Chimeric receptor analyses of the interactions of the ectodomains of ErbB-1 with epidermal growth factor and of those of ErbB-4 with neuregulin.

A series of chimeric receptors was generated between the epidermal growth factor (EGF) receptor, ErbB-1, and its homologue, ErbB-4, to investigate the roles of the extracellular domains (I-IV) in the ligand specificities. As compared with ErbB-1 and the chimeras with both domains I and III of ErbB-1, the chimeras with only one of these domains exhibited reduced binding of 125I-labeled EGF. Particularly, the contribution of domain III was appreciably larger than that of domain I of ErbB-1 in 125I-labeled EGF binding. Nevertheless, the chimeras with domain III of ErbB-1 and domain I of ErbB-4 were prevented from binding to 125I-labeled EGF competitively by the ErbB-4 ligand, neuregulin (NRG). On the other hand, NRG did not compete with 125I-labeled EGF for binding to the chimeras with the ErbB-1 domain I and the ErbB-4 domain III. Therefore, NRG binding to ErbB-4 depends much more on domain I than on domain III. With respect to autophosphorylation and subsequent ERK activation, EGF activated the chimeras with either domain I or III of ErbB-1. In contrast, NRG activated the chimeras with the ErbB-4 domain I and the ErbB-1 domain III, but not those with the ErbB-1 domain I and the ErbB-4 domain III. Therefore, the relative contributions between domains I and III of ErbB-4 in the NRG signaling are different from those of ErbB-1 in the EGF signaling.     

Crystal structure of the complex of human epidermal growth factor and receptor extracellular domains

Epidermal growth factor (EGF) regulates cell proliferation and differentiation by binding to the EGF receptor (EGFR) extracellular region, comprising domains I-IV, with the resultant dimerization of the receptor tyrosine kinase. In this study, the crystal structure of a 2:2 complex of human EGF and the EGFR extracellular region has been determined at 3.3 A resolution. EGFR domains I-III are arranged in a C shape, and EGF is docked between domains I and III. The 1:1 EGF*EGFR complex dimerizes through a direct receptor*receptor interaction, in which a protruding beta-hairpin arm of each domain II holds the body of the other. The unique "receptor-mediated dimerization" was verified by EGFR mutagenesis.     

Functional analysis of the ligand binding site of EGF-receptor utilizing chimeric chicken/human receptor molecules.

The epidermal growth factor (EGF)-receptor is composed of an extracellular ligand-binding region connected by a single transmembrane region to the cytoplasmic kinase domain. In spite of its importance for understanding signal transduction, the ligand-binding domain of the EGF-receptor is not yet defined. We describe the identification of a major ligand-binding domain of the EGF-receptor by utilizing chimeras between the human EGF-receptor and the chicken EGF-receptor. This approach is based on the fact that murine EGF binds to the chicken EGF-receptor with 100-fold lower affinity as compared to the human EGF-receptor. Hence, the substitution of various domains of the chicken EGF-receptor by domains of the human EGF-receptor may restore the higher binding affinity towards EGF, characteristic of the human receptor. We show that chimeric chicken/human EGF-receptor, which contains domain III of the extracellular region of the human receptor, behaves like the human EGF-receptor with respect to EGF binding affinity and biological responsiveness. However, a chimeric chicken/human EGF-receptor containing domains I and II of the human receptor behaves like the chicken rather than the human EGF-receptor. Moreover, two different monoclonal antibodies which compete for the binding of EGF to EGF-receptor recognize specifically domain III of the human EGF-receptor. It is concluded that domain III which is flanked by the two cysteine-rich domains is a major ligand-binding domain of the EGF-receptor.     

Photolabeling identifies position 172 of the human AT(1) receptor as a ligand contact point: receptor-bound angiotensin II adopts an extended structure

An angiotensin II (AngII) peptidic analogue in which the third residue (valine) was substituted with the photoreactive p-benzoyl-L-phenylalanine (Bpa) was used to identify ligand-binding sites of the human AT(1) receptor. High-affinity binding of the analogue, (125)I-[Bpa(3)]AngII, to the AT(1) receptor heterologously expressed in COS-7 cells enabled us to efficiently photolabel the receptor. Chemical and enzymatic digestions of the (125)I-[Bpa(3)]AngII-AT(1) complex were performed, and receptor fragments were analyzed in order to define the region of the receptor with which the ligand interacts. Results show that CNBr hydrolysis of the photolabeled receptor gave a glycosylated fragment which, after PNGase-F digestion, migrated as a 11.4 kDa fragment, circumscribing the labeled domain between residues 143-243 of the AT(1) receptor. Digestion of the receptor-ligand complex with Endo Lys-C or trypsin followed by PNGase-F treatment yielded fragments of 7 and 4 kDa, defining the labeling site of (125)I-[Bpa(3)]AngII within residues 168-199 of the AT(1) receptor. Photolabeling of three mutant receptors in which selected residues adjacent to residue 168 were replaced by methionine within the 168-199 fragment (I172M, T175M, and I177M) followed by CNBr cleavage revealed that the bound photoligand (125)I-[Bpa(3)]AngII forms a covalent bond with the side chain of Met(172) of the second extracellular loop of the AT(1) receptor. These data coupled with previously obtained results enable us to propose a model whereby AngII adopts an extended beta-strand conformation when bound to the receptor and would orient itself within the binding domain by having its N-terminal portion interacting with the second extracellular loop and its C-terminus interacting with residues of the seventh transmembrane domain.     

The membrane proximal disulfides of the EGF receptor extracellular domain are required for high affinity binding and signal transduction but do not play a role in the localization of the receptor to lipid rafts

The EGF receptor is a transmembrane receptor tyrosine kinase that is enriched in lipid rafts. Subdomains I, II and III of the extracellular domain of the EGF receptor participate in ligand binding and dimer formation. However, the function of the cysteine-rich subdomain IV has not been elucidated. In this study, we analyzed the role of the membrane-proximal portion of subdomain IV in EGF binding and signal transduction. A double Cys-->Ala mutation that breaks the most membrane-proximal disulfide bond (Cys600 to Cys612), ablated high affinity ligand binding and substantially reduced signal transduction. A similar mutation that breaks the overlapping Cys596 to Cys604 disulfide had little effect on receptor function. Mutation of residues within the Cys600 to Cys612 disulfide loop did not alter the ligand binding or signal transducing activities of the receptor. Despite the fact that the C600,612A EGF receptor was significantly impaired functionally, this receptor as well as all of the other receptors with mutations in the region of residues 596 to 612 localized normally to lipid rafts. These data suggest that the disulfide-bonded structure of the membrane-proximal portion of the EGF receptor, rather than its primary sequence, is important for EGF binding and signaling but is not involved in localizing the receptor to lipid rafts.     

Identification of a heregulin binding site in HER3 extracellular domain

HER3 (also known as c-Erb-b3) is a type I receptor tyrosine kinase similar in sequence to the epidermal growth factor (EGF) receptor. The extracellular segment of this transmembrane receptor contains four domains. Domains I and II are similar in sequence to domains III and IV, respectively, and domains II and IV are cysteine-rich. We show that the EGF-like domain of heregulin (hrg) binds to domains I and II of HER3, in contrast to the EGF receptor, for which prior studies have shown that a construct consisting of domains III and portions of domain IV binds EGF. Next, we identified a putative hrg binding site by limited proteolysis of the recombinant extracellular domains of HER3 (HER3-ECD(I-IV)) in both the presence and absence of hrg. In the absence of hrg, HER3-ECD(I-IV) is cleaved after position Tyr(50), near the beginning of domain I. Binding of hrg to HER3-ECD(I-IV) fully protects position Tyr(50) from proteolysis. To confirm that domain I contains a hrg binding site, we expressed domains I and II (HER3-ECD(I-II)) and find that it binds hrg with 68 nm affinity. These data suggest that domains I and II of HER3-ECD(I-IV) act as a functional unit in folding and binding of hrg. Thus, our biochemical findings reinforce the structural hypothesis of others that HER3-ECD(I-IV) is similar to the insulin-like growth factor-1 receptor (IGF-1R), as follows: 1) The protected cleavage site in HER3-ECD(I-IV) corresponds to a binding footprint in domain I of IGF-1R; 2) HER3-ECD(I-II) binds hrg with a 68 nm dissociation constant, supporting the hypothesis that domain I is involved in ligand binding; and 3) the large accessible surface area (1749 A) of domain L1 of IGF-1R that is buried by domain S1, as well as the presence of conserved contacts in this interface of type 1 RTKs, suggests that domains L1 and S1 of IGF-1R function as a unit as observed for HER3-ECD(I-II). Our results are consistent with the proposal that HER3 has a structure similar to IGF-1R and binds ligand at a site in corresponding domains.     

Platelet receptor recognition domains on the alpha chain of human fibrinogen: structure-function analysis

We have previously shown that the alpha chain of human fibrinogen interacts directly with ADP-activated human platelets [Hawiger, J., Timmons, S., Kloczewiak, M., Strong, D. D., & Doolittle, R. F. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 2068]. Now, we report that platelet receptor recognition domains are localized on two CNBr fragments of the human fibrinogen alpha chain. They encompass residues 92-147 and 518-584, which inhibit 125I-fibrinogen binding to ADP-stimulated platelets. The inhibitory CNBr fragment alpha 92-147 contains the RGD sequence at residues 95-97. Synthetic peptides encompassing this sequence were inhibitory while peptide 99-113 lacking the RGD sequence was inactive. The synthetic peptide RGDF, corresponding to residues alpha 95-98, inhibited the binding of 125I-fibrinogen to ADP-treated platelets (IC50 = 2 microM). However, the peptides containing sequence RGDF, with residues preceding Arg95 or following Phe98, were less inhibitory. It appears that the sequence alpha 95-98 constitutes a platelet receptor recognition domain which is constrained by flanking residues. The second inhibitory CNBr fragment, alpha 518-584, also contains the sequence RGD at positions 572-574. Synthetic peptides overlapping this sequence were inhibitory, while peptides lacking the sequence RGDS were not reactive. Thus, another platelet reactive site on the alpha chain encompasses residues 572-575 containing sequence RGDS. In conclusion, the platelet receptor recognition domains on the human fibrinogen alpha chain in the amino-terminal and in the carboxy-terminal zones contain the ubiquitous cell recognition sequence RGD shared with other known adhesive proteins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanisms for Kinase-mediated Dimerization of the Epidermal Growth Factor Receptor

We study a mechanism by which dimerization of the EGF receptor (EGFR) cytoplasmic domain is transmitted to the ectodomain. Therapeutic and other small molecule antagonists to the kinase domain that stabilize its active conformation, but not those that stabilize an inactive conformation, stabilize ectodomain dimerization. Inhibitor-induced dimerization requires an asymmetric kinase domain interface associated with activation. EGF and kinase inhibitors stimulate formation of identical dimer interfaces in the EGFR transmembrane domain, as shown by disulfide cross-linking. Disulfide cross-linking at an interface in domain IV in the ectodomain was also stimulated similarly; however, EGF but not inhibitors stimulated cross-linking in domain II. Inhibitors similarly induced noncovalent dimerization in nearly full-length, detergent-solubilized EGFR as shown by gel filtration. EGFR ectodomain deletion resulted in spontaneous dimerization, whereas deletion of exons 2–7, in which extracellular domains III and IV are retained, did not. In EM, kinase inhibitor-induced dimers lacked any well defined orientation between the ectodomain monomers. Fab of the therapeutic antibody cetuximab to domain III confirmed a variable position and orientation of this domain in inhibitor-induced dimers but suggested that the C termini of domain IV of the two monomers were in close proximity, consistent with dimerization in the transmembrane domains. The results provide insights into the relative energetics of intracellular and extracellular dimerization in EGFR and have significance for physiologic dimerization through the asymmetric kinase interface, bidirectional signal transmission in EGFR, and mechanism of action of therapeutics.     

Mapping of a ligand-binding site for the human thromboxane A2 receptor protein

The human thromboxane A(2) (TP) receptor, a member of the G protein-coupled receptor superfamily, consists of seven transmembrane segments. Attempts to elucidate the specific segment(s) that define the receptor ligand-binding pocket have produced less than definitive and sometimes conflicting results. On this basis, the present work identified an amino acid sequence of the TP receptor that is directly involved in ligand binding. Mapping of this domain was confirmed by two separate approaches: photoaffinity labeling and site-specific antibodies. The newly synthesized, biotinylated photoaffinity probe, SQBAzide, was first shown to specifically label TP receptor protein. Sequential digestion of this protein with CNBr/trypsin revealed photolabeling of a 2.9-kDa peptide. Using anti-peptide antibodies directed against different regions of the receptor protein, it was established that this peptide represents the predicted cleavage product for CNBr/trypsin and corresponds to amino acids Arg(174)-Met(202) of the receptor protein. Furthermore, antibody screening revealed that inhibition of the amino acid region Cys(183)-Asp(193) was critical for radioligand binding and platelet aggregation, whereas inhibition of Gly(172)-Cys(183) was not. Collectively these findings provide evidence that ligands interact with amino acids contained within the C-terminal portion of the third extracellular domain (ED3) of the receptor protein. This information should be of significant value in the study of TP receptor structure and signaling.     

Epidermal growth factor receptor cytoplasmic domain mutations trigger ligand-independent transformation.

The transforming gene product of avian erythroblastosis virus, v-erbB, is derived from the epidermal growth factor (EGF) receptor but has lost its extracellular ligand-binding domain and was mutated in its cytoplasmic portion, which is thought to be responsible for biological signal generation. We have repaired the deletion of extracellular EGF-binding sequences and investigated the functional consequences of cytoplasmic erbB mutations. Within the resulting EGF receptors, the autophosphorylation activities of the cytoplasmic domains of v-erbB-H and v-erbB-ES4 were fully ligand dependent in intact cells. However, the mitogenic and transforming signaling activities of an EGF receptor carrying v-erbB-ES4 (but not v-erbB-H) cytoplasmic sequences remained ligand independent, whereas those of a receptor with a v-erbB-H cytoplasmic domain were regulated by EGF or transforming growth factor alpha. Thus, structural alterations in the cytoplasmic domain of growth factor receptor tyrosine kinases may induce constitutive signaling activity without autophosphorylation. These findings provide new insight into the mechanism of receptor-mediated signal transduction and suggest a novel alternative for subversion of cellular control mechanisms and proto-oncogene activation.     

Dimerization of the extracellular domain of the receptor for epidermal growth factor containing the membrane-spanning segment in response to treatment with epidermal growth factor

A recombinant fragment of the human receptor for epidermal growth factor containing both its extracellular domain and its membrane-spanning segment, when dissolved with Triton X-100, was observed to dimerize in response to addition of epidermal growth factor (EGF) even at the lowest concentration of this fragment that could be assayed (4 nM). Consequently, the dissociation constant for the dimer of this fragment is at least 10,000-fold smaller than that for dimers of soluble, recombinant forms of the extracellular domain lacking the membrane-spanning segment. The second-order rate constant for dimerization of the fragment containing the extracellular domain and the membrane-spanning segment was estimated to be greater than 0.3 nM(-1) min(-1), more than 10-fold that of the native enzyme under the same conditions. This result suggests that the cytoplasmic domain of the intact enzyme sterically hinders its dimerization. When EGF is removed from the dimer of the fragment, the rate constant for its dissociation is greater than 0.2 min(-1), more than 40-fold that of the native enzyme. This result suggests that interfaces between cytoplasmic domains of intact EGF receptor impart significant stabilization to the dimer of the enzyme.     

The Drosophila EGF receptor gene homolog: conservation of both hormone binding and kinase domains

Chicken v-erB probe was used to isolate a unique clone of Drosophila melanogaster DNA. It maps by in situ hybridization to position 57F on chromosome 2. A complete nucleotide sequence of the coding region has been obtained. The putative Drosophila EGF receptor protein is similar in overall organization to the human homolog. It shows three distinct domains: an extracellular putative EGF binding domain, a hydrophobic transmembrane region, and a cytoplasmic kinase domain. The overall amino acid homology is 41% in the extracellular domain and 55% in the kinase domain. Two cysteine-rich regions, a hallmark of the human ligand-binding domain, have also been conserved. Fusion of the coding sequences of the kinase and extracellular domains generating the receptor gene must have occurred over 800 million years ago.     

Primary structure of the mannose receptor contains multiple motifs resembling carbohydrate-recognition domains

Macrophages express a cell surface receptor which mediates phagocytosis and pinocytosis of particles and solutes containing mannose (fucose and N-acetylglucosamine are also ligands for the receptor). An apparently identical protein has been isolated from human placenta. Proteolytic fragments of the placental receptor were sequenced so that oligonucleotide probes complementary to the receptor cDNA could be generated. These probes were used to isolate cDNA clones covering the entire coding portion of the mRNA for the receptor. Confirmation that these clones encode the mannose receptor was obtained by expression in rat fibroblasts. The expressed protein mediates uptake and degradation of mannose-conjugated serum albumin. The deduced amino acid sequence of the receptor reveals that it is most likely to be a type I transmembrane protein (COOH terminus on the cytoplasmic side of the membrane) since the mature polypeptide is preceded by a signal sequence and a hydrophobic stop transfer sequence is located 45 amino acids from the COOH terminus. The extracellular portion of the receptor polypeptide consists of three types of domains. The first 139 amino acids constitute a cysteine-rich segment which does not resemble other known sequences. There follows a domain which closely resembles fibronectin type II repeats. The remainder of the extracellular portion of the receptor is composed of eight segments homologous with the C-type carbohydrate-recognition domains of the asialoglycoprotein receptor, mannose binding proteins, and other Ca2(+)-dependent animal lectins. This structure suggests that the receptor may contain multiple ligand-binding domains thus accounting for its tight binding to highly multivalent ligands.     

Functional domains of the granulocyte colony-stimulating factor receptor.

The granulocyte colony-stimulating factor (G-CSF) receptor has a composite structure consisting of an immunoglobulin(Ig)-like domain, a cytokine receptor-homologous (CRH) domain and three fibronectin type III (FNIII) domains in the extracellular region. Introduction of G-CSF receptor cDNA into IL-3-dependent murine myeloid cell line FDC-P1 and pro-B cell line BAF-B03, which normally do not respond to G-CSF, enabled them to proliferate in response to G-CSF. On the other hand, expression of the G-CSF receptor cDNA in the IL-2-dependent T cell line CTLL-2 did not enable it to grow in response to G-CSF, although G-CSF could transiently stimulate DNA synthesis. Mutational analyses of the G-CSF receptor in FDC-P1 cells indicated that the N-terminal half of the CRH domain was essential for the recognition of G-CSF, but the Ig-like, FNIII and cytoplasmic domains were not. The CRH domain and a portion of the cytoplasmic domain of about 100 amino acids in length were indispensable for transduction of the G-CSF-triggered growth signal.     

Identification and primary structure of a calmodulin binding domain of the Ca2+ pump of human erythrocytes

Exposure of the purified Ca2+ pump of human erythrocytes to chymotrypsin led to the rapid loss of calmodulin activation. A fragment of about 12 kDa was removed from the ATPase in 1-2 min. Blotting experiments with 125I-labeled calmodulin showed that this fragment contains the calmodulin binding region. The remainder of the ATPase molecule was degraded to a number of fragments ranging from 3 to 120 kDa; none of them bound calmodulin. To isolate the calmodulin binding domain, calmodulin which had been coupled to the Denny-Jaffe reagent (a cleavable radioactive photoaffinity cross-linker) was allowed to bind to the Ca2+ pump. After illumination to couple the cross-linker to the pump, the cleavable bond was split and the calmodulin removed, leaving the pump radioactively labeled. This pump was digested with chymotrypsin, and the products were separated by gel permeation chromatography. The only radioactive peak (migrating at about 12 kDa) was further purified on reverse-phase high pressure liquid chromatography (HPLC). Amino acid analysis showed the fragment to have a minimal molecular mass of 12.4 kDa and to contain a single methionine. After attempts to sequence the peptide directly failed. CNBr digestion was carried out on the labeled ATPase, producing both soluble and insoluble labeled material. After reverse-phase HPLC purification of the soluble material, a single radioactive peak was collected. Its sequence was (Formula: see text). A portion of this peak was passed through a microcalmodulin column; it bound in the presence of Ca2+ and was eluted by EDTA, and by a mixture of EDTA and urea. Staphylococcal V8 protease digestion of the eluted peak produced the same sequence as shown above, but starting at Leu-2 and ending at Glu-32. Structural analysis of this peptide showed that it shares features with the calmodulin binding domains of other enzymes which are regulated by calmodulin.     

Characterization of structural domains of the human epidermal growth factor receptor obtained by partial proteolysis

Partial cleavage with trypsin has been used to study the structure of the epidermal growth factor (EGF) receptor purified from human carcinoma cells. Following affinity labeling of the receptor with 125I-EGF or the ATP analogue 5'-p-fluorosulfonyl benzoyl[14C]adenosine, metabolic labeling with [35S]methionine, [3H]glucosamine, or [32P]orthophosphate, or in vitro autophosphorylation with [gamma-32P]ATP, tryptic cleavage defines the following three regions of the 180-kDa receptor protein: 1) a 125-kDa trypsin-resistant domain which contains sites of glycosylation, EGF binding, and an EGF-specific threonine phosphorylation site; 2) an adjacent 40-kDa fragment which contains serine and threonine phosphorylation sites and is further cleaved to a 30-kDa trypsin-resistant domain; and 3) a terminal 15-kDa portion of the receptor that contains the sites of tyrosine phosphorylation and is degraded to small fragments in the presence of trypsin. Both the 125- and 40-kDa regions of the EGF receptor appear to be required for receptor-associated protein kinase activity since separation of these regions by tryptic cleavage abolishes this activity, and both regions are specifically labeled with an ATP affinity analogue, suggesting that both are involved in ATP binding. Additional 63- and 48-kDa phosphorylated fragments are generated upon trypsin treatment of EGF receptor from EGF-treated cells. The potential usefulness of partial tryptic cleavage in studying the EGF receptor and the possible biological function of the 30-kDa trypsin-resistant fragment of the receptor are discussed.     

Perinuclear location and recycling of epidermal growth factor receptor kinase: immunofluorescent visualization using antibodies directed to kinase and extracellular domains

This paper describes studies on the migratory behavior of epidermal growth factor (EGF) receptor kinase using antibodies that are specific for either the kinase domain or the extracellular domain of the receptor. Antiserum was raised to a 42,000-D subfragment of EGF receptor, which was shown earlier to carry the kinase catalytic site but not the EGF-binding site. Another antiserum was raised to the pure intact 170,000-D EGF receptor. The specificities of these antibodies were established by immunoprecipitation and immunoblotting experiments. The domain specificity was examined by indirect immunofluorescent staining of fixed cells. The anti-42-kD peptide antibody could bind specifically to EGF receptors of both human and murine origin and was found to be directed to the cytoplasmic part of the molecule. It did not bind to EGF receptor-negative cells, which contained other types of tyrosine kinases. The antibodies raised against the intact receptor recognized only EGF receptor-specific epitopes and were directed to the extracellular part of the molecule. The anti-receptor antibodies described above were used to visualize the cyclic locomotory behavior of EGF receptor kinase under various conditions of EGF stimulation and withdrawal. The receptor was examined in fixed and permeabilized cells by indirect immunofluorescent staining. The results demonstrate the following: (a) the receptor kinase domain migrates to the perinuclear region upon challenge with EGF; (b) both extracellular and cytoplasmic domains of the receptor are involved in migration as a unit; (c) withdrawal of EGF results in rapid recycling of the perinuclear receptors to the plasma membrane; (d) this return to the cell surface is inhibited by methylamine, chloroquine, and monensin; and (e) neither the internal migration nor the recycling process is blocked by inhibitors of protein biosynthesis.     

Characterization of a comparative model of the extracellular domain of the epidermal growth factor receptor

The Epidermal Growth Factor (EGF) receptor is a tyrosine kinase that mediates the biological effects of ligands such as EGF and transforming growth factor alpha. An understanding of the molecular basis of its action has been hindered by a lack of structural and mutational data on the receptor. We have constructed comparative models of the four extracellular domains of the EGF receptor that are based on the structure of the first three domains of the insulin-like growth factor-1 (IGF-1) receptor. The first and third domains of the EGF receptor, L1 and L2, are right-handed beta helices. The second and fourth domains of the EGF receptor, S1 and S2, consist of the modules held together by disulfide bonds, which, except for the first module of the S1 domain, form rod-like structures. The arrangement of the L1 and S1 domains of the model are similar to that of the first two domains of the IGF-1 receptor, whereas that of the L2 and S2 domains appear to be significantly different. Using the EGF receptor model and limited information from the literature, we have proposed a number of regions that may be involved in the functioning of the receptor. In particular, the faces containing the large beta sheets in the L1 and L2 domains have been suggested to be involved with ligand binding of EGF to its receptor.     

Epidermal growth factor (EGF) induces oligomerization of soluble, extracellular, ligand-binding domain of EGF receptor. A low resolution projection structure of the ligand-binding domain.

Ligand-induced oligomerization is a universal phenomenon among growth factor receptors. Although the mechanism involved is yet to be defined, much evidence indicates that receptor oligomerization plays a crucial role in receptor activation and signal transduction. Here we show that epidermal growth factor (EGF) is able to stimulate the oligomerization of a recombinant, soluble, extracellular ligand-binding domain of EGF receptor. Covalent cross-linking experiments, analysis by sodium dodecyl sulfate-gel electrophoresis, size exclusion chromatography, and electron microscopy demonstrate that receptor dimers, trimers and larger multimers are formed in response to EGF. This establishes that receptor oligomerization is an intrinsic property of the extracellular ligand-binding domain of EGF receptor. Ligand-induced conformational change in the extracellular domain will stimulate receptor-receptor interactions. This may bring about the allosteric change involved in signal transduction from the extracellular domain across the plasma membrane, resulting in the activation of the cytoplasmic kinase domain. Electron microscopic images of individual extracellular ligand-binding domains appear as clusters of four similarly-sized stain-excluding areas arranged around a central, relatively less stain-excluded area. This suggests that the extracellular ligand-binding domain is structurally composed of four separate domains.