The epidermal growth factor receptor (EGFR) is a multisited and multifunctional transmembrane glycoprotein with intrinsic tyrosine kinase activity. Upon ligand binding, the monomeric receptor undergoes dimerization resulting in kinase activation. The consequences of kinase stimulation are the phosphorylation of its own tyrosine residues (autophosphorylation) followed by association with and activation of signal transducers. Deregulation of signaling resulting from aberrant expression of the EGFR has been implicated in a number of neoplasms including breast, brain, and skin tumors. A mutant epidermal growth factor (EGF) receptor missing 267 amino acids from the exoplasmic domain is common in human glioblastomas. The truncated receptor (EGFRvIII/⌬EGFR) lacks EGF binding activity; however, the kinase is constitutively active, and cells expressing the receptor are tumorigenic. Our studies revealed that the high kinase activity of the ⌬EGFR is due to self-dimerization, and contrary to earlier reports, the kinase activity per molecule of the dimeric ⌬EGFR is comparable to that of the EGF-stimulated wild-type receptor. Furthermore, the phosphorylation patterns of both receptors are similar as determined by interaction with a conformation-specific antibody and by phosphopeptide analysis. This eliminates the possibility that the defective down-regulation of the ⌬EGFR is due to its altered phosphorylation pattern as has been suggested previously. Interestingly, the receptor-receptor self-association is highly dependent on a conformation induced by N-linked glycosylation. We have identified four potential sites that might participate in self-dimerization; these sites are located in a domain that plays an important role in EGFR functioning. The human epidermal growth factor receptor (EGFR)1 is a transmembrane glycoprotein with a cysteine-rich extracellular region and an intracellular domain containing uninterrupted kinase site and multiple autophosphorylation sites clustered at the C-terminal tail (see Ref (Fig. 1). On the basis of internal sequence identity, the extracellular portion of the EGFR has been subdivided into four domains. Domains I (amino acids 1-165) and III (aa 310 -481) have 37% sequence identity, whereas domains II (aa 166 -309) and IV (aa 482-621) are rich in cysteines (3) (see Fig. 1). These cysteines are linked by intra-chain disulfide bonding (4). Domain III has been shown to bind directly with EGF, and then two molecules of the monomeric receptor-ligand complex interact to form a dimeric complex. Domain I is believed to be involved in the second interaction (3, 4). The receptor dimerization results in kinase activation. The earliest consequence of kinase activation is the phosphorylation of its own tyrosine residues (autophosphorylation), and this is followed by its association with and activation/phosphorylation of signal transducers leading to mitogenesis. In addition, we have demonstrated a phosphorylation-induced conformational alteration of the EGFR (5). Such conformational change agrees well with...

Two types of PDGF receptors have been cloned and sequenced. Both receptors are transmembrane glycoproteins with a ligand-stimulatable tyrosine kinase site. We have shown earlier that ligand-induced activation of the beta-type PDGF receptor is due to the conversion of the monomeric form of the receptor to the dimeric form [Bishayee et al. (1989) J. Biol. Chem. 264, 11699-11705]. In the present studies, we have established the ligand-binding specificity of two receptor types and extended it further to investigate the ligand-induced association state of the alpha-receptor and the role of alpha-receptor in the activation of beta-receptor. These studies were conducted with cells that express one or the other type of PDGF receptor as well as with cells that express both types of receptors. Moreover, ligand-binding characteristics of the receptor were confirmed by immunoprecipitation of the receptor-125I-PDGF covalent complex with type-specific anti-PDGF receptor antibodies. These studies revealed that all three isoforms of PDGF bind to alpha-receptor, and such binding leads to dimerization as well as activation of the receptor. In contrast, beta-receptor can be activated only by PDGF BB and not by PDGF AB or PDGF AA. However, by using antipeptide antibodies that are specific for alpha- or beta-type PDGF receptor, we demonstrated that in the presence of alpha-receptor, beta-receptor kinase can be activated by PDGF AB. We present here direct evidence that strongly suggests that such PDGF AB induced activation of beta-receptor is due to the formation of a noncovalently linked alpha-beta receptor heterodimer.

No abstract

Abstract— Lipid peroxide formation as measured by the thiobarbituric acid reaction was demonstrated in subcellular fractions of rat brain. The ascorbic acid induced nonenzymic lipid peroxidation was distributed in all the subcellular fractions with a maximum in microsomes. The NADPH dependent enzymic lipid peroxidation occurred mainly in microsomes and to a smaller extent in synaptosomes; NADH could replace NADPH for the enzymic lipid peroxidation under the assay conditions employed. Fe2+ but not Fe3+ stimulated the NADPH or NADH dependent lipid peroxide formation. The optimum conditions with respect to pH, ascorbic acid or NADPH concentration, time of incubation and protein concentration were studied. Heating the microsomes at 100oCdid not influence the ascorbate‐induced lipid peroxidation but completely abolished the NADPH linked peroxidation. Several heavy metal ions, surface active agents and EDTA were inhibitory to lipid peroxidation. The effect of thiol agents indicated that ‐SH groups were involved in the enzymic lipid peroxidation. Studies on subcellular fractions of developing rat brain showed an increasing trend in lipid peroxidation with the advancing age of the animal. No significant difference in lipid peroxidation was observed between brains from normal rats and those from rats affected by experimental allergic encephalomyelitis.

Two site-specific anti-peptide antibodies (AbPj and AbP2) were raised against the platelet-derived growth factor (PDGF) receptor. These two sites correspond to amino acid residues 977 through 988 (peptide 1) and 932 through 947 (peptide 2) of the murine PDGF receptor. Both antibodies recognized human and murine PDGF receptors in immunoprecipitation and immunoblotting analyses. None of the antibodies was directed to phosphotyrosine. One of the antibodies (AbP2) showed unusual antigen recognition specificity. This antibody specifically recognized the tyrosine-phosphorylated PDGF receptor and not the unphosphorylated native receptor, suggesting that recognition by this antibody requires a specific conformation that is induced by PDGF-stimulated autophosphorylation.The human platelet-derived growth factor (PDGF) consists of a family of glycoproteins (Mr, 28,000 to 35,000) composed of two homologous but nonidentical polypeptides (A and B) of Mr 14,000 to 18,000 linked by disulfide bonds (1,7,11,17); the B chain of PDGF is a product of c-sis proto-oncogene (8, 23). PDGF initiates cellular replication and transformation by binding to a specific high-affinity cell surface receptor, a transmembrane glycoprotein of 180 kilodaltons (kDa). The PDGF receptor has recently been purified from various sources (3,6,19). The purified receptor displays PDGF-stimulated tyrosine kinase activity, suggesting that tyrosine kinase is an integral part of the receptor molecule (3,19). This conclusion has recently been confirmed by cloning of cDNA for the murine PDGF receptor; the deduced sequence has considerable homology with known tyrosine kinases (24).Unlike the well-studied receptors for epidermal growth factor and insulin, the PDGF receptor is relatively uncharacterized. Studies on biosynthesis and processing of this receptor have recently been aided by the availability of anti-PDGF-receptor antibodies (4, 10, 13). In this communication, we report the generation and characterization of a conformation-specific novel anti-PDGF-receptor antibody. It recognizes a peptide epitope (residues 932 through 947 of murine PDGF receptor) that is normally cryptic but becomes accessible upon receptor phosphorylation. This antibody, unlike other reported reagents, may be useful in future structure-function analyses of the receptor. ed by the chloramine T procedure, and '25I-PDGF was purified as described previously (3). Platelet-poor plasma, which lacks PDGF, was prepared as described previously (21).[_y-32P]ATP. Labeled ATP was prepared with 32pi (ICN) and a -y-prep A kit (Promega Biotec, Madison, Wis.) according to the manufacturers' directions. The [_y-32P]ATP was diluted with unlabeled ATP to a final specific activity of 40 to 60 Ci/mmol. Peptide synthesis. The peptides Glu-Gly-Tyr-Lys-LysLys-Tyr-Gln-Gln-Val-Asp-Glu-Glu-Phe-Leu-Arg (P2) and Tyr-Thr-Ala-Val-Gln-Pro-Asn-Glu-Ser-Asp-Asn-Asp (Pl), corresponding to the residues 932 through 947 and 977 through 988, respectively, of the murine PDGF-receptor sequence (24), were synthesized with an ...