Beta 2-microglobulin induces intracellular transport of human class I transplantation antigen heavy chains in Xenopus laevis oocytes.

Human class I transplantation antigens are cell-surface-expressed molecules composed of one glycosylated, membrane-integrated heavy chain and one nonglycosylated, water-soluble subunit, beta 2-microglobulin (beta 2m). We have examined the intracellular transport of the two subunits by microinjecting mRNA into Xenopus laevis oocytes. Beta 2m, translated in oocytes, was transported and secreted into the medium in the absence of heavy chains whereas heavy chains were retained in the endoplasmic reticulum if not cotranslated with beta 2m. In the presence of beta 2m, heavy chains resisted digestion by endoglycosidase H (Endo H), suggesting that beta 2m promotes the transport of heavy chains from endoplasmic reticulum to the Golgi compartment. Pulse-chase experiments confirmed this notion. The possibility that heavy chains aggregate irreversibly when synthesized in the absence of beta 2m was ruled out and it is demonstrated that performed heavy chains will become transported once beta 2m is available. It is suggested that intracellular transport is controlled by structural features that are part of the transported polypeptide. If so, beta 2m but not heavy chains may possess such features.

Abrogation of cell surface expression of human class I transplantation antigens by an adenovirus protein in Xenopus laevis oocytes.

Class I transplantation antigens form complexes with a virus protein encoded in the early region E3 of the adenovirus-2 genome. The interaction between this viral glycoprotein, E19, and nascent human class I antigens has been examined by microinjecting purified mRNA into Xenopus laevis oocytes. Both E19 and the two class I antigen subunits, the heavy chain and beta 2-microglobulin (beta 2M), were efficiently translated. The heavy chains did not become terminally glycosylated, as monitored by endoglycosidase H digestion, and were not expressed on the oocyte surface unless they were associated with beta 2M. The E19 protein did not become terminally glycosylated, and we failed to detect this viral protein on the surface of the oocytes. Co-translation of heavy chain and E19 mRNA demonstrated that the two proteins associate intracellularly. However, neither protein appeared to be transported to the trans-Golgi compartment. Similar observations were made in adenovirus-infected HeLa cells. Heavy chains bound to beta 2M became terminally glycosylated in oocytes in the presence of low concentrations of E19. At high concentrations of the viral protein, no carbohydrate modifications and no cell surface expression of class I antigens were apparent. Thus, beta 2M and E19 have opposite effects on the intracellular transport of the heavy chains. These data suggest that adenovirus-2 may impede the cell surface expression of class I antigens to escape immune surveillance.

Deletion of the kinase insert sequence of the platelet-derived growth factor beta-receptor affects receptor kinase activity and signal transduction.

A characteristic feature of the platelet-derived growth factor (PDGF) beta-receptor is the presence of an insert sequence in the protein tyrosine kinase domain. A receptor mutant which lacks the entire insert of 98 amino acids was expressed in CHO cells, and its functional characteristics were compared with those of the wild-type receptor. The mutant receptor bound PDGF-BB with high affinity and mediated internalization and degradation of the ligand with efficiency similar to that of the wild-type receptor but did not transduce a mitogenic signal. It was found to display a decreased autophosphorylation after ligand stimulation and had a decreased ability to phosphorylate exogenous substrates; phosphofructokinase was not phosphorylated at all, whereas a peptide substrate was phosphorylated, albeit at a lower rate compared with phosphorylation by the wild-type receptor. Furthermore, the mutant receptor did not mediate actin reorganization but mediated an increase in c-fos expression. The data indicate that the insert in the kinase domain of the PDGF beta-receptor is important for the substrate specificity or catalytic efficiency of the kinase; the deletion of the insert interferes with the transduction of some, but not all, of the signals that arise after activation of the receptor.

cDNA cloning and expression of a human platelet-derived growth factor (PDGF) receptor specific for B-chain-containing PDGF molecules.

The structure of the human receptor for platelet-derived growth factor (PDGF) has been deduced through cDNA cloning. A 5.45-kilobase-pair cDNA clone predicts a 1,106-amino-acid polypeptide, including the cleavable signal sequence. The overall amino acid sequence similarity with the murine PDGF receptor is 85%. After transcription of the cDNA and translation in vitro, a PDGF receptor antiserum was used to immunoprecipitate a product of predicted size, which also could be phosphorylated in vitro. Stable introduction of the cDNA into Chinese hamster ovary (CHO) cells led to the expression of a 190-kilodalton component, which was immunoprecipitated by the PDGF receptor antiserum; this most probably represents the mature PDGF receptor. Binding assays with different 125I-labeled dimeric forms of PDGF A and B chains showed that the PDGF receptor expressed in CHO cells bound PDGF-BB and, to a lesser extent, PDGF-AB, but not PDGF-AA.

Adenovirus proteins and MHC expression.

Adenoviruses are able to specifically down-regulate the cell surface expression of MHC class I antigens. Most viral serotypes achieve these ends by synthesizing a protein that binds to class I antigens in the endoplasmic reticulum (ER) and impedes the transport of these molecules to the cell surface. However, viruses belonging to the highly oncogenic subgenus A do not affect the class I antigen expression during acute infection. Instead, they are distinct from other adenoviruses in that they specifically down-regulate the level of mRNAs, encoding MHC class I antigens, in virally transformed cells. The virus-induced reduction of class I antigen expression drastically diminishes the ability of CTLs to recognize cells infected or transformed by adenovirus. A number of issues concerning these viral mechanisms for class I antigen modulation need to be addressed. The molecular mechanism by which the E1A gene product of subgenus A viruses diminishes class I mRNA levels has not been elucidated. Also, the details of the interaction between the E19 protein and class I molecules should be studied, preferably by X-ray crystallography of the complexes. This would clarify the role of the antigen-binding site as well as other portions of the class I molecule in the binding to the E19 protein. Of general importance for our understanding of the sorting and intracellular transport of proteins is the exact delimitation of the signal for ER localization, which is present in the COOH-terminus of the E19 protein. The putative interaction of this peptide sequence with components of the ER membrane should also be studied. Finally, the study of the pathophysiological role of the MHC class I down-regulation will undoubtedly yield new insights into how the immune system combats virally infected and transformed cells.

Retrovirus-mediated gene transfer of the human gamma-IFN gene: a therapy for cancer.

A retroviral vector-mediated gene transfer system was used to introduce m gamma-IFN and h gamma-IFN genes into mouse and human tumor cells, respectively. Murine tumor cell lines and primary human melanoma tumor cells were successfully transduced with gamma-IFN vector, and these transduced cells secreted measurable levels of biologically active m gamma-IFN and h gamma-IFN, respectively. Both murine and human tumor cell lines that expressed gamma-IFN exhibited increased surface expression of HLA class I antigens when tested by Western blot and FACS analysis. gamma-IFN--transduced human melanoma cells were more active in stimulating tumor-specific cytolytic activity of CTLs from melanoma patients in vitro. m gamma-IFN--transduced tumor cells were substantially less tumorigenic than the corresponding parent tumor cell lines in immune-competent mice. In addition, injection of m gamma-IFN--transduced tumor cells resulted in activation of tumor-specific CTL in vivo. We plan to use gamma-IFN--transduced autologous tumor cells to boost host immune responses as a potential therapy for human melanoma.

A B-type PDGF receptor lacking most of the intracellular domain escapes degradation after ligand binding.

The characteristics of the human B-type platelet-derived-growth-factor (PDGF) receptor expressed in Chinese hamster ovary (CHO) cells, were compared with those of a mutant receptor lacking all but 19 amino acids of the intracellular domain. The transfected wild-type receptor was synthesized as a 160-kDa precursor that was processed to 190 kDa. Each CHO cell expressed 30,000-100,000 receptors which bound PDGF-BB with a Kd of about 0.5 nM. Analysis of PDGF-AB binding yielded non-linear Scatchard plots; the major part of the binding sites had a Kd of 6 nM. PDGF-AA was not bound. The receptors expressed in CHO cells were down-regulated after binding of PDGF-BB, and mediated degradation of 125I-PDGF-BB with similar efficiency as PDGF-B-type receptors in human fibroblasts. The transfected receptor also transduced a mitogenic signal. The mutant receptor was synthesized as a 90-kDa precursor and was processed to 120 kDa with a slightly faster rate than the wild-type receptor. Cells expressing the mutant receptor generally had around 10(6) ligand-binding sites/cell, with a Kd for binding of PDGF-BB of 3 nM. The mutant receptor, which did not transduce a mitogenic response, mediated degradation of 125I-PDGF-BB, albeit less efficiently compared to the wild-type receptor. In contrast to the wild-type receptor, it was down-regulated only to a limited extent and not degraded in response to ligand binding. These findings indicate a role for the intracellular part of the receptor, not only in mitogenic signaling, but also in receptor internalization and intracellular routing.

Differential association between two human MHC class I antigens and an adenoviral glycoprotein.

The glycoprotein E19, encoded in early region 3 of adenovirus-2, forms complexes with major histocompatibility complex class I antigens. As a result of the complex formation, the intracellular transport of the class I antigens is abrogated, and adenovirus-infected cells display gradually diminishing quantities of cell surface-expressed class I molecules. To assess whether the E19 protein interacts equally well with different class I antigens, the associations between the viral protein and HLA-A2 and HLA-B7 antigens have been estimated. By infecting transfected HeLa cells expressing various amounts of HLA-A2 and HLA-B7 molecules, respectively, with various infectious doses of adenovirus-2, experimental conditions could be established that allowed quantitative estimates of the interactions to be determined. It was found that HLA-A2 molecules and the E19 protein interacts with a binding constant that is more than twice as high as that for HLA-B7 antigens and the viral protein. It is suggested that the pathogenicity of the virus may be dependent on the HLA-type of the infected individual.

Growth factor-binding proteases in the murine submaxillary gland: isolation of a cDNA clone.

The submaxillary gland of the adult male mouse contains a number of serine proteases, several of which are involved in the proteolytic processing of precursors to growth factors and other biologically active polypeptides. Here we report the isolation and identification of a cDNA clone corresponding to one of the proteases, the type B of the epidermal growth factor-binding protein. A pronounced sequence homology was found between the predicted activation peptide of this protease and the NH2-terminal extension of the nerve growth factor alpha subunit, suggesting that the latter protein has an uncleaved activation peptide attached to its NH2 terminus.

Complex formation of class I transplantation antigens and a viral glycoprotein.

Indirect immunoprecipitations of labeled glycoproteins from the adenovirus-transformed rat cell line A2T2C4 and from adenovirus-infected HeLa cells revealed that the class I major histocompatibility antigens co-precipitated with the viral E19 protein. The degree of co-precipitation was highly dependent on the antiserum used. The identity of the co-precipitated components was verified by peptide mapping and radiochemical amino acid sequencing. Cell-free translation of mRNA for the E19 protein and the class I antigen heavy chains demonstrated that the E19 protein-class I antigen interaction is an inherent property of the participating components. In intact cells the virus protein and the transplantation antigens form large complexes, held together by weak, noncovalent interactions.