Biochemical properties of site-directed mutants of human epidermal growth factor: importance of solvent-exposed hydrophobic residues of the amino-terminal domain in receptor binding

Eight analogues of human epidermal growth factor (hEGF) having specific amino acid substitutions in the beta-sheet structure (residues 19-31) of the amino-terminal domain were generated by site-directed mutagenesis. Affinity of the epidermal growth factor (EGF) receptor for each of these mutant hEGF analogues was measured by both radioreceptor competition binding and receptor tyrosine kinase stimulation assays. The relative binding affinities obtained by these two methods were generally in agreement for each hEGF species. The results indicate that hydrophobic residues on the exposed surface of the beta-sheet structure of the amino-terminal domain of hEGF have an important role in the formation of the active EGF-receptor complex. The substitution of hydrophobic amino acid residues, Val-19----Gly, Met-21----Thr, Ile-23----Thr, and Leu-26----Gly, resulted in decreased binding affinity, with the most severe reductions observed with the last two mutants. The mutations Ala-25----Val and Lys-28----Arg introduced amino acid residues resulting in slightly increased receptor binding affinity. Similar to previous results with acidic residues in this region [Engler, D.A., Matsunami, R.K., Campion, S.R., Stringer, C.D., Stevens, A., & Niyogi, S.K. (1988) J. Biol. Chem. 263, 12384-12390], removal of the positive charge in the Lys-28----Leu substitution had almost no effect on binding affinity, indicating the lack of any absolute requirement for ionic interactions at this site. Substitution of Tyr-22, which resulted in decreased receptor binding affinity, provides further indication of the importance of aromatic residues in this region of the molecule, as found earlier with Tyr-29 (cf. reference above).(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of residues in GTPase-activating protein Src homology 2 domains that control binding to tyrosine phosphorylated growth factor receptors and p62.

Ras GTPase-activating protein (GAP) contains two Src homology 2 (SH2) domains which are implicated in binding to tyrosine-phosphorylated sites in specific activated growth factor receptors and to a cytoplasmic tyrosine-phosphorylated protein, p62. We have used site-directed mutagenesis of the two GAP SH2 domains (SH2-N and SH2-C) to identify residues involved in receptor and p62 binding. A bacterial fusion protein containing the precise SH2-N domain, as defined by sequence homology, associated with both the activated beta platelet-derived growth factor receptor and epidermal growth factor receptor, and p62 in vitro. However, short deletions at either the N or C termini of the SH2-N domain abolished binding, suggesting that the entire SH2 sequence is required for formation of an active domain. Conservative substitutions of 2 highly conserved basic residues in the SH2-N domain, an arginine and a histidine, resulted in complete loss of receptor and p62 binding, whereas other basic residues, and residues at variable SH2 sites, were more tolerant of substitution. The conserved arginine and histidine therefore appear critical for association with phosphotyrosine-containing proteins, possibly through an interaction with phosphotyrosine. The GAP SH2-C domain, unlike SH2-N, does not bind efficiently to activated receptors or p62 in vitro. The SH2-C domain lacks 3 residues which are otherwise well conserved, and contribute to high affinity SH2-N binding. Replacement of 1 of these residues, a cysteine, with the consensus glycine, conferred SH2-C binding activity toward tyrosine-phosphorylated p62 and epidermal growth factor receptor. Loss-of-function and gain-of-function mutations in the GAP SH2 domains can therefore be used to identify residues that are critical for receptor and p62 binding.     

Role of the first extracellular loop in the functional activation of CCR2. The first extracellular loop contains distinct domains necessary for both agonist binding and transmembrane signaling.

The physiological cellular responses to monocyte chemoattractant protein-1 (MCP-1), a potent chemotactic and activating factor for mononuclear leukocytes, are mediated by specific binding to CCR2. The aim of this investigation is to identify receptor microdomains that are involved in high affinity agonist binding and receptor activation. The results from our functional studies in which we utilized neutralizing antisera against CCR2 are consistent with a multidomain binding model, previously proposed by others. The first extracellular loop was of particular interest, because in addition to a ligand-binding domain it contained also information for receptor activation, crucial for transmembrane signaling. Replacement of the first extracellular loop of CCR2 with the corresponding region of CCR1 decreased the MCP-1 binding affinity about 10-fold and prevented transmembrane signaling. A more detailed analysis by site-directed mutagenesis revealed that this receptor segment contains two distinct microdomains. The amino acid residues Asn(104) and Glu(105) are essential for high affinity agonist binding but are not involved in receptor activation. In contrast, the charged amino acid residue His(100) does not contribute to ligand binding but is vital for receptor activation and initiation of transmembrane signaling. We hypothesize that the interaction of agonist with this residue initiates the conformational switch that allows the formation of the functional CCR2-G protein complex.     

Binding areas of urokinase-type plasminogen activator-plasminogen activator inhibitor-1 complex for endocytosis receptors of the low-density lipoprotein receptor family, determined by site-directed mutagenesis

Some endocytosis receptors related to the low-density lipoprotein receptor, including low-density lipoprotein receptor-related protein-1A, very-low-density lipoprotein receptor, and sorting protein-related receptor, bind protease-inhibitor complexes, including urokinase-type plasminogen activator (uPA), plasminogen activator inhibitor-1 (PAI-1), and the uPA-PAI-1 complex. The unique capacity of these receptors for high-affinity binding of many structurally unrelated ligands renders mapping of receptor-binding surfaces of serpin and serine protease ligands a special challenge. We have mapped the receptor-binding area of the uPA-PAI-1 complex by site-directed mutagenesis. Substitution of a cluster of basic residues near the 37-loop and 60-loop of uPA reduced the receptor-binding affinity of the uPA-PAI-1 complex approximately twofold. Deletion of the N-terminal growth factor domain of uPA reduced the affinity 2-4-fold, depending on the receptor, and deletion of both the growth factor domain and the kringle reduced the affinity sevenfold. The binding affinity of the uPA-PAI-1 complex to the receptors was greatly reduced by substitution of basic and hydrophobic residues in alpha-helix D and alpha-helix E of PAI-1. The localization of the implicated residues in the 3D structures of uPA and PAI-1 shows that they form a continuous receptor-binding area spanning the serpin as well as the A-chain and the serine protease domain of uPA. Our results suggest that the 10-100-fold higher affinity of the uPA-PAI-1 complex compared with the free components depends on the bonus effect of bringing the binding areas on uPA and PAI-1 together on the same binding entity.     

Structure-based analysis of the herpes simplex virus glycoprotein D binding site present on herpesvirus entry mediator HveA (HVEM)

Binding of herpes simplex virus (HSV) envelope glycoprotein D (gD) to a cell surface receptor is an essential step of virus entry. We recently determined the crystal structure of gD bound to one receptor, HveA. HveA is a member of the tumor necrosis factor receptor family and contains four characteristic cysteine-rich domains (CRDs). The first two CRDs of HveA are necessary and sufficient for gD binding. The structure of the gD-HveA complex reveals that 17 amino acids in HveA CRD1 and 4 amino acids in HveA CRD2 directly contact gD. To determine the contribution of these 21 HveA residues to virus entry, we constructed forms of HveA mutated in each of these contact residues. We determined the ability of the mutant proteins to bind gD, facilitate virus entry, and form HveA oligomers. Our results point to a binding hot spot centered around HveA-Y23, a residue that protrudes into a crevice on the surface of gD. Both the hydroxyl group and phenyl group of HveA-Y23 contribute to HSV entry. Our results also suggest that an intermolecular beta-sheet formed between gD and HveA residues 35 to 37 contributes to binding and that a C37-C19 disulfide bond in CRD1 is a critical component of HveA structure necessary for gD binding. The results argue that CRD2 is required for gD binding mainly to provide structural support for a gD binding site in CRD1. Only one mutant, HveA-R75A, exhibited enhanced gD binding. While some mutations influenced complex formation, the majority did not affect HSV entry, suggesting that most contact residues contribute to HveA receptor function collectively rather than individually. This structure-based dissection of the gD-HveA binding site highlights the contribution of key residues within HveA to gD binding and HSV entry and defines a target region for the design of small-molecule inhibitors.     

Superagonistic activation of ErbB-1 by EGF-related growth factors with enhanced association and dissociation rate constants

Epidermal growth factor (EGF) and transforming growth factor-alpha (TGFalpha) are mitogenic hormones that exert their activity primarily by binding to the EGF receptor, also known as ErbB-1. We have recently characterized a set of EGF/TGFalpha chimeric molecules with similar high affinity for ErbB-1 as EGF and TGFalpha and shown that three of these chimeras induce mitogenic cell stimulation at already a 10-fold lower concentration than their wild-type counterparts (Lenferink, A. E., Kramer, R. H., van Vugt, M. J., K?nigswieser, M., DiFiore, P. P., van Zoelen, E. J., and van de Poll, M. L. (1997) Biochem. J. 327, 859-865). In the present study we show that these so-called superagonistic chimeras do not differ from EGF and TGFalpha in their ability to induce ErbB-1 tyrosine phosphorylation but are considerably more potent in activation of mitogen-activated protein kinase phosphorylation. Direct cell binding studies and analysis of ligand-receptor interaction by surface plasmon resonance measurements revealed that both the association rate constant (k(on)) and the dissociation rate constant (k(off)) of these superagonists is 3-5-fold higher in comparison with the wild-type ligands and nonsuperagonistic chimeras. These data indicate that the dynamic on and off rate constants for receptor binding may be more specific parameters for determining the mitogenic activity of peptide hormones than their constants for equilibrium receptor binding.     

NMR dynamics-derived insights into the binding properties of a peptide interacting with an SH2 domain

The signal transduction protein phospholipase C-gamma1 (PLC-gamma1) is activated when its C-terminal SH2 domain (PLCC) binds the phosphorylated Tyr-1021 site (pTyr-1021) in the beta-platelet-derived growth factor receptor (PDGFR). To better understand the contributions that dynamics make to binding, we have used NMR relaxation experiments to investigate the motional properties of backbone amide and side chain methyl groups in a peptide derived from the pTyr-1021 site of PDGFR, both free and in complex with the PLCC SH2 domain. The free peptide has relaxation properties that are typical for a small, unstructured polymer, while the backbone of the bound peptide is least flexible for residues in the central portion of the binding site with the amplitude of pico- to nanosecond time scale motions increasing toward the C-terminus of the peptide. The increase in large amplitude motion toward the end of the pY1021 peptide is consistent with the bound peptide existing as an ensemble of states with C-terminal residues having the broadest distribution of backbone conformations, while residues in the central binding site are the most restricted. Deuterium spin relaxation experiments establish that the protein-peptide interface is highly dynamic, and this mobility may play an important role in modulating the affinity of the interaction.     

Modeling the epidermal growth factor -- epidermal growth factor receptor l2 domain interaction: implications for the ligand binding process

Signaling from the epidermal growth factor (EGF) receptor is triggered by the binding of ligands such as EGF or transforming growth factor alpha (TGF-alpha) and subsequent receptor dimerization. An understanding of these processes has been hindered by the lack of structural information about the ligand-bound, dimerized EGF receptor. Using an NMR-derived structure of EGF and a homology model of the major ligand binding domain of the EGF receptor and experimental data, we modeled the binding of EGF to this EGF receptor fragment. In this low resolution model of the complex, EGF sits across the second face of the EGF receptor L2 domain and EGF residues 10-16, 36-37, 40-47 bind to this face. The structural model is largely consistent with previously published NMR data describing the residues of TGF-alpha which interact strongly with the EGF receptor. Other EGF residues implicated in receptor binding are accounted by our proposal that the ligand binding is a two-step process with the EGF binding to at least one other site of the receptor. This three-dimensional model is expected to be useful in the design of ligand-based antagonists of the receptor.     

The chemical synthesis and binding affinity to the EGF receptor of the EGF-like domain of heparin-binding EGF-like growth factor (HB-EGF).

Heparin-binding EGF-like growth factor (HB-EGF), which belongs to the EGF-family of growth factors, was isolated from the conditioned medium of macrophage-like cells. To investigate the effect of N- and C-terminal residues of the EGF-like domain of HB-EGF in the binding affinity to the EGF receptor on A431 cell. We synthesized HB-EGF(44-86) corresponding to the EGF-like domain of HB-EGF and its N- or C-terminal truncated peptides. Thermolytic digestion demonstrated three disulfide bond pairings of the EGF-like domain in HB-EGF is consistent with that of human-EGF and human-TGF-alpha. HB-EGF(44-86) showed high binding affinity to EGF-receptor, like human-EGF. The truncation of the C-terminal Leu86 residue from HB-EGF(44-86), HB-EGF(45-86) or HB-EGF(46-86) caused a drastic reduction in the binding affinity to the EGF receptor. These results suggest that the EGF-like domain of HB-EGF plays an important role in the binding to the EGF receptor, and its C-terminal Leu86 residue is necessary for binding with the EGF-receptor. In addition, the deletion of the two N-terminal residues (Asp44-Pro45) from HB-EGF(44-86) caused a 10-fold decrease in relative binding affinity to the EGF receptor. This indicates that the two N-terminal residues of the EGF-like domain of HB-EGF are necessary for its optimal binding affinity to the EGF receptor.     

Structural analysis of an epidermal growth factor/transforming growth factor-alpha chimera with unique ErbB binding specificity

Various chimeras of the ErbB1-specific ligands epidermal growth factor (EGF) and transforming growth factor-alpha (TGFalpha) display an enlarged repertoire as activators of ErbB2.ErbB3 heterodimers. Mutational analysis indicated that particularly residues in the N terminus and B-loop region of these ligands are involved in the broadened receptor specificity. In order to understand the receptor specificity of T1E, a chimeric ligand constructed by the introduction of the linear N-terminal region of TGFalpha into EGF, we determined in this study the solution structure and dynamics of T1E by multidimensional NMR analysis. Subsequently, we studied the structural characteristics of T1E binding to both ErbB1 and ErbB3 by superposition modeling of its structure on the known crystal structures of ErbB3 and liganded ErbB1 complexes. The results show that the overall structure of T1E in solution is very similar to that of native EGF and TGFalpha but that its N terminus shows an extended structure that is appropriately positioned to form a triple beta-sheet with the large antiparallel beta-sheet in the B-loop region. This conformational effect of the N terminus together with the large overall flexibility of T1E, as determined by 15N NMR relaxation analysis, may be a facilitative property for its broad receptor specificity. The structural superposition models indicate that hydrophobic and electrostatic interactions of the N terminus and B-loop of T1E are particularly important for its binding to ErbB3.     

Modeling the Epidermal Growth Factor—Epidermal Growth Factor Receptor L2 Domain Interaction: Implications for the Ligand Binding Process

Abstract

Signaling from the epidermal growth factor (EGF) receptor is triggered by the binding of lig-ands such as EGF or transforming growth factor alpha (TGF-α) and subsequent receptor dimerization. An understanding of these processes has been hindered by the lack of structural information about the ligand-bound, dimerized EGF receptor. Using an NMR-derived structure of EGF and a homology model of the major ligand binding domain of the EGF receptor and experimental data, we modeled the binding of EGF to this EGF receptor fragment. In this low resolution model of the complex, EGF sits across the second face of the EGF receptor L2 domain and EGF residues 10–16, 36–37, 40–47 bind to this face. The structural model is largely consistent with previously published NMR data describing the residues of TGF-α which interact strongly with the EGF receptor. Other EGF residues implicated in receptor binding are accounted by our proposal that the ligand binding is a two-step process with the EGF binding to at least one other site of the receptor. This three-dimensional model is expected to be useful in the design of ligand-based antagonists of the receptor.     

Mutations in the immunoglobulin-like domain of gp190, the leukemia inhibitory factor (LIF) receptor,increase or decrease its affinity for LIF

The leukemia inhibitory factor (LIF) receptor comprises the low affinity binding chain gp190 and the high affinity converter gp130. The ectodomain of gp190 is among the most complex in the hematopoietin receptor family, because it contains two typical cytokine receptor homology domains separated by an immunoglobulin-like (Ig-like) domain. Human and murine gp190 proteins share 76% homology, but murine gp190 binds human LIF with a much higher affinity, a property attributed to the Ig-like domain. Using alanine-scanning mutagenesis of the Ig-like domain, we mapped a LIF binding site at its carboxyl terminus, mainly involving residue Phe-328. Mutation of selected residues into their orthologs in the murine receptor (Q251E and N321D) significantly increased the affinity for human LIF. Interestingly, these residues, although localized at both the amino and carboxyl terminus, make a spatially unique LIF binding site in a structural model of the Ig-like module. These results demonstrate definitively the role of the Ig-like domain in LIF binding and the potential to modulate receptor affinity in this family with very limited amino acid changes.     

Selective formation of ErbB-2/ErbB-3 heterodimers depends on the ErbB-3 affinity of epidermal growth factor-like ligands

EGF-like growth factors activate their ErbB receptors by promoting receptor-mediated homodimerization or, alternatively, by the formation of heterodimers with the orphan ErbB-2 through an as yet unknown mechanism. To investigate the selectivity in dimer formation by ligands, we have applied the phage display approach to obtain ligands with modified C-terminal residues that discriminate between ErbB-2 and ErbB-3 as dimerization partners. We used the epidermal growth factor/transforming growth factor alpha chimera T1E as the template molecule because it binds to ErbB-3 homodimers with low affinity and to ErbB-2/ErbB-3 heterodimers with high affinity. Many phage variants were selected with enhanced binding affinity for ErbB-3 homodimers, indicating that C-terminal residues contribute to the interaction with ErbB-3. These variants were also potent ligands for ErbB-2/ErbB-3 heterodimers despite negative selection for such heterodimers. In contrast, phage variants positively selected for binding to ErbB-2/ErbB-3 heterodimers but negatively selected for binding to ErbB-3 homodimers can be considered as "second best" ErbB-3 binders, which require ErbB-2 heterodimerization for stable complex formation. Our findings imply that epidermal growth factor-like ligands bind ErbB-3 through a multi-domain interaction involving at least both linear endings of the ligand. Apparently the ErbB-3 affinity of a ligand determines whether it can form only ErbB-2/ErbB-3 complexes or also ErbB-3 homodimers. Because no separate binding domain for ErbB-2 could be identified, our data support a model in which ErbB heterodimerization occurs through a receptor-mediated mechanism and not through bivalent ligands.     

High affinity ligand binding is not essential for granulocyte-macrophage colony-stimulating factor receptor activation.

The high affinity receptor of the cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) is a heterodimer composed of two members of the cytokine receptor superfamily. GM-CSF binds to the alpha-subunit (GM-R alpha) with low affinity and to the receptor alpha beta complex (GM-R alpha beta) with high affinity. The GM-CSF.GM-R alpha beta complex is responsible for biological activity. Interactions of the N-terminal helix of mouse GM-CSF with mGM-R alpha beta were examined by introducing single alanine substitutions of hydrophilic residues in this region of mGM-CSF. The consequences of these substitutions were evaluated by receptor binding and biological assays. Although all mutant proteins exhibited near wild-type biological activity, most were defective in high affinity receptor binding. In particular, substitution of Glu-21 with alanine abrogated high affinity binding leaving low affinity binding unaffected. Despite near wild-type biological activity, no detectable binding interaction of this mutant with mGM-R beta in the context of mGM-R alpha beta was observed. Cross-linking studies showed an apparent interaction of this mutant protein with mGM-R alpha beta. The deficient receptor binding characteristics and near wild-type biological activity of this mutant protein demonstrate that mGM-CSF receptor activation can occur independently of high affinity binding, suggesting that conformational changes in the receptor induced by mGM-CSF binding generate an active ligand-receptor complex.     

Proteolytic processing of TGFalpha redirects its mitogenic activity: the membrane-anchored form is autocrine, the secreted form is paracrine

Wild-type transforming growth factor alpha (TGFalpha) expression in lactotrope cells in the pituitary gland led to lactotrope-specific pituitary hyperplasia and adenomata. To indicate whether the EGF receptor is involved in this TGFalpha-mediated phenotype, we bred TGFalpha mice with mice expressing the cytoplasmic truncated-EGF receptor (EGFR-tr), which is dominant-negative in other models. These bitransgenic mice developed pituitary pathology despite expression of the dominant-negative receptor. To further characterize this observation, we generated two lineages of transgenic mice that overexpress mutant forms of TGFalpha: a processed soluble form (s TGFalpha) and a cytoplasmic-deleted form (TGFalphaDeltaC). While sTGFalpha expression in lactotrope cells failed to induce autocrine lactotrope hyperplasia, the pituitary became very enlarged due to proliferation of neighboring interstitial cells. In contrast, the TGFalphaDeltaC mice did not develop a phenotype, although the mRNA and protein were present in the pituitary and this form of TGFalpha was confirmed to be biologically active and targeted properly to the plasma membrane of cultured CHO cells. The results suggest that the cytoplasmic domain of TGFalpha is required for autocrine parenchymal tumor formation in the pituitary gland. This signal cannot be inhibited by the EGFR-tr. Conversely, the released form of TGFalpha appears to have primarily paracrine activity.     

Structural determinants of the Na+/H+ exchanger regulatory factor interaction with the beta 2 adrenergic and platelet-derived growth factor receptors

The Na(+)/H(+) exchanger regulatory factor (NHERF) binds through its PDZ1 domain to the carboxyl-terminal sequences NDSLL and EDSFL of the beta(2) adrenergic receptor (beta(2)AR) and platelet-derived growth factor receptor, respectively, and plays a critical role in the membrane localization and physiological regulation of these receptors. The crystal structures of the human NHERF PDZ1 domain bound to the sequences NDSLL and EDSFL have been determined at 1.9- and 2.2-A resolution, respectively. The beta(2)AR and platelet-derived growth factor receptor ligands insert into the PDZ1 binding pocket by a beta-sheet augmentation process and are stabilized by largely similar networks of hydrogen bonds and hydrophobic contacts. In the PDZ1-beta(2)AR complex, the side chain of asparagine at position -4 in the beta(2)AR peptide forms two additional hydrogen bonds with Gly(30) of PDZ1, which contribute to the higher affinity of this interaction. Remarkably, both complexes are further stabilized by hydrophobic interactions involving the side chains of the penultimate amino acids of the peptide ligands, whereas the PDZ1 residues Asn(22) and Glu(43) undergo conformational changes to accommodate these side chains. These results provide structural insights into the mechanisms by which different side chains at the position -1 of peptide ligands interact with PDZ domains and contribute to the affinity of the PDZ-ligand interaction.     

Epidermal growth factor contains both positive and negative determinants for interaction with ErbB-2/ErbB-3 heterodimers

Epidermal growth factor (EGF) and transforming growth factor (TGF)-alpha are potent activators of the ErbB-1 receptor, but, unlike TGF-alpha, EGF is also a weak activator of ErbB-2/ErbB-3 heterodimers. To understand the specificity of EGF-like growth factors for binding to distinct ErbB members, we used EGF/TGF-alpha chimeras to examine the requirements for ErbB-2/ErbB-3 activation. Here we show that in contrast to these two wild-type ligands, distinct EGF/TGF-alpha chimeras are potent activators of ErbB-2/ErbB-3 heterodimers. On the basis of differences in the potency of these various chimeras, specific residues in the linear N-terminal region and the so-called B-loop of these ligands were identified to be involved in interaction with ErbB-2/ErbB-3. A chimera consisting of human EGF sequences with the linear N-terminal region of human TGF-alpha was found to be almost as potent as the natural ligand neuregulin (NRG)-1beta in activating 32D cells expressing ErbB-2/ErbB-3 and human breast cancer cells. Binding studies revealed that this chimera, designated T1E, has high affinity for ErbB-2/ErbB-3 heterodimers, but not for ErbB-3 alone. Subsequent exchange studies revealed that introduction of both His2 and Phe3 into the linear N-terminal region was already sufficient to make EGF a potent activator of ErbB-2/ErbB-3 heterodimers, indicating that these two amino acids contribute positively to this receptor binding. Analysis of the B-loop revealed that Leu26 in EGF facilitates interaction with ErbB-2/ErbB-3 heterodimers, while the equivalent Glu residue in TGF-alpha impairs binding. Since all EGF/TGF-alpha chimeras tested have maintained high binding affinity for ErbB-1, it is concluded that the diversity of the ErbB signaling network is determined by specific amino acids that facilitate binding to one receptor member, in addition to residues that impede binding to other ErbB family members.     

Identification of the ligand-binding site of the BMP type IA receptor for BMP-4

Bone morphogenetic proteins (BMPs) belong to the transforming growth factor-beta (TGF-beta) superfamily of multifunctional cytokines. BMP induces its signal to regulate growth, differentiation, and apoptosis of various cells upon trimeric complex formation with two distinct type I and type II receptors on the cell surface: both are single-transmembrane serine/threonine kinase receptors. To identify the amino acid residues on BMP type I receptor responsible for its ligand binding, the structure-activity relationship of the extracellular ligand-binding domain of the BMP type IA receptor (sBMPR-IA) was investigated by alanine-scanning mutagenesis. The mutant receptors, as well as sBMPR-IA, were expressed as fusion proteins with thioredoxin in Escherichia coli, and purified using reverse phase high performance liquid chromatography (RP-HPLC) after digestion with enterokinase. Structural analysis of the parent protein and representative mutants in solution by CD showed no detectable differences in their folding structures. The binding affinity of the mutants to BMP-4 was determined by surface plasmon resonance biosensor. All the mutant receptors examined, with the exception of Y70A, displayed reduced affinities to BMP-4 with the rank order of decreases: I52A (17-fold) approximately F75A (15-fold) > T64A (4-fold) = T62A (4-fold) approximately E54A (3-fold). The decreases in binding affinity observed for the latter three mutants are mainly due to decreased association rate constants while alterations in rate constants both, for association and dissociation, result in the drastically reduced affinities for the former two mutants. These results allow us to conclude that sBMPR-IA recognizes the ligand using the concave face of the molecule. The major ligand-binding site of the BMP type IA receptor consists of Phe75 in loop 2 and Ile52, Glu54, Thr62 and Thr64 on the three-stranded beta-sheet. These findings should provide a general basis for the ligand/type I receptor recognition in the TGF-beta superfamily.