Richard Kris

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.

Kinetic parameters of the protein tyrosine kinase activity of EGF-receptor mutants with individually altered autophosphorylation sites.

Epidermal growth factor (EGF)‐receptor mutants in which individual autophosphorylation sites (Tyr1068, Tyr1148 or Tyr1173) have been replaced by phenylalanine residues were expressed in NIH‐3T3 cells lacking endogenous EGF‐receptors. Kinetic parameters of the kinase of wild‐type and mutant receptors were compared. Both wild‐type and mutant EGF‐receptors had a Km(ATP) 1‐3 microM for the autophosphorylation reaction, and a Km(ATP) of 3‐7 microM for the phosphorylation of a peptide substrate. These are similar to the Km(ATP) values reported for EGF‐receptor of A431 cells. A synthetic peptide representing the major in vitro autophosphorylation site Tyr1173 of the EGF‐receptor (KGSTAENAEYLRV) was phosphorylated by wild‐type receptor with a Km of 110‐130 microM, and the peptide inhibited autophosphorylation with a Ki of 150 microM. Mutant EGF‐receptors phosphorylated the peptide substrate with a Km of 70‐100 microM. A similar decrease of Km (substrate) was obtained when the phosphorylation experiments were performed with the commonly applied substrates angiotensin II and a peptide derived from c‐src. The Km of angiotensin II phosphorylation was reduced from 1100 microM for wild‐type receptor to 890 microM for mutant receptor and for c‐src peptide from 1010 microM to 770 microM respectively. The Vmax of the kinase was dependent on receptor concentration, but was not significantly affected by the mutation. Analogs of the Tyr1173 peptide in which the tyrosine residue was replaced by either a phenylalanine or an alanine residue also inhibited autophosphorylation with Ki of 650‐750 microM. These analyses show that alterations of individual autophosphorylation sites do not have a major effect on kinase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Cloning and chromosomal localization of a human endothelin ETA receptor

A cDNA clone encoding a human endothelin receptor was isolated from a placenta cDNA library. The deduced amino acid sequence of the clone is 94% identical to the bovine endothelin ETA receptor and represents the human homologue. The human endothelin ETA receptor gene was localized to chromosome 4 by analysis of its segregation pattern in rodent-human hybrids.

The Structure and Function of the Epidermal Growth Factor Receptor Studied by Using Antisynthetic Peptide Antibodies

The human epidermal growth factor receptor has been purified and partial amino acid sequence obtained. A synthetic oligonucleotide was used to select complementary DNA clones from placental and A431 clone banks. The nucleotide sequence of a 5.8 kilobase transcript was determined and used to predict the total amino acid sequence of the receptor. We have predicted a model for the receptor which has an external ligand binding domain of 621 amino acids, a transmembrane region of 23 amino acids, and a cytoplasmic domain of 542 amino acids having protein tyrosine kinase activity. The kinase autophosphorylation sites have been mapped onto the primary amino acid sequence. Analysis of protein sequence databases have shown that the erb-B oncogene of avian erythroblastosis virus has acquired part of the avian EGF receptor gene. The hypothesis has been proposed that transformation by this virus is the result of expression of a truncated EGF receptor which lacks the majority of the EGF binding domain and delivers a continuous proliferation signal to transformed cells. We describe here the production of polyclonal and monoclonal antibodies to selected synthetic peptides from the EGF receptor and v-erb B sequences. Antisera to sequences encompassing the three major sites of autophosphorylation and the putative ATP binding site all recognize the native EGF receptor molecule. We have used these reagents to test our model of EGF receptor structure and v-erb B function.

Activation of c-erbB in avian leukosis virus-induced erythroblastosis leads to the expression of a truncated EGF receptor kinase

Chicken erythroblastosis caused by avian leukosis virus (ALV) is thought to be mediated by activation of the c‐erbB/EGF receptor oncogene by a promoter‐insertion mechanism. Here we study the proteins expressed by two ALV‐induced leukemias and compare them with the avian EGF receptor and with the oncogene product of avian erythroblastosis virus (v‐erbB) which was shown to be a truncated EGF receptor. It appears that the two leukemias express truncated EGF receptors of slightly different sizes with intrinsic tyrosine kinase activity. Hence, acute and chronic retroviruses utilize a common pathway for transformation. Moreover, the proteins expressed in the leukemias are similar to the avian EGF receptor with respect to their phosphopeptide maps, suggesting that they do not carry the C‐terminal deletion characteristic of v‐erbB.

Subcellular distribution of the external and internal domains of the EGF receptor in A-431 cells

Using specific antibodies directed against the external and internal domains of the epidermal growth factor (EGF) receptor, we have directly localized by the protein A gold technique at the electron microscopic level these receptor regions in A-431 epidermoid carcinoma cells. With all antibodies tested, 80-85% of the EGF receptors are found inside the cells, where they preferentially associate with lysosome-like structures, a tubulovesicular system, the rough endoplasmic reticulum and the nuclear envelope. The same distribution pattern is observed for antibodies directed against the external carbohydrate region of the receptor, an antibody against the protein core of the external segment of the receptor, and an antibody reacting with the internal kinase domain of the receptor, suggesting that both receptor segments are similarly distributed intracellularly.

Characterization of receptors for bombesin/gastrin-releasing peptide in human and murine cells

Publisher Summary Bombesin (BN), a 14-amino-acid peptide initially isolated from frog skin, and gastrin-releasing peptide (GRP), a 27-amino-acid peptide isolated from porcine stomach tissue, represent one class of peptides biologically active in the mammalian central nervous system (CNS), periphery, and in tumor cells. This chapter discusses the characterization of receptors for bombesin/gastrin-releasing peptide in human and murine cells. BN/GRP-like peptides have been detected in CNS neurons, peripheral neurons, ganglia, and endocrine as well as tumor cells. GRP is derived from a 148-amino-acid precursor that has been cloned from a human lung carcinoid biopsy specimen. Cross-linking studies were performed to identify the cellular binding component for BN/GRP. Because BN has no functional groups available for cross-linking, 125 I-GRP, with a free amino group at Lys and an unblocked amino terminus, was used as a ligand. Ligand binding to BN/GRP receptors stimulates phosphatidylinositol turnover.

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

The primary strudure 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 transfeded 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-substanceK and three different chemical cross-linking reagents (disuccinimidyl suberate, disuccinimidyl tartrate, or 1-ethyl-3-(3dimethylaminopropyl)carbodiimide-HCI) 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 transfeded cells led to a rapid increase in the produdion of total inositol phosphates and the release of Ca2� from internal stores. Growth of the cells transfeded 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 fador receptor when expressed in mouse 3T3 cells.

Characterization of the detergent solubilized receptor for gastrin-releasing peptide

Properties of detergent solubilized gastrin-releasing peptide receptor were investigated. Swiss 3T3 membranes were covalently labeled with [125I]GRP and homobifunctional cross-linkers. A major labeled protein of 75 kDa was resolved using SDS-polyacrylamide gel electrophoresis. When the same preparation was solubilized with zwitterionic detergent and analyzed under nondenaturing conditions the protein bound radioactivity was resolved in two different peaks, a major one of apparent molecular weight 220,000 (peak 1) and a minor one of 80,000 (peak 2) both containing the 75 kDa protein. Specific ligand binding activity also eluted with peak 1. These results indicate that the active form of bombesin/GRP receptor is a large complex containing the 75 kDa ligand binding domain.

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.