-tants that enhance binding at site 1. Ala-nine-scanning mutagenesis of all contactresidues at the site 1 interface shows thatonly a small and complementary set ofside chains clustered near the center ofthe interface affects binding. The mostimportant contacts are hydrophobic, andthese are surrounded by polar andcharged interactions of lesser impor-tance. Kinetic analysis shows for the mostpart that the important side chains func-tion to maintain the complex, not to guidethe hormone to the receptor. Hormone-induced homodimerization or het-erodimerization reactions are turning outto be pervasive mechanisms for signaltransduction. Moreover, the molecularrecognition principles seen in the hGH-receptor complex are likely to generalizeto other protein-protein complexes.

How do hormones find their receptors?Once there, what forces allow the hor-mone to bind? How does hormone bind-ing lead to receptor activation? Mostbiological processes are regulated orstructured by these reactions involvingnoncovalent associations. Thus, an under-standing of how hormones bind their re-ceptors is broadly relevant to many othermolecular recognition events in biology.

This minireview will begin to addressthese questions from the perspective of acomplex between human growth hormone(hGH) and the extracellular domain of itsreceptor, called the hGHbp (for generalreviews see refs. 1 and 2). This complexhas been intensively studied by mutationaland structural methods for several rea-

sons. First, the pharmacology of hGH israther complex (3). For example it canbind and activate at least two differentcloned receptors, the hGH (4) and pro-lactin receptors (5). Moreover, hGH is amember of the cytokine receptor super-family (6), and thus an understanding ofhow it binds its receptor may shed light onthe entire family. Finally, it is hoped thatthrough a detailed understanding of thestructure and chemistry involved in thebinding reaction, one would be in a betterposition to rationally design small mole-cules that could mimic the large interfacesthat are typical of protein-protein com-plexes.

Basic Methods and Approaches

Any detailed structural and functionalanalysis is greatly facilitated by having anabundant and recombinant source of thecomponent molecules. We were particu-larly fortunate to have Escherichia colisecretion systems for both hGH (7) andthe hGHbp (8). These expression systemsallowed rapid and high-level production ofthese proteins in forms that interactedwith virtually the same affinity as thosefrom natural or recombinant mammaliansources (Kd 0.3 nM).

Determining the stoichiometry of thecomplex is fundamental to characterizingany binding reaction. Initially, we pre-sumed that growth hormone bound toonly one receptor in solution, becauseScatchard analysis from an RIA showed a1:1 stoichiometry. As it turned out, how-ever, the receptor antibody (MAb5, ref. 9)used to precipitate the hGHhGHbp com-plex sterically excluded the second recep-tor molecule from binding (10). This wasa blessing in disguise because more com-plex stoichiometries (such as the 1:2 stoi-chiometry that was later discovered)would have made interpretation more dif-ficult than was the case for the single inter-face that was initially discovered (11, 12).A variety of biophysical methods were

used to determine that the stoichiometryof the hGH-hGHbp complex in solutionwas 1:2 (10). One of the most powerfulwas titration calorimetry, which showedthat the binding reaction was completewhen 1 equivalent of hormone was addedto 2 equivalents of the hGHbp. Gel filtra-tion showed that mixtures containing 1equivalent of hGH (22 kDa) plus 2 equiv-

alents of hGHbp (28 kDa) ran as a singlepeak (-75 kDa) with no evidence of ex-cess free components. SDS/PAGE anddensitometry of the 75-kDa peak showedit had a composition of one hGH moleculeper two hGHbp molecules. Furthermore,crystals of the complex were obtained andwhen dissociated gave a composition ofone hGH molecule per two hGHbp mol-ecules (10, 13).Two mutagenesis strategies, homolog-

scanning (11) and alanine-scanning (12),were employed to define binding determi-nants in the hGH and the hGHbp (forreview see ref. 14). Initially, these studieswere performed without the aid of thestructures of the complex or of the indi-vidual components. In homolog-scanning,segments (7-30 residues long) derivedfrom nonbinding homologs, such as pro-lactin or porcine growth hormone, weresubstituted into hGH. From the set ofsegment-substituted molecules that dis-rupted binding affinity, we could inferregions of the hormone that may containbinding determinants. These regions werethen subjected to alanine-scanning mu-tagenesis in which each residue within thedisruptive segment was replaced by ala-nine and the consequences for bindingaffinity were measured. In this way therole of side-chain atoms beyond the /3-car-bon could be assessed. A panel of mono-clonal antibodies that reacted with thenative components, but not the unfoldedforms, were used to verify that these mu-tations did not affect the overall fold of theprotein.Two different assays were used to de-

termine the effects of hGH mutants onbinding. Using MAb5 to precipitate 1:1complexes, we found a patch of mutationsthat disrupted binding to a region wecalled site 1. The second assay followedthe hGH-induced dimerization of hGHbpmolecules in solution by the quenching ofa fluorescent tag placed near the C terminusof the hGHbp (10). The patch of alaninemutations outside the site 1 patch that hadreduced affinity in this assaywe called site 2.The solution of the x-ray structure of

the 1:2 hGH-hGHbp complex (15) per-

Abbreviations: hGH, human growth hormone;hGHbp, the extracellular domain of the hGHreceptor; variant proteins are indicated by thesingle-letter code for the wild-type residue fol-lowed by its position in the mature proteinsequence and the substituted residue.

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A + B .A*B (Ka =kon/koff) [1] lized hGHbp. This causes a change in

data to be interpreted in structural con- kThfaff refractive index of the medium which is

text for the first time. This was an enor- detected by a change in surface plasmonmous advance. Not only did it reveal the What role do contact side chains play in resonance of the gold film (called reso-

1:2 natu're of the complex and the struc- determining on-rate (kon) or off-rate nance units or RU). The stoichiometry of

tures of the bound components, it iden- (koff)? To address this question we focused binding can be calculated from the total

tified all the contact residues at the two on the binding of hGH via site to the first change in RU, as this is related to the mass

hormone-receptor interfaces as well as bound receptor. bound to the chip. On-rates are measured

showing that the receptors contact each To study the kinetics of binding we used by the rate of change in RU as a functionother (Fig. 1). a BlAcore (Pharmacia) (16). This device of hGH concentration. Off-rates are de-

Side Chains on the hGH Mostly Affect has a flow cell with a gold film layered on termined from rate of the decrease in RU

Dissociation, Not Association the outside. Attached to the gold film is a upon release of hGH from a saturated

layer of dextran fibers which extend into chip.

Binding affinity can be considered a sim- the flow cell to which we covalently at- To study the binding of hGH at site 1

ple balance of two reactions, association tached the hGHbp (17). As hGH flows without the complication of forming 1:2

and dissociation (Eq. 1). through the cell, it binds to the immobi- complexes, or binding through site 2, we

FIG. 1. Structural model of the 1:2 hGH-hGHbp complex taken from ref 15. hGH is shown in red, while the hGHbpl and hGHbp2 are shownin green and blue, respectively. The membrane bilayer is modeled below. Reprinted with permission from Science 255,257. Copyright 1992 AmericanAssociation for the Advancement of Science.

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prepared a receptor that could bind hGHonly at site 1 (17). This was done byintroducing a mutation in the receptorstem region (S201C) and fixing this by a

disulfide linkage to the flow chip. Not onlydid this prevent receptor dimerization, italso presented the hGHbp in a uniformfashion to the hormone. Using this con-struction, we determined a stoichiometryof binding of hGH-hGHbp of 0.84, a ko, of3 x 105 s-I.M-1, and a koff of 2.7 x 10-4M- 1. These values were virtually the samefor a variant of hGH (G120R) which isblocked in binding to a second receptor bya mutation in site 2, indicating that themeasured kinetic parameters reflectedbinding at site 1. Moreover, the affinityconstant was in close agreement with thatdetermined by RIA using MAbS.The side chains of 31 residues of hGH

become buried to various degrees uponbinding the first receptor (15). We con-

verted each of these residues to alanineand measured their on-rates and off-rates(17). The largest effects were for six mu-tants (P61A, R64A, K172A, T175A,F176A, and R178A) which individuallycaused a 5- to 30-fold increase in off-rate,but only a 1.1- to 2.5-fold decrease inon-rate. The subtle effects on the on-ratewere best correlated to changes in theelectrostatics of the binding site. For ex-

ample, the largest decreases in on-ratewere for mutating positively charged res-idues, and the largest increases in on-rate(up to 1.5-fold) were for mutating twonegatively charged groups (E65A andE174A) at the binding site.Northrup and Erickson (18) have noted

that protein-protein association generallyoccurs at rates that are 103 to 104 times fasterthan would be expected from simple con-siderations of collision frequencies (- 109s-1.M-1) and strict orientation effects thatassume productive binding occurs onlywhen the molecules collide within 2 A oftheir final binding site. These orientationeffects are predicted to reduce the rate ofproductive collisions by _106 to produceon-rates of 103 s-' M-1. They argue thatlong-lived collisions, or Brownian diffusion,would greatly accelerate the association. Inother words, when the proteins collide, theydo not diffuse away immediately but roll onone another and thereby sample much moresurface area than would be the case for a

single elastic collision. They suggest thatassociation rates between proteins are dom-inated by considerations of diffusion eventsalone.We can conclude that only a small set of

side chains modulate these effects on theaffinity between hGH and the hGHbp andthat their major role is to slow the off-rate.This suggests there are many paths togetting the hormone to the receptor. Therole of side chains is to keep the hormonebound once it has reached the binding site,not to get it there in the first place. Al-though electrostatic interactions can sub-

tly influence the rate of association of thehGH to the receptor, association is con-trolled by diffusion, which is independentof the side-chain composition of the in-terface. Thus, the hormone finds its re-ceptor mostly by a random but rapid col-lision process; the side chains function tokeep it bound once it has reached itsreceptor target.

Binding Affinity Is Maintained by aSmall Cluster of Contact Residues

From the ratio of on- and off-rates, wecalculated the effects of the contact sidechains in site 1 on affinity. From this itbecame clear that only a small set of theburied side chains were necessary for tightbinding affinity. In fact, alanine substitu-tions at only 8 of the 31 positions (K41,L45, P61, R64, K172, T175, F176, andR178) could account for -85% of thebinding energy. These formed two smallpatches near the center of the contactinterface that we call the functionalepitope (Fig. 2).A similar analysis (19, 20) was per-

formed on the receptor side, where 33 sidechains become buried at the interface.Each of these residues (except G168 andthe C108-C122 disulfide) were convertedto alanine, and affinities of the mutatedreceptors to hGH were measured by RIA.Nine of these residues (R43, E44, 1103,W104, 1105, P106, 1164, D165, and W169)could account for virtually all the bindingaffinity. These residues cluster at the cen-ter of the contact interface (Fig. 2).What structural features explain why

some residues are important while othersare not? The functionally important resi-dues are those located near the center ofthe contact epitope and contact thosefound to be functionally important resi-dues on the other side. The most criticalinteractions tend to be well-packed hydro-phobic contacts. This is obvious on thereceptor side, where W104 and W169 areby far the most important residues. Thealiphatic portions of important chargedand polar side chains from hGH, such asD171, K172, and T175, pack against thetryptophans on the receptor. No (or few)buried waters are seen between the func-tional epitopes (20).The unimportant residues tend toward

the periphery of the contact or structuralepitope. These are often polar and incom-pletely dehydrated. Thus, bridging watermolecules are seen between polar orcharged contact residues in regions of thecontact interface that are less important.The extent of side-chain burial, or numberof van der Waals contacts on their own,are generally weak predictors of relativeeffects on affinity when side chains areconverted to that of alanine (17, 20).These correlations are much better, how-ever, when one considers only the burial ofwell-packed hydrophobic side chains.

The hGH Receptor Is Activated bySequential Dimerization

Receptor oligomerization is a common, ifnot pervasive, means by which extracellu-lar hormones transmit their signals to theinside of a cell without ever passingthrough the membrane (for reviews seerefs. 21-23). By a hormone simply bring-ing together two or more transmembranemembrane receptors, the intracellular do-mains of these can be juxtaposed so thatthey may interact with other cellular com-ponents or catalyze a reaction (typicallyprotein phosphorylation).The most striking aspect of the hGH-

receptor complex is that one hormonebinds and dimerizes two receptors. Muta-tional and biophysical studies (10, 24)have shown that these sites do not reactrandomly with the receptor but do so in asequential fashion (Fig. 3). That is, hGHreacts first with a receptor by using site 1and then with a second receptor by usingsite 2. The basis for proposing this mech-anism was that mutations in site 2 do nothave an impact on the ability to form 1:1complexes with hGH, whereas mutationsin site 1 do.The structure provided additional sup-

port for the sequential dimerization model(15). For example, the contact epitopebetween hGH and hGHbp1 buried about1300 A2, whereas that for hGH withhGHbp2 buried only -850 A2. If, however,hGHbp2 binds to the hGH hGHbpl com-Rlex, then 1350 A2 becomes buried-850A2 for the hGH-hGHbp2 interface plus500 A2 for the hGHbp1lhGHbp2 interface.Thus, the binding of hGHbp2 requiresprior binding of hGHbpl because its bind-ing site is composed of determinants fromsite 2 on hGH as well as the stem portionof the bound hGHbpl. The reason that thehormone does not form higher oligomerswith the receptor is that the receptor usesvirtually the same site to bind either site 1or site 2 on hGH (15). Thus, one receptorcan bind only one hormone molecule at atime.On the basis of this sequential dimer-

ization mechanism and the structure ofthe complex it was possible to explain thebell-shaped dose-response curve for acti-vation of receptors by hGH (24-26). Atlow concentrations, hGH can bind to re-ceptors and easily find an empty receptorto form a dimer and effect a signal. How-ever, at high concentrations receptors be-come occupied to greater extents as 1:1complexes, and thus fewer empty recep-tors are available to dimerize.

This principle of sequential assemblymay be general to other hormone-receptor complexes for the simple reasonthat it reduces the degrees of freedom forassociation (for general review see ref.27). In the hGH mechanism, the hormoneneeds only to diffuse once in three dimen-sions to reach the first receptor. The next

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FIG. 2. A series of space-filling models showing the complementary structural and functional epitopes on site 1 of hGH (dark gray, right) andthe first bound receptor, hGHbpi (light gray, left). All contact residues are colored according to the effects that alanine substitutions have on bindingaffinity: light blue, <0.5 kcal/mol reduction in affinity (1 kcal = 4.18 U); dark blue, 0.5 to 1.5 kcal/mol reduction in affinity; and red, >1.5 kcal/molreduction in affinity. Reprinted with permission from Science 267, 301 (see ref. 20). Copyright 1995 American Association for the Advancementof Science.

association event occurs in the mem-brane, which is two-dimensional. Fur-thermore, because the receptor is teth-ered by its transmembrane domain thisfurther limits two of the three degrees ofrotational freedom for the receptors todimerize.

Contrast the sequential 1:2 dimeriza-tion model with one in which two separatehormone molecules need to bind to dimer-ize and signal (a 2:2 model, ref. 21). In thiscase, two three-dimensional diffusionevents would be required. The second

membrane diffusion event would be sim-ilar except that a 1:1 hormone-receptorcomplex would require finding anotherloaded receptor. For both steps, forming a2:2 complex is far less efficient than form-ing a 1:2 complex.A bell-shaped dose-response curve,

which is consistent with formation of 1:2complexes, may be difficult to see in otherhormone/receptor systems. To observethe inhibitory phase requires extremelyhigh hormone concentrations. Some hor-mones may not be readily available or

even soluble at the concentrations re-quired. Even for hGH, the half-maximalinhibitory concentration (IC5o) occurs at-2 ,M, whereas the half-maximal excita-tory concentration (EC5o) is 20 pM. This10,000-fold difference can be rationalizedif the step to dimerize and signal is muchmore efficient than the initial bindingevent. In other words, it is difficult tosaturate the receptors as 1:1 complexesbefore some have dimerized. Further-more, the difference between EC50 andIC50 will be further expanded if only a

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hGH

Active (agonist) Inactive (antagonist)

FIG. 3. Sequential dimerization model for activation of the hGH receptor taken from ref. 24.Copyright 1992 American Association for the Advancement of Science. hGH first binds via site1 to receptor 1 and then through site 2 to the receptor 2. This dimerization event leads to signaling.

small number of receptors need to dimer-ize in order to signal (25).

Mathematical models to describe theoligomerization reactions on cell mem-branes with the appropriate dimensionalconstraints would be of use in trying to de-convolute dose-response data in terms ofoligomerization parameters. Among otherfactors, the dimerization reaction will de-pend on receptor concentration or ten-dency for the receptor to preassociate, aswell as on the association rate for the 1:1hormone-receptor complex (25). Theseparameters will surely vary from one celltype to another and from one hormone/receptor system to another. For these rea-sons and perhaps more, it may be difficultto observe a bell-shaped dose-responsecurve. Such behavior applies to receptorsystems which form homodimers; het-erodimeric (or oligomeric) systems wherethe hormone has a single site for twodifferent receptors and binds them se-quentially may not be antagonized by highhormone concentrations because saturat-ing the first receptor would not reducebinding of the second.The sequential dimerization mecha-

nism predicted that antagonists of hGHcould be produced by allowing (or en-

hancing) binding by site 1 while preventingdimerization by site 2 (24). Indeed, potentantagonists of hGH have been generatedby introducing a mutation (G120R) intosite 2 that blocks dimer formation (24, 28).This same strategy has been used to createantagonists to the prolactin receptorbased on hGH or a homolog, humanplacental lactogen (29).As the literature accumulates on other

helical cytokines it is becoming apparentthat a number of their receptors such asthose for interleukin (IL)-2, IL-3, IL-4,IL-5, IL-6, granulocyte colony-stimulatingfactor (G-CSF), granulocyte-macrophage(GM)-CSF, and erythropoietin undergoligand-induced receptor homo- or hetero-oligomerization (for reviews see refs. 23,24, and 30). Moreover, mutational analy-ses of the monomeric cytokines IL-2 (31),IL-3 (32, 33), IL-4 (34), GM-CSF (35, 36),IL-5 (35), and IL-6 (37) suggest they mayhave at least two receptor binding sites, acircumstance that has allowed the gener-ation of hormone antagonists for IL-2,IL-3, IL-4, and GM-CSF.

Conclusions

The helical cytokines and their cognatereceptors form a family of related struc-

tures and likely share a common bindingmechanism. Each hormone has at leasttwo sites for binding and oligomerizing itsreceptor(s). Such stoichiometry econo-mizes on the diffusion events-after dif-fusion to the first membrane-bound re-ceptor the second reaction with a receptoris facilitated by diffusion in two dimen-sions instead of three. This mechanismshould provide a common strategy forgenerating antagonists to these hormones.That is, one can produce hormone vari-ants that allow the first binding reactionbut prevent downstream binding reac-tions. Such antagonists may be useful indisease states where excessive hormonelevels are a problem, such as in the case ofacromegaly produced by excessive levelsof growth hormone.The growth hormone receptor inter-

faces are large, but only a few residues arecritical in binding. The interface generallyresembles the core of a folded protein:well-packed hydrophobic contacts arecrucial and they are inside, while hydro-philic interactions appear less importantand they are outside. Although the polarresidues appear less important for affinity,they may be important for solubility of thehormone and for specificity of the bindinginteraction. We believe the implications ofthese findings are important for rationaldrug design. Perhaps small molecules canbe built to bind to these large interfaces ifthey are designed to bind the small func-tional epitopes.Are these findings general to protein-

protein interactions? Structures of anti-body-antigen complexes show these inter-faces to be large and it is likely that only asmall number of contact side chains areimportant in binding [see Davies and Co-hen review in this issue (38)]. Trappedwater molecules are also present at theseinterfaces, whose functional importancehas yet to be determined. Althoughcharged and polar side chains can becritical in binding, it is noted that hydro-phobic side chains in these and otherprotein-protein complexes [see Jones andThornton review in this issue (39)] arevery important. Whether these findingsare general to most protein-protein inter-faces remains an open question-one thatwill surely be tested as more complexesare brought to the same level of structuraland functional understanding.

I thank my colleagues in the Protein Engi-neering Department at Genentech for theirsupport and useful discussions, Kerri Andowand Wayne Anstine for their graphics genius,and Brian Cunningham, Bart de Vos, and TonyKossiakoff for critical review of the manuscript.

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