Synthetic peptide approach to the analysis of kinase activities of avian EGF receptor and v-erbB protein.

Analysis of the structure and function of a protein such as the epidermal growth factor receptor is facilitated by the use of antibodies directed against discrete portions of the protein. Here, we describe the characterization and use of antibodies directed against synthetic peptides corresponding to specific portions of the epidermal growth factor receptor and/or v-erbB protein. In particular, one useful antiserum has allowed us to compare the protein kinase activities of the epidermal growth factor receptor and the v-erbB proteins and to conclude that the v-erbB protein is a protein-tyrosine specific kinase as is its homologue the avian epidermal growth factor receptor.

The carbohydrate specificities of the monoclonal antibodies 29.1, 455 and 3C1B12 to the epidermal growth factor receptor of A431 cells.

Sixteen hybridoma-derived antibodies to the epidermal growth factor receptor of A431 cells were studied with respect to their reactions with blood group-related carbohydrate structures. Twelve of these were assessed as recognizing carbohydrate determinants on the basis of their immunostaining of reference blood group substances on nitrocellulose paper. Three of these antibodies were further investigated by inhibition of binding assays with glycoproteins and structurally defined oligosaccharides or by haemagglutination of erythrocytes before and after treatment with endo-beta-galactosidase. Two of the antibodies, 29.1 and 455, were shown to have blood group A-related specificities which differed from one another and from those of monoclonal anti-A antibodies described previously. The third antibody, 3C1B12, which was shown to recognize a determinant based on alpha 1----3 fucosylated Type 2 chains on linear and branched backbone sequences, also differs from previously described monoclonal antibodies of 3-fucosyl-N-acetyllactosamine type, such as anti-SSEA-1 (anti-mouse embryo) and several antibodies to human myeloid cells. While these antibodies are invaluable in providing structural information on the carbohydrate chains of the receptor glycoprotein and should help to elucidate their functions, their use as 'anti-receptor' reagents in cell biology will be influenced by the knowledge that the determinants they recognize are shared by other glycoproteins and glycolipids of diverse cell types.

Desensitization of human endothelin A receptor.

The response of G-protein-coupled receptors is modulated by homologous desensitization. Because endothelin A receptor (ETA) plays a part in vasoconstriction, the extent of desensitization and resensitization of endothelin responsiveness was studied. A cDNA clone encoding human ETA receptor was isolated based on similarities to bovine endothelin A receptor. Xenopus laevis oocytes injected with cloned mRNA transcripts were used as a model system for analyzing desensitization. Human neurokinin A (NKA) receptor was used for comparison with ETA receptor in desensitization experiments. Xenopus laevis oocytes injected with the two receptor mRNAs show homologous desensitization but variable rates of recovery. Human NKA receptors desensitize within 5 min and resensitize after 20-30 min. Human ETA receptors also desensitize within 5 min but have a prolonged resensitization period lasting 1.5-2 h. Such a prolonged recovery period is unique to this class of receptors and may serve to regulate some of the deleterious effects mediated by the ETA receptor. The mechanism of differential desensitization is being investigated using genetically engineered mutants of human ETA receptor.

Prolonged desensitization of the human endothelin A receptor in Xenopus oocytes. Comparative studies with the human neurokinin A receptor.

Human endothelin (ET) A receptor (hETAR) is a G-protein-mediated receptor that binds ET1 with high affinity and ET2 and ET3 with lower affinities. ET1 is the most potent endogenous vasoconstrictor known at this time. When expressed in Xenopus laevis oocytes, hETAR is rapidly desensitized after stimulation with ET1. This desensitization lasts 90-110 min. Human neurokinin A (hNKAR) and human serotonin type 2 receptors were also expressed in the Xenopus system for comparison to hETAR. hNKAR desensitizes for 25-35 min, while the serotonin receptor does not appear to desensitize. To examine the role of the cytoplasmic tail of hETAR in desensitization, deletion mutations were constructed which remove 11, 36, and 51 amino acids from the cytoplasmic tail. The mutations removing 11 and 36 residues were functional, but the mutation removing 51 amino acids was not functional. The two functional mutations have a resensitization time similar to that of hETAR. In summary, the prolonged desensitization time of hETAR is unique for G-protein-mediated receptors and may attenuate the adverse physiological effects of the endothelin family. In addition, the cytoplasmic tail of hETAR does not appear to play a role in desensitization or resensitization of this receptor.

Passive serum antibody causes temporary recovery from influenza virus infection of the nose, trachea and lung of nude mice.

BALB/c normal and nude mice were infected with a non-lethal mouse-passaged A/PC/1/73 (H3N2) influenza virus in order to assess the role of T cells on the course of disease of the nose, trachea and lung. The tracheal epithelium of both mouse strains was desquamated by 3 days after infection. Although normal regeneration began, nude mice never completed that regeneration whereas normal mice had fully regenerated tracheas by Day 14. This failure to complete the recovery was also evident from the continued virus shedding by the nude mouse. In order to assess the role of serum antibody on recovery from infection, ferret, goat or mouse antibody to H3N2 influenza virus was passively administered to nude mice after infection. It resulted in a transient decrease in virus shedding from the nose, trachea and lung, and complete but temporary regeneration of the tracheal epithelium. However, later in the course of the infection, when serum antibody levels were no longer detectable, the tracheal epithelium of these animals redesquamated and large amounts of virus were again shed from nose, trachea and lungs. We conclude that: (i) desquamation of the ciliated epithelium of the trachea is not T-cell dependent; and (ii) serum antibody can contribute to temporary recovery from infection, but by itself is insufficient for permanent recovery of the nose, trachea or lung.

Evidence that autophosphorylation of solubilized receptors for epidermal growth factor is mediated by intermolecular cross-phosphorylation.

Structurally distinguishable mutants of human epidermal growth factor receptor (EGFR) were used to investigate the mechanism of EGFR autophosphorylation. Mutant receptors generated by site-directed mutagenesis were expressed in transfected NIH 3T3 cells lacking endogenous receptors. After coincubation of cell lysates in the presence or absence of EGF, receptor immunoprecipitates were incubated with [gamma-32P]ATP. A kinase-negative mutant EGFR (K721A), in which Lys-721 in the ATp binding site was replaced by an alanine residue, was shown to be phosphorylated in an EGF-dependent manner by an enzymatically active EGFR deletion mutant lacking two autophosphorylation sites. A mutant EGFR lacking the EGF-binding domain as well as the phosphorylation sites also phosphorylated the kinase-negative mutant. In both cases the kinase-negative mutant K721A was phosphorylated on sites virtually identical to the sites that are autophosphorylated by wild-type recombinant or native human EGFRs. With four different site-specific anti-EGFR antibodies, it was shown that deletion mutants devoid of epitopes recognized by the antibodies were coimmunoprecipitated together with wild-type or mutant receptors recognized by the antibodies. This indicates that EGFR oligomers were preserved during immunoprecipitation. On the basis of these results, we propose that autophosphorylation of solubilized EGFR is mediated by intermolecular cross-phosphorylation, probably facilitated by receptor oligomerization.

Protection and recovery in influenza virus-infected mice immunosuppressed with anti-IgM.

BALB/c mice, immunosuppressed from birth with goat anti-mouse IgM, were able to recover from influenza virus infection in the absence of detectable serum and nasal antibody. Recovery was delayed a few days when compared with control animals. Antibody-deficient mice, that had recovered from an initial influenza virus infection, i.e., convalescent mice, were subsequently rechallenged with homologous influenza virus in order to study the importance of nasal and serum antibody in prevention of infection. Convalescent mice were susceptible to reinfection when nasal and serum antibody were not detectable. The mice were resistant to reinfection when serum and/or nasal antibody was detectable by radioimmunoassay. Normal mice that were passively immunized with high titer mouse anti-influenza virus serum were susceptible to challenge with homologous influenza virus. The serum antibody levels in these mice were higher than most of those found in the immune convalescent mice suppressed with anti-IgM, thereby suggesting that the serum antibody, found in convalescent suppressed mice, is not protective. We conclude that 1) mice can recover from influenza virus infection in the absence of detectable levels of nasal and serum antibody, thus indirectly confirming the role of cell-mediated immunity in recovery; 2) serum IgM, IgG2A, IgG2B, IgG3, and probably IgG1 antibody levels are not responsible for protection against influenza virus infection of the upper respiratory tract; and 3) nasal IgA antibody correlates best with protection against reinfection of the upper respiratory tract, but some other locally protective agent cannot be excluded.

Casein kinase II enhances the DNA binding activity of serum response factor.

Serum response factor (SRF) is a mammalian transcription factor that binds to the serum response element in the enhancer of the c-fos proto-oncogene and thus may mediate serum-induction of c-fos transcription. We report here that the DNA binding activity of recombinant SRF made in Escherichia coli can be greatly enhanced by incubation of the protein with HeLa cell nuclear extract. The enhancing activity is ATP or GTP dependent and cofractionates with a protein kinase that phosphorylates SRF on a specific tryptic peptide. Coincubation with phosphatase blocks the enhancing activity, further suggesting that the enhanced binding activity is due to phosphorylation. The specific tryptic phosphopeptide phosphorylated in vitro is also phosphorylated in vivo, demonstrating that this phosphorylation is physiologically important. We have localized the phosphorylation site by a small deletion mutant. Finally, we show that the kinase activity is provided by casein kinase II (CKII) or a close variant. The potential role of CKII as either a regulatory or constitutive modifier of SRF in vivo will be discussed.

Maternal-infant transfer of influenza-specific immunity not detectable by haemagglutination inhibition.

Evidence was sought for the transfer from mother to infant mouse of influenza-specific immunity other than that associated with haemagglutination inhibition (HI) specific antibody. Data were analysed for infant mice born to three groups of mothers: influenza immunized mothers with high levels of HI antibody. influenza immunized mothers in whom influenza-specific HI antibody was suppressed to undetectable levels by passive administration of antibody prior to influenza immunization, and non-immunized control mothers. At 4 weeks of age, infants were given either a lethal or a non-lethal influenza challenge. After a lethal influenza challenge, infants of immunized mothers with HI antibody showed no mortality. Infants of immunized mothers in whom HI antibody was suppressed showed a mortality of 8 to 33%, which was significantly lower than the 71% mortality found in infants of non-immunized control mothers (P less than 0.001). The lower mortality in infants of immunized mothers without HI antibody appeared to be associated with breast feeding, but could not be attributed to the transfer from mother to infant of a local immune response, an influenza specific cytotoxic response, or a secondary antibody response after non-lethal influenza infection. When sera were additionally tested for anti-influenza IgG by ELISA, this lower mortality was found to correlate with anti-influenza serum IgG measurable by ELISA but not detectable by HI. Protective immunity in infants undetectable by HI could be attributable to very low levels of antibody detected by more sensitive ELISA.

Heterologous desensitization of the human endothelin A and neurokinin A receptors in Xenopus laevis oocytes.

Endothelin 1 (ET1) desensitizes endothelin A receptor for 90-110 min while neurokinin A (NKA) desensitizes neurokinin A receptor for 25-35 min in Xenopus laevis oocytes. In the present study, endothelin A receptor and neurokinin A receptor were coexpressed in Xenopus laevis oocytes in an effort to characterize heterologous desensitization of the receptors that activate phospholipase C-beta. ET1 desensitizes both the endothelin A receptor and the neurokinin A receptor for 90-110 min, whereas stimulation with NKA desensitizes the same two receptors for only 25-35 min. Homologous and heterologous desensitization experiments were also carried out with endothelin 3 (ET3), a ligand that exhibits lower affinity to the endothelin A receptor and a quicker dissociation rate than ET1. ET3 was unable to desensitize endothelin A receptor and the neurokinin A receptor; this is in contrast to ET1 that desensitizes both receptors. These results suggests that the receptors that undergo homologous desensitization are able to heterologously desensitize other receptors that activate PLC-beta. Furthermore, the agonist-specific dissociation constant dictates the extent of desensitization and time of recovery of the receptor-mediated response.

Identification of the bombesin receptor on murine and human cells by cross-linking experiments.

The bombesin receptor present on the surface of murine and human cells was identified using 125I-labeled gastrin-releasing peptide as a probe, the cross-linking agent disuccinimidyl suberate, and sodium dodecyl sulfate gels. A clone of NIH-3T3 cells which possesses approximately 80,000 bombesin receptors/cell with a single binding constant of approximately 1.9 X 10(-9) M was used in these studies. In addition, we used Swiss 3T3 cells and a human glioma cell line which possesses approximately 100,000 and approximately 55,000 bombesin receptors/cell, respectively. Under conditions found optimal for binding, it is demonstrated that 125I-labeled gastrin-releasing peptide can be cross-linked specifically to a glycoprotein of apparent molecular mass of 65,000 daltons on the surface of the NIH-3T3 cells. Similar results were obtained when the cross-linked product was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing or non-reducing conditions. Moreover, the cross-linking reaction is specific and saturable and the 65,000-dalton polypeptide is not observed when the cross-linking experiments were performed with a NIH-3T3 cell line which is devoid of bombesin receptors. Interestingly, glycoproteins with apparent molecular weights of 75,000 were labeled specifically by 125I-labeled gastrin-releasing peptide when similar experiments were performed with Swiss 3T3 cells and with human glioma cell line GM-340. These different molecular weights may indicate differential glycosylation as treatment with the enzyme N-glycanase reduced the apparent molecular weight of the cross-linked polypeptide to 45,000. On the basis of these results it is concluded that the cross-linked polypeptides represent the bombesin receptor or the ligand-binding subunit of a putative larger bombesin receptor expressed on the surface of these cells.

Intranasal immunization with proteoliposomes protects against influenza.

Worldwide, influenza virus remains a serious disease which has successfully eluded numerous attempts to design a consistently effective vaccine. In part, these attempts have been thwarted because of a lack of basic understanding of the mechanisms which mediate protection and recovery from influenza infection. A better understanding of the roles of secretory antibody, serum antibody and cell mediated immunity vis-à-vis protection and recovery from influenza infection has allowed us more rationally to approach the design and administration of a vaccine for influenza. We have constructed a vaccine composed of glycoproteins from the envelopes of either influenza of Sendai virus embedded in a lipid bilayer (immunosomes) mimicking the presentation of the virus to the cells during natural infection. Intranasal immunization with these immunosomes induces an adequate systemic Ir compared with intramuscular immunization and a superior local IgA response. These animals were specifically protected from virus challenge.

Antibodies against a synthetic peptide as a probe for the kinase activity of the avian EGF receptor and v-erbB protein.

The transforming protein v-erbB of avian erythroblastosis virus (AEV) displays extensive sequence homology with the presumptive protein-tyrosine kinase domain of the human EGF receptor and with the src protein-tyrosine kinase family of oncogenes. However, no kinase activity has previously been demonstrated for the v-erbB protein. Here antibodies generated against a synthetic peptide from the C terminus of human EGF receptor are shown to immunoprecipitate the EGF receptor from human and avian cells, as well as the v-erbB proteins from AEV-transformed cells that become phosphorylated on tyrosine residues upon the addition of gamma-32P-ATP. The immunoprecipitates are also able to phosphorylate exogenous tyrosine-containing substrates. Hence, it is likely that both avian EGF receptor and v-erbB proteins are protein tyrosine-specific protein kinases. Since the kinase activity of v-erbB protein cannot be regulated by EGF, it is proposed that the tyrosine protein kinase function of v-erbB may be constitutively activated.

Expression of ki-ras oncogene in tumor cell variants exhibiting different metastatic capabilities.

The expression of oncogenes in well-characterized B16 melanoma and UV-2237 fibrosarcoma cell variants that exhibit distinct metastatic properties was explored. The search for the expression of 11 different oncogenes revealed that the major oncogene in those two tumor systems is the Kirsten-ras (ki-ras). The results indicate that the amounts of specific ki-ras mRNA and the p21 protein are similar in both low- and high-metastatic counterparts. These results suggest that in these systems there is no apparent direct correlation between the amount and expression of the major cellular oncogene so far identified and the metastatic potential of these tumor cells.

Regions of the human neurokinin A receptor involved in the generation of second messengers and in receptor desensitization.

Deletion analysis was used to study sites of human Neurokinin A receptor (HNKAR) necessary for signal transduction in CHO cells. Deletion of 62 and 81 amino acids from the c-terminus of HNKAR forms mutant receptors HNKAR delta 62 and HNKAR delta 81, which bind neurokinin A with high affinity but are functionally different. Wild type HNKAR and HNKAR delta 62 are functionally active whereas HNKAR delta 81 is functionally inactive. In addition, HNKAR and HNKAR delta 62 were both desensitized to the neurokinin A signal within 5 minutes. The data indicates: 1) an intact cytoplasmic tail of the HNKAR is not critical for signal transduction, but the n-terminal amino acids of the cytoplasmic tail are necessary for signaling and 2) the c-terminal 62 amino acids are not necessary for desensitization.

Lung carcinoid cell lines have bombesin-like peptides and EGF receptors.

The biochemical properties of lung cancer cell lines were investigated. Bombesin-like peptides were present in three small cell lung cancer (SCLC) cell lines examined and three of four lung carcinoids but not in five non-small cell lung cancer (NSCLC) cell lines. Therefore SCLC and some lung carcinoids, but not NSCLC, are enriched in neuroendocrine properties. In contrast, 125I-EGF bound with high affinity to all five NSCLC cell lines and three of four lung carcinoids but not to the three SCLC cell lines examined. For lung carcinoid cell line NCI-H727, 125I-EGF bound with high affinity (Kd = 6 nM) to a single class of sites (Bmax = 110,000/cell). The 125I-EGF bound was rapidly internalized at 37 degrees C but not 4 degrees C. Using Western blot techniques and antiphosphotyrosine antibodies, EGF induced phosphorylation of a major 170 Kd protein. Using immunoprecipitation techniques and anti-EGF receptor antibodies a major 170 Kd protein was labeled. These data indicate that biologically active EGF receptors are present on NSCLC and lung carcinoid cell lines.

Cloning and expression of the human substance K receptor and analysis of its role in mitogenesis.

The primary structure of the human substance K receptor was established from the sequences of complementary DNA clones isolated from a human jejunal complementary DNA library. It consists of 398 amino acids, including seven putative transmembrane regions. The gene for the human substance K receptor was localized to chromosome region 10p13-10q23, a region with frequent chromosomal abnormalities. The human substance K receptor was expressed in transfected NIH-3T3 cells lacking endogenous substance K receptors, and Scatchard analysis of 125I-labeled substance K binding indicates approximately 100,000 receptors/cell with a single dissociation constant of 12 nM. Covalent cross-linking experiments utilizing 125I-substance K and three different chemical cross-linking reagents (disuccinimidyl suberate, disuccinimidyl tartrate, or 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide-HCl) demonstrate an apparent molecular weight of 45,000, consistent with little or no N-linked glycosylation. The binding of substance K to its receptor on transfected cells led to a rapid increase in the production of total inositol phosphates and the release of Ca2+ from internal stores. Growth of the cells transfected with the human substance K receptor is stimulated by the addition of substance K to the medium to a level similar to 10% serum. Therefore, the human substance K receptor can function as a growth factor receptor when expressed in mouse 3T3 cells.