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The addition of epidermal growth factor (EGF) to cultured rat pituitary cells (GH3) leads to increased synthesis of prolactin and to partial inhibition of cell proliferation. Monoclonal antibodies generated against EGF receptor from human epidermoid carcinoma (A-431) cells were used to characterize the EGF receptor kinase system of GH3 cells and to investigate the role of the hormone-receptor complex in the expression of the prolactin gene in these cells. The EGF receptor of GH3 cells is a 170,000-dalton protein associated with a protein kinase. It is similar but not identical to the EGF receptor identified in other tissues. The immunoprecipitated membrane receptor is phosphorylated on both serine and tyrosine residues. The monoclonal antibody denoted 2G2-IgM binds to EGF receptor on GH3 cells. Like EGF, the monoclonal antibody induced the synthesis of prolactin and morphological changes in these cells. Hence, EGF receptor in GH3 cells, when properly triggered, contains all of the biological attributes necessary for the induction of EGF-induced gene expression and morphological changes in GH3 cells.

Epidermal growth factor (EGF) receptor from A-431 cells was purified by affinity chromatography with monoclonal anti-receptor antibodies. The purified radiolabeled receptor was incubated with EGF and then analyzed by gel electrophoresis under nondenaturing conditions. In these gels, the EGF receptor migrates in two forms: a fast-migrating (low) form and an EGF-induced slow-migrating (high) form. On the basis of the various control and calibration experiments described, it is concluded that the low form represents the monomeric 170-kilodalton EGF receptor and the high form represents an EGF receptor dimer. The binding of EGF causes a rapid, temperature-sensitive dimerization of the EGF receptor. Receptor dimerization is fully reversible and involves saturable, noncovalent interactions that are stable at neutral pH and in nonionic detergents. Both the monomeric and dimeric forms of the receptor bind EGF and undergo self-phosphorylation. The dimeric form of the receptor may possess higher ligand binding affinity, and it seems to be phosphorylated earlier than the monomeric form following the addition of EGF and [gamma-32P]ATP. On the basis of these results, it is concluded that receptor oligomerization is an intrinsic property of the occupied EGF receptor and that it may play a role in the activation of the kinase function and the subsequent transmembrane signaling process.

The primary structure of the receptor for platelet-derived growth factor (PDGF), determined by means of cloning a cDNA that encodes the murine pre-PDGF receptor, is closely related to that of the v-kit oncogene product and the receptor for macrophage colony stimulating factor (CSF-1). Common structural features include the presence of long sequences that interrupt the tyrosine-specific protein kinase domains of each molecule. The PDGF and CSF-1 receptors also share a characteristic distribution of extracellular cysteine residues. Ubiquitin is covalently bound to the purified PDGF receptor, the human gene for which is on chromosome 5.

Partial amino acid sequence information allowed the isolation of cDNA clones encoding the turkey erythrocyte beta-adrenergic receptor. Antisera raised against synthetic peptides encoded by the cDNA crossreacted with the purified receptor and appropriate tryptic fragments, confirming the identity of the cDNA. The receptor is composed of 483 amino acids and has a molecular mass of 54 kDa. Its sequence suggests that it is arranged predominantly in seven membrane-spanning sequences and a long cytoplasmic carboxyl-terminal domain. The extracellular amino-terminal domain contains a consensus sequence for N-glycosylation. The beta-adrenergic receptor displays overall structural similarity and weak sequence homology with rhodopsin. Because both proteins act by regulating GTP-binding proteins, a compact structure based on seven membrane-spanning regions may be a general model for receptors that act on G proteins.

The rotational diffusion of epidermal growth factor (EGF)--receptor complexes on living human epidermoid carcinoma cells (A-431) has been measured by phosphorescence emission and anisotropy in the mu s time domain. A biologically active phosphorescent conjugate of EGF, erythrosin-EGF, was applied to living cells. The hormone--receptor complexes were mobile with rotational correlation times in the range 25--90 mu s when labeled and measured at 4 degrees C. Prolonged incubation and exposure to higher temperatures (23 and 37 degrees C) resulted in longer times up to 350 mu s, indicative of the progressive formation of microclusters, estimated to contain 10-50 receptors. Upon internalization of the hormone--receptor complexes, visible patches were observed by fluorescence microscopy, and the rotational correlation times were shorter, indicating a decrease in size of the dynamic unit. The sign of the rotational relaxation also varied with localization and processing of the hormones. The rate of lateral diffusion of EGF--receptor complexes, measured under similar conditions by fluorescence photobleaching recovery, increased with temperature in contrast to the rotational motion.

There is a growing list of oncogenes encoding transmembrane tyrosine kinases that have structures reminiscent of growth factor receptors. In most cases, the ligands for these putative receptors are unknown. Using the neu oncogene as a model system, we have developed several experimental approaches for the detection of such hypothetical ligands. The following lines of evidence collectively imply that a candidate ligand of the neu-encoded oncoprotein is secreted by ras-transformed fibroblasts: Medium conditioned by ras transformants is able to induce down-modulation of the neu-encoded p185 and to activate its intrinsic tyrosine kinase activity in vitro. In addition, a rapid increase in the phosphorylation in vivo of tyrosine residues of the neu-encoded protein is induced by the conditioned medium. Finally, transfer of the neu gene into hematopoietic cells renders them mitogenically responsive to the conditioned medium. The possibility of indirect activation of the oncoprotein through other known receptors, especially the receptor for the epidermal growth factor, was experimentally excluded.

Monoclonal anti-epidermal growth factor (EGF) receptor antibodies were used as a diagnostic tool for the investigation of the role of the receptor molecule in the transduction of the biological effects mediated by EGF. The specificity of the antibodies was established by immunoprecipitation of the receptor from biosynthetically labeled cells. The previously described (Schreiber, A. B., Lax, I., Yarden, Y., Eshhar, Z., and Schlessinger, J. (1981) Proc. Natl. Acad. Sci U. S. A. 78, 7535-7539) 2F2-IgM antibody binds to or close to the binding site of the growth factor on the receptor molecule; it induces receptor clustering and internalization and triggers early and delayed effects of EGF. A monovalent Fab fragment of the 2G2-IgM antibody stimulates the EGF-receptor sensitive protein kinase, but does not induce receptor clustering and DNA synthesis. When cell-bound 2G2-Fab fragments are cross-linked with anti-mouse Ig antibodies, both receptor clustering and mitogenic activity are restored. A second monoclonal antibody against EGF-receptor denoted TL5-IgG does not interfere with the binding of EGF to the receptor, fails to induce receptor clustering, and does not possess any intrinsic bioactivity. The cross-linking of cell-bound TL5-IgG with anti-mouse Ig antibodies leads to the stimulation of DNA synthesis. It is concluded that the EGF-receptor, when properly triggered, contains all the biochemical attributes necessary for the initiation of biological effects. Receptor clustering, even when mediated via domains distinct from the hormone binding site on the receptor molecule, appears as a necessary and sufficient signal for the induction of DNA synthesis.

The rotational diffusion of the complexes of epidermal growth factor (EGF) with its specific receptor on plasma membrane vesicles prepared from human epidermoid carcinoma A431 cells was studied using the time-resolved polarization of phosphorescence of erythrosin-labeled hormone. The measured rotational correlation times of 16-20 microseconds at 4 degrees C are consistent with monomeric freely diffusing EGF receptor. Upon increasing the temperature to 37 degrees C, the rate of rotational diffusion slows down as evidenced by an increase in the correlation time to 75 microseconds. This finding suggests that small clusters of the occupied EGF receptor (microaggregation) form at the higher temperature, a property we have reported previously for occupied receptors on living A431 cells. Subsequent cooling of the membranes leads to a partial reversal of the microaggregation. We conclude that clustering of occupied EGF receptors can proceed at 37 degrees C in the absence of metabolic energy and external interactions, e.g. with components of the cytoskeleton, and thus reflects inherent properties of the receptor protein in its natural environment. A lag phase in the time course of microaggregation observed with the isolated membrane preparations may reflect cooperativity in the process of receptor association.