Highly conserved eight amino acid sequence in SH2 is important for recognition of phosphotyrosine site.

Src homology region 2(SH2) has been demonstrated to recognize phosphotyrosine site. To clarify the precise mechanism of the recognition, we developed in vitro binding assay system using EGF receptor and SH2/SH3 region of phospholipase C(PLC) gamma 1. Phosphorylated EGF receptor bound to immobilized SH2/SH3 of PLC gamma 1 in Sepharose beads, while nonphosphorylated EGF receptor did not bind. In SH2 domain of PLC gamma 1, there are several highly conserved amino acid sequences that are common in a variety of SH2-containing proteins. Especially the eight amino acid sequence, G(S/T)FLVR(E/D)S is highly conserved in these proteins. We synthesized several peptides related to these sequences and examined the effect of peptides on the binding of EGF receptor to SH2 of PLC gamma 1. P1, GSFLVRES was the most effective inhibitor to suppress the binding. P2, GSFLVAES in which one amino acid, arginine of P1 is substituted by alanine is still effective. But a peptide, P3, SFLVRE in which two amino acids are deleted from P1 did not inhibit markedly. Moreover, P1 peptide immobilized in Sepharose beads also bound phosphorylated EGF receptor. These data suggest that highly conserved amino acid sequence GSFLVRES is the minimum essential unit to recognize tyrosine phosphorylated site.     

Expression and characterization of hepatocyte growth factor receptor-IgG fusion proteins. Effects of mutations in the potential proteolytic cleavage site on processing and ligand binding.

The receptor for hepatocyte growth factor (HGF) is the product of the c-met proto-oncogene, a membrane-spanning tyrosine kinase receptor. To facilitate analysis of HGF and its receptor (HGFr), we expressed and purified a chimeric protein containing the extracellular domain (ECD) of the HGFr fused to the constant region of IgG heavy chain. This soluble form of the HGFr (sHGFr) bound HGF with an affinity similar to that of the authentic, membrane-associated receptor. The sHGFr also neutralized the binding of HGF to the HGFr expressed on A549 cells. Like the mature form of the HGFr, sHGFr is a heterodimer which arises by proteolytic processing within the ECD. In order to characterize the requirements for proteolytic processing of the ECD and the effects of cleavage on ligand binding, we expressed sHGFr variants containing amino acid substitutions in the putative processing site. Replacement of the P1 or P4 arginine, but not the P3 lysine, with alanine inhibited conversion to the alpha/beta heterodimer. This suggests that maturation is mediated by furin or a furin-like protease. Finally, we showed that processing of the sHGFr into the alpha/beta form is not required for high affinity binding to either pro- or mature HGF.     

Hepsin activates prostasin and cleaves the extracellular domain of the epidermal growth factor receptor

The epithelial extracellular serine protease activation cascade involves matriptase (PRSS14) and prostasin (PRSS8), capable of modulating epidermal growth factor receptor (EGFR) signaling. Matriptase activates prostasin by cleaving in the amino-terminal pro-peptide region of prostasin, presumably at the Arg residue of position 44 (R44) of the full-length human prostasin. Using an Arg-to-Ala mutant (R44A) human prostasin, we showed in this report that the cleavage of prostasin by matriptase is at Arg44. This prostasin proteolytic activation site is also cleaved by hepsin (TMPRSS1) to produce active prostasin capable of forming a covalent complex with protease nexin 1 (PN-1). An amino-terminal truncation of EGFR in the extracellular domain (ECD) was observed when the receptor was co-expressed with hepsin. Hepsin and matriptase appear to cleave the EGFR ECD at different sites, while the hepsin cleavage is not affected by active prostasin, which enhances the matriptase cleavage of EGFR. Using hepsin as the prostasin-activating protease in cells co-transfected with EGFR, we showed that active prostasin does not cleave the EGFR ECD directly in the cellular context. Purified active prostasin also does not cleave purified EGFR. Hepsin cleavage of EGFR is not dependent on receptor tyrosine phosphorylation, while the hepsin-cleaved EGFR is phosphorylated at Tyr1068 and no longer responsive to EGF stimulation. The cleavage of EGFR by hepsin does not result in increased phosphorylation of the downstream extracellular signal-regulated kinases (Erk1/2), an event inducible by the matriptase–prostasin cleavage of EGFR. The role of hepsin serine protease should be considered in future studies of epithelial biology concerning matriptase, prostasin, and EGFR.     

Antibodies to the autophosphorylation sites of the epidermal growth factor receptor protein-tyrosine kinase as probes of structure and function.

Antisera were prepared against three synthetic peptides with amino acid sequences identical to those surrounding the three major autophosphorylation sites of the epidermal growth factor (EGF) receptor. The affinity-purified antibodies reacted strongly in an enzyme-linked immunosorbent assay against the immunizing peptide but showed little cross-reaction with the other two phosphorylation site peptides. EGF receptors labelled by autophosphorylation could be specifically precipitated by each of the phosphorylation site antibodies. The antibodies recognised EGF receptors labelled at each of the autophosphorylation sites, indicating that they could bind to the immunizing sequences irrespective of their states of phosphorylation. The antibodies were able to inhibit EGF receptor autophosphorylation without affecting EGF-stimulated tyrosine kinase activity towards exogenous peptide substrates, suggesting that the kinase and autophosphorylation sites were in distinct domains. Immunofluorescent staining of A431 cells showed that the autophosphorylation site sequences resided inside the cell. The autophosphorylation sites were shown to be within a domain of 20 000 mol. wt. which could be cleaved from the receptor through limited proteolysis by the calcium-dependent protease, calpain. The position of cleavage of the EGF receptor by the protease was mapped to lie between residues 996 and 1059. These results are discussed in the context of a model for the structure and function of the human EGF receptor.     

Molecular cloning of a functional thrombin receptor reveals a novel proteolytic mechanism of receptor activation.

We isolated a cDNA encoding a functional human thrombin receptor by direct expression cloning in Xenopus oocytes. mRNA encoding this receptor was detected in human platelets and vascular endothelial cells. The deduced amino acid sequence revealed a new member of the seven transmembrane domain receptor family with a large amino-terminal extracellular extension containing a remarkable feature. A putative thrombin cleavage site (LDPR/S) resembling the activation cleavage site in the zymogen protein C (LDPR/I) was noted 41 amino acids carboxyl to the receptor's start methionine. A peptide mimicking the new amino terminus created by cleavage at R41 was a potent agonist for both thrombin receptor activation and platelet activation. "Uncleavable" mutant thrombin receptors failed to respond to thrombin but were responsive to the new amino-terminal peptide. These data reveal a novel signaling mechanism in which thrombin cleaves its receptor's amino-terminal extension to create a new receptor amino terminus that functions as a tethered ligand and activates the receptor.     

Antibodies to the ATP-binding site of the human epidermal growth factor (EGF) receptor as specific inhibitors of EGF-stimulated protein-tyrosine kinase activity

A region of the primary amino acid sequence of the epidermal growth factor receptor (EGF) protein-tyrosine kinase, which is involved in ATP binding, was identified using chemical modification and immunological techniques. EGF receptor was 14C-labelled with the ATP analogue 5'-p-fluorosulphonylbenzoyladenosine and from a tryptic digest a single radiolabelled peptide was isolated. The amino acid sequence was determined to be residues 716-724 and hence lysine residue 721 is located within the ATP-binding site. Antisera were elicited in rabbits to a synthetic peptide identical to residues 716-727 of the EGF receptor and the homologous sequence in v-erb B transforming protein from avian erythroblastosis virus. The affinity-purified antibodies precipitated human ECF receptor from A431 cells and placenta, and the v-erb B protein from erythroblasts. The antibodies inhibited EGF-stimulated receptor protein-tyrosine kinase autophosphorylation and phosphorylation of an exogenous peptide substrate containing tyrosine. The antibodies did not immunoprecipitate the transforming proteins pp60v-src or P120gag-abl or cAMP-dependent protein kinase, proteins which have homologous but not identical sequences surrounding the lysine residue within the ATP-binding site, nor did they react with the platelet-derived growth factor receptor. The antibodies had no effect on the kinase activity of purified v-abl protein in solution. The antibodies may therefore be a specific inhibitor of the tyrosine kinase of the EGF receptor.     

Molecular determinants of substrate specificity for Semliki Forest virus nonstructural protease

The C-terminal cysteine protease domain of Semliki Forest virus nonstructural protein 2 (nsP2) regulates the virus life cycle by sequentially cleaving at three specific sites within the virus-encoded replicase polyprotein P1234. The site between nsP3 and nsP4 (the 3/4 site) is cleaved most efficiently. Analysis of Semliki Forest virus-specific cleavage sites with shuffled N-terminal and C-terminal half-sites showed that the main determinants of cleavage efficiency are located in the region preceding the cleavage site. Random mutagenesis analysis revealed that amino acid residues in positions P4, P3, P2, and P1 of the 3/4 cleavage site cannot tolerate much variation, whereas in the P5 position most residues were permitted. When mutations affecting cleavage efficiency were introduced into the 2/3 and 3/4 cleavage sites, the resulting viruses remained viable but had similar defects in P1234 processing as observed in the in vitro assay. Complete blockage of the 3/4 cleavage was found to be lethal. The amino acid in position P1' had a significant effect on cleavage efficiency, and in this regard the protease markedly preferred a glycine residue over the tyrosine natively present in the 3/4 site. Therefore, the cleavage sites represent a compromise between protease recognition and other requirements of the virus life cycle. The protease recognizes at least residues P4 to P1', and the P4 arginine residue plays an important role in the fast cleavage of the 3/4 site.     

Further definition of the substance P (SP)/neurokinin-1 receptor complex. MET-174 is the site of photoinsertion p-benzoylphenylalanine4 SP

The covalent attachment site of a substance P (SP) analogue containing the photoreactive amino acid p-benzoyl-l-phenylalanine (Bpa) in position 8 of the C-terminal portion of the peptide was identified previously as Met-181 on the neurokinin-1 (NK-1) receptor. In this study, a second photoreactive SP analogue, Bpa(4)-SP, in which the Bpa residue is located in the N-terminal portion of the peptide, was used to define further the peptide-receptor interface. The NK-1 receptor expressed in Chinese hamster ovary cells was specifically and efficiently photolabeled with a radioiodinated derivative of Bpa(4)-SP. Fragmentation analysis of the photolabeled receptor restricted the site of photoincorporation of Bpa(4)-SP to an amino acid within the sequence Thr-173 to Arg-177 located on the N-terminal side of the E2 loop. To identify the specific amino acid in this sequence that serves as the covalent attachment site for Bpa(4)-SP, a small photolabeled receptor fragment was generated by chemical cleavage with cyanogen bromide. Matrix-assisted laser desorption/ionization time of flight mass spectrometric analysis of the purified fragment identified a single protonated molecular ion with a molecular mass of 1801.3 +/- 1.8, indicating that upon irradiation, the bound photoligand covalently attaches to the terminal methyl group of a methionine residue. This result, taken together with the results of the peptide mapping studies, establishes that the site of Bpa(4)-SP covalent attachment to the NK-1 receptor is Met-174.     

EMR4, a novel epidermal growth factor (EGF)-TM7 molecule up-regulated in activated mouse macrophages,binds to a putative cellular ligand on B lymphoma cell line A20

A novel member of the EGF-TM7 family, mEMR4, was identified and characterized. The full-length mouse EMR4 cDNA encodes a predicted 689-amino acid protein containing two epidermal growth factor (EGF)-like modules, a mucin-like spacer domain, and a seven-transmembrane domain with a cytoplasmic tail. Genetic mapping established that mEMR4 is localized in the distal region of mouse chromosome 17 in close proximity to another EGF-TM7 gene, F4/80 (Emr1). Similar to F4/80, mEMR4 is predominantly expressed on resident macrophages. However, a much lower expression level was also detected in thioglycollate-elicited peritoneal neutrophils and bone marrow-derived dendritic cells. The expression of mEMR4 is up-regulated following macrophage activation in Biogel and thioglycollate-elicited peritoneal macrophages. Similarly, mEMR4 is over-expressed in TNF-alpha-treated resident peritoneal macrophages, whereas interleukin-4 and -10 dramatically reduce the expression. mEMR4 was found to undergo proteolytic processing within the extracellular stalk region resulting in two protein subunits associated noncovalently as a heterodimer. The proteolytic cleavage site was identified by N-terminal amino acid sequencing and located at the conserved GPCR (G protein-coupled receptor) proteolytic site in the extracellular region. Using multivalent biotinylated mEMR4-mFc fusion proteins as a probe, a putative cell surface ligand was identified on a B lymphoma cell line, A20, in a cell-binding assay. The mEMR4-ligand interaction is Ca2+-independent and is mediated predominantly by the second EGF-like module. mEMR4 is the first EGF-TM7 receptor known to mediate the cellular interaction between myeloid cells and B cells.     

Shedding of epidermal growth factor receptor is a regulated process that occurs with overexpression in malignant cells

Soluble isoforms of the epidermal growth factor receptor (sEGFR) previously have been identified in the conditioned culture media (CCM) of the vulvar adenocarcinoma cell line, A431 and within exosomes of the keratinocyte cell line HaCaT. Here, we report that the extracellular domain (ECD) of EGFR is shed from the cell surface of human carcinoma cell lines that express 7 × 10⁵ receptors/cell or more. We purified this proteolytic isoform of EGFR (PI-sEGFR) from the CCM of MDA-MB-468 breast cancer cells. The amino acid sequence of PI-sEGFR was determined by reverse-phase HPLC nano-electrospray tandem mass spectrometry of peptides generated by trypsin, chymotrypsin or GluC digestion. The PI-sEGFR protein is identical in amino acid sequence to the EGFR ECD. The release of PI-sEGFR from MDA-MB-468 cells is enhanced by phorbol 12-myristate 13-acetate, heat-inactivated fetal bovine serum, pervanadate, and EGFR ligands (i.e., EGF and TGF-α). In addition, 4-aminophenylmercuric acetate, an activator of metalloproteases, increased PI-sEGFR levels in the CCM of MDA-MB-468 cells. Inhibitors of metalloproteases decreased the constitutive shedding of EGFR while the PMA-induced shedding was inhibited by metalloprotease inhibitors, by the two serine protease inhibitors leupeptin and 3,4-dichloroisocoumarin (DCI), and by the aspartyl inhibitor pepstatin. These results suggest that PI-sEGFR arises by proteolytic cleavage of EGFR via a mechanism that is regulated by both PKC- and phosphorylation-dependent pathways. Our results further suggest that when proteolytic shedding of EGFR does occur, it is correlated with a highly malignant phenotype.     

Assignment of conserved amino acid residues to the ATP site in the protein kinase domain of the receptor for epidermal growth factor

The ATP substrate site in the epidermal growth factor (EGF) receptor was mapped by using a series of 26 ATP derivatives with modifications at the base, ribose or triphosphate moiety. Ki values for these derivatives were determined by competition with [gamma-32P]ATP. The enzyme seems to interact specifically with the beta-phosphate in an ion-pair bond with the N-6 amino group at the adenine in a hydrogen bond. With ribosyl-2-aminopurine triphosphate and GTP, the enzyme most likely recognizes the 2-amino group in a hydrogen bond. This high specificity for ATP and GTP is unique for the ATP site in the EGF receptor among all investigated protein kinases. The available data on the interaction between ATP derivatives and protein kinases were used to assign conserved amino acid residues found in diverse protein kinases to the ATP site in this type of enzyme.     

Elucidation of the role of peptide linker in calcium-sensing receptor activation process

Family 3 G-protein-coupled receptors (GPCRs), which includes metabotropic glutamate receptors (mGluRs), sweet and "umami" taste receptors (T1Rs), and the extracellular calcium-sensing receptor (CaR), represent a distinct group among the superfamily of GPCRs characterized by large amino-terminal extracellular ligand-binding domains (ECD) with homology to bacterial periplasmic amino acid-binding proteins that are responsible for signal detection and receptor activation through as yet unresolved mechanism(s) via the seven-transmembrane helical domain (7TMD) common to all GPCRs. To address the mechanism(s) by which ligand-induced conformational changes are conveyed from the ECD to the 7TMD for G-protein activation, we altered the length and composition of a 14-amino acid linker segment common to all family 3 GPCRs except GABA(B) receptor, in the CaR by insertion, deletion, and site-directed mutagenesis of specific highly conserved residues. Small alterations in the length and composition of the linker impaired cell surface expression and abrogated signaling of the chimeric receptors. The exchange of nine amino acids within the linker of CaR with the homologous sequence of mGluR1, however, preserved receptor function. Ala substitution for the four highly conserved residues within this amino acid sequence identified a Leu at position 606 of the CaR critical for cell surface expression and signaling. Substitution of Leu(606) for Ala resulted in impaired cell surface expression. However, Ile and Val substitutions displayed strong activating phenotypes. Disruption of the linker by insertion of nine amino acids of a random-coiled structure uncoupled the ECD from regulating the 7TMD. These data are consistent with a model of receptor activation in which the peptide linker, and particularly Leu(606), provides a critical interaction for the CaR signal transmission, a finding likely to be relevant for all family 3 GPCRs containing this conserved motif.     

Phosphorylation of tyrosine 992, 1068, and 1086 is required for conformational change of the human epidermal growth factor receptor c-terminal tail

We reported previously that a conformation-specific antibody, Ab P2, to a 16-amino acid peptide (Glu-Gly-Tyr-Lys-Lys-Lys-Tyr-Gln-Gln-Val-Asp-Glu-Glu-Phe-Leu-Arg) of the cytoplasmic domain of the beta-type platelet-derived growth factor receptor also recognizes the epidermal growth factor (EGF) receptor. Although the antibody is not directed to phosphotyrosine, it recognizes in immunoprecipitation the activated and hence phosphorylated form of both receptors. In P2 peptide, there are two tripeptide sequences, Asp-Glu-Glu and Tyr-Gln-Gln, that are also present in the EGF receptor. Our present studies using either EGF receptor C-terminal deletion mutants or point mutations (Tyr-->Phe) and our previous studies on antibody inhibition by P2-derived peptides suggest that Gln-Gln in combination with Asp-Glu-Glu forms a high-affinity complex with Ab P2 and that such complex formation is dependent on tyrosine phosphorylation. Of the five phosphate acceptor sites in the EGF receptor, clustered in the extreme C-terminal tail, phosphorylation of three tyrosine residues (992, 1068, and 1086) located between Asp-Glu-Glu and Gln-Gln is necessary for Ab P2 binding. In contrast, the acceptor sites Tyr 1173 and 1148 play no role in the conformation change. Asp-Glu-Glu and Gln-Gln are located 169 amino acids apart, and it is highly likely that the interactions among three negatively charged phosphotyrosine residues in the receptor C terminus may result in the bending of the peptide chain in such a way that these two peptides come close to each other to form an antibody-binding site. Such a possibility is also supported by our finding that receptor dephosphorylation results in complete loss of Ab P2-binding activity. In conclusion, we have identified a domain within the cytoplasmic part of the EGF receptor whose conformation is altered by receptor phosphorylation; furthermore, we have identified the tyrosine residues that positively regulate this conformation.     

Interaction of the extracellular domain of the epidermal growth factor receptor with gangliosides.

Ganglioside GM3 inhibits epidermal growth factor (EGF)-dependent cell proliferation in a variety of cell lines. Both in vitro and in vivo, this glycosphingolipid inhibits the kinase activity of the EGF receptor (EGFR). Furthermore, membrane preparations containing EGFR can bind to GM3-coated surfaces. These data suggest that GM3 may interact directly with the EGFR. In this study, the interaction of gangliosides with the extracellular domain (ECD) of the EGFR was investigated. The purified human recombinant ECD from insect cells bound directly to ganglioside GM3. The ganglioside interaction site appears to be distinct from the EGF-binding site. In agreement with previous reports on the effects of specific gangliosides on EGFR kinase activity, the ECD preferentially interacted with GM3. The order of relative binding of other gangliosides investigated was as follows: GM3 GM2, GD3, GM4 > GM1, GD1a, GD1b, GT1b, GD2, GQ1b > lactosylceramide. These data suggest that NeuAc-lactose is essential for binding and that any sugar substitution reduces binding. In agreement with the specificity of soluble ECD binding to gangliosides, GM3 specifically inhibited EGFR autophosphorylation. Identification of a ganglioside interaction site on the ECD of the EGFR is consistent with the hypothesis that endogenous GM3 may function as a direct modulator of EGFR activity.     

In silico analysis of ORF1ab in coronavirus HKU1 genome reveals a unique putative cleavage site of coronavirus HKU1 3C-like protease

Recently we have described the discovery and complete genome sequence of a novel coronavirus associated with pneumonia, coronavirus HKU1 (CoV-HKU1). In this study, a detailed in silico analysis of the ORF1ab, encoding the 7,182-amino acid replicase polyprotein in the CoV-HKU1 genome showed that the replicase polyprotein of CoV-HKU1 is cleaved by its papain-like proteases and 3C-like protease (3CL(pro)) into 16 polypeptides homologous to the corresponding polypeptides in other coronaviruses. Surprisingly, analysis of the putative cleavage sites of the 3CL(pro) revealed a unique putative cleavage site. In all known coronaviruses, the P1 positions at the cleavage sites of the 3CL(pro) are occupied by glutamine. This is also observed in CoV-HKU1, except for one site at the junction between nsp10 (helicase) and nsp11 (member of exonuclease family), where the P1 position is occupied by histidine. This amino acid substitution is due to a single nucleotide mutation in the CoV-HKU1 genome, CAG/A to CAT. This probably represents a novel cleavage site because the same mutation was consistently observed in CoV-HKU1 sequences from multiple specimens of different patients; the P2 and P1'-P12' positions of this cleavage site are consistent between CoV-HKU1 and other coronaviruses; and as the helicase is one of the most conserved proteins in coronaviruses, cleavage between nsp10 and nsp11 should be an essential step for the generation of the mature functional helicase. Experiments, including purification and C-terminal amino acid sequencing of the CoV-HKU1 helicase and trans-cleavage assays of the CoV-HKU1 3CL(pro) will confirm the presence of this novel cleavage site.     

Primary structure of bovine endothelin ETB receptor and identification of signal peptidase and metal proteinase cleavage sites.

A cDNA clone corresponding to the entire coding region of the bovine ETB endothelin receptor mRNA was isolated from a lung cDNA library and sequenced. The cDNA encodes 441 amino acids: 26 constituting an NH2-terminal signal peptide and 415 constituting the mature receptor. The signal peptidase cleavage site was determined by direct amino acid sequencing of purified receptor. A comparison of the predicted amino acid sequence with the available bovine ETA and rat ETB endothelin receptor sequences revealed 63 and 85% homology, respectively. Endothelin receptors of various species are known to be very sensitive to a certain metal proteinase(s) and have been shown to be converted to a lower Mr form in the absence of EDTA. The metal proteinase cleavage site was also determined by direct protein sequencing of the proteolysis product. The amino acid sequence (Ala-Gly-X-Pro-Pro-Arg) surrounding the cleavage site (between Ala-79 and Gly-80) is conserved among the ETB endothelin receptors, explaining the above mentioned proteolytic conversion from the higher to lower Mr forms observed in various species.     

Analysis of N-terminal processing of hepatitis C virus nonstructural protein 2.

We determined the partial amino (N)-terminal amino acid sequence of hepatitis C virus p21 (nonstructural protein 2 [NS2]). Cleavage at the p21 (NS2) N terminus depended on the presence of microsomal membranes. The amino-terminal position of p21 (NS2) was assigned to amino acid 810 of the hepatitis C virus strain IIJ precursor polyprotein. Mutation of the alanine residue at position P1 of the putative cleavage site inhibited membrane-dependent processing. This alteration in processing together with the fact that hydrophobic amino acid residues are clustered upstream of the putative cleavage site suggested the involvement of a signal peptidase(s) in the cleavage. Furthermore, mutation analysis of this possible cleavage site revealed the presence of another microsome membrane-dependent cleavage site upstream of the N terminus of p21 (NS2).     

All autophosphorylation sites of epidermal growth factor (EGF) receptor and HER2/neu are located in their carboxyl-terminal tails. Identification of a novel site in EGF receptor

Activation of the epidermal growth factor (EGF) receptor kinase leads to autophosphorylation and to the phosphorylation of various cellular substrates. The three known autophosphorylation sites of EGF receptor are located at the carboxyl-terminal tail where they probably act to compete with and thus modulate substrate phosphorylation. Mutational analysis and microsequencing techniques have been used to localize and identify new autophosphorylation site(s) of the EGF receptor. We have compared the phosphopeptide maps of human EGF receptor, and two deletion mutants lacking 63 and 126 amino acids from the carboxyl-terminal tail with the phosphopeptide maps of HER/neu and a chimeric EGF receptor containing the carboxyl-terminal tail of HER2/neu. HER2/neu is highly homologous to the EGF receptor, and it probably functions as a growth factor receptor for as yet unidentified growth factor. On the basis of this analysis, we have concluded that all autophosphorylation sites of EGF receptor and HER2/neu are located in their carboxyl-terminal tails. Utilizing the EGF receptors with carboxyl-terminal deletions, we were also able to identify tyr1086 as an additional autophosphorylation site of EGF receptor. Direct microsequencing of a phosphorylated tryptic peptide from the human EGF receptor confirmed this assignment.     

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.