The cytokine erythropoietin (Epo) is tissue-protective in preclinical models of ischemic, traumatic, toxic, and inflammatory injuries. We have recently characterized Epo derivatives that do not bind to the Epo receptor (EpoR) yet are tissue-protective. For example, carbamylated Epo (CEpo) does not stimulate erythropoiesis, yet it prevents tissue injury in a wide variety of in vivo and in vitro models. These observations suggest that another receptor is responsible for the tissue-protective actions of Epo. Notably, prior investigation suggests that EpoR physically interacts with the common ␤ receptor (␤cR), the signal-transducing subunit shared by the granulocyte-macrophage colony stimulating factor, and the IL-3 and IL-5 receptors. However, because ␤cR knockout mice exhibit normal erythrocyte maturation, ␤cR is not required for erythropoiesis. We hypothesized that ␤cR in combination with the EpoR expressed by nonhematopoietic cells constitutes a tissueprotective receptor. In support of this hypothesis, membrane proteins prepared from rat brain, heart, liver, or kidney were greatly enriched in EpoR after passage over either Epo or CEpo columns but covalently bound in a complex with ␤cR. Further, antibodies against EpoR coimmunoprecipitated ␤cR from membranes prepared from neuronal-like P-19 cells that respond to Epo-induced tissue protection. Immunocytochemical studies of spinal cord neurons and cardiomyocytes protected by Epo demonstrated cellular colocalization of Epo ␤cR and EpoR. Finally, as predicted by the hypothesis, neither Epo nor CEpo was active in cardiomyocyte or spinal cord injury models performed in the ␤cR knockout mouse. These data support the concept that EpoR and ␤cR comprise a tissue-protective heteroreceptor.

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The transforming protein of Rous sarcoma virus (pp6OV'rC) and its normal cellular homologue (pp6Occ) appear to be protein kinases that phosphorylate tyrosine in a variety of protein substrates. In addition, pp6Ov-tc and pp60)-CrC are themselves phosphorylated on serine and tyrosine. It is likely that these phosphorylations serve to regulate the function(s) of pp60v87c and pp6COr. We The transforming gene (v-src) of Rous sarcoma virus (RSV) encodes a 60,000-dalton protein (pp6V-rC) that is capable ofphosphorylating tyrosine in a variety of protein substrates (1-7).Uninfected vertebrate cells contain a similar protein [pp6Ocrc (8)(9)(10)(11)(12)]. Both pp6Ov-rc and pp60c-*c are phosphorylated on serine and tyrosine; these phosphorylations may regulate the enzymatic activity of the proteins. Phosphorylation of cellular proteins by pp60v-rc presumably plays a major role in the establishment and maintenance of the neoplastic phenotype induced by infection with RSV. It is therefore important to explore all of the means by which the action of this protein might be controlled.We have characterized and compared the sites of tyrosine phosphorylation in pp60v-src and pp60CslC. Phosphorylation of the proteins was carried out in two ways: by metabolic labeling of cells in culture (4) and by phosphotransfer in vitro, using partially purified preparations ofpp60v'rcand pp60CS-rC (13,14).Our strategy exploited the availability ofa predicted amino acid sequence for the viral protein (15). Tryptic peptides containing phosphotyrosine were isolated by high-performance liquid chromatography (HPLC) and then subjected to sequential Edman degradation to locate the phosphorylated residue in the peptide. The results indicate that phosphorylation of tyrosine in pp60VS-C occurs on the same amino acid residue either in vitro or in vivo and an apparently identical site is also phosphorylated in pp60Cl-SC in vitro. By contrast, phosphorylation of pp6Ocsrc in vivo apparently occurs at a different site in the protein (16,17). The significance ofthis difference remains to be elucidated. MATERIALS AND METHODSGeneral Procedures. We have described our procedures for the propagation and isotopic labeling ofcultured cells, the preparation of antisera from rabbits bearing tumors induced by the Schmidt-Ruppin strain of RSV, the immunoprecipitation of virus-specific proteins, partial proteolysis by staphylococcus V8 protease, the assay ofthe protein kinase associated with pp6Osrc, the purification ofpp60v-rc by immunoaffinity chromatography, and the fractionation of proteins by electrophoresis in polyacrylamide gels (4,9,13,14). For metabolic labeling of pp6OV-t, chicken embryo fibroblasts were infected with a virus stock that was generated by transfection with cloned SRA-2 DNA (18). Hydrolysis of Proteins with Trypsin. 32P-Labeled polypeptides were visualized by autoradiography ofdried gels, excised, oxidized with performic acid, and digested with N-tosylphenylalanine chloromethyl ketone-trypsin while still in the gel slice, as described by S...

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Human factor VIII has been isolated from a high purity factor VIII concentrate by immunoaffmity chromatography and HPLC on Mono Q gel. Two fractions of factor VIII were obtained with a specific activity of w7000 units/mg.The major fraction contained eight peptide chains of 200, 180, 160, 150, 135, 130, 115, and 105 kDa plus one doublet chain of 80 kDa. The minor fraction contained one peptide chain of 90 kDa plus the chain of 80 kDa. Both fractions were activated by thrombin to the same extent. Amino-terminal amino acid sequence analysis was performed on the 180-kDa, 130-kDa, and 90-kDa chains and showed an identical amino-terminal sequence in these chains. Each chain from 200 kDa to 90 kDa was linked to one 80-kDa chain by a metal-ion bridge(s).Studies on factor VIII in plasma and cryoprecipitate, prepared and gel ifitered in the presence of protease inhibitors, showed that one 200-kDa plus one 80-kDa chain were the only or dominating chains in the materials and may represent native factor VIII. The results indicated that all chains from 180 kDa to 90 kDa are fragments of the 200-kDa chain. All of these more or less fragmented chains form active factor VIII complexes with the 80-kDa chain.Factor VIII (antihemophilic factor) is the protein that is deficient or absent in individuals with classic hemophilia, an X-chromosome-linked bleeding disorder. It participates in the intrinsic pathway of blood coagulation as a cofactor in the activation of factor X by factor IXa, in the presence of phospholipid and calcium (1-7). In plasma, factor VIII is noncovalently bound to von Willebrand factor (vWF), a high molecular weight protein involved in primary hemostasis. Due to the low concentration of factor VIII in plasma and to the fact that factor VIII is highly susceptible to degradation by serine proteases, it is only recently that highly purified factor VIII, free of vWF, has been obtained. Bovine factor VIII, as isolated from plasma, is composed of three peptide chains of 93 kDa, 88 kDa, and 85 kDa (8). Factor VIII purified from porcine plasma has been shown to consist of subunits of 160 kDa, 130 kDa, 82 kDa, and 76 kDa (9). Purified human factor VIII has been prepared from commercial factor VIII concentrate and had a specific activity of 2294 units/mg (10). NaDodSO4/PAGE showed several peptide chains having molecular sizes of 188-79 kDa. The majority of the material was related to factor VIII as shown by immunoblot technique with use of monoclonal antibodies against factor VIII (11). A preparation of human factor VIII from cryoprecipitate, in the presence of seine protease inhibitors, has also been reported with the specific activity of 4740 units/mg (12). Unreduced NaDodSO4/PAGE showed predominant bands at 360 kDa, 210 kDa, and 90 kDa and an 80 kDa/79 kDa doublet band.Immunoblotting using monoclonal antibodies suggested that the 360 kDa component was a precursor to the other components. Thus different results are obtained when different starting materials and procedures for purification are used.The human fac...