Interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF ) receptors share a common β chain (βc), and both cytokines enhance erythropoietin (Epo)-dependent in vitro erythropoiesis by primary hematopoietic progenitors and factor-dependent cells. These data suggest that the Epo receptor and βc may functionally interact. To determine whether such interactions can be documented, we studied a murine factor-dependent cell line (Ba/F3), which endogenously expresses IL-3R. First, Ba/F3 cells were transfected with murine EpoR, making them responsive to both IL-3 and Epo. Next, the EpoR expressing cells were transfected with murine βc. This resulted in an enhanced sensitivity of these cells to Epo, which was especially pronounced at low Epo concentrations. Ba/F3-EpoR were then treated with antisense oligodeoxynucleotides to the murine β. Control sense and nonsense had no effect on Epo-dependent growth, but the antisense markedly and specifically inhibited Epo-dependent growth. In contrast, the antisense did not affect β-globin message levels (another Epo-responsive effect in these cells) detectable by Northern blot. Finally, Western blot analysis of proteins immunoprecipitated from cells expressing both receptors with antibody against β and blotted with antibody against EpoR, or immunoprecipitated with antibody against EpoR and blotted with antibody against β, showed that EpoR and β coimmunoprecipitate. These data show that the β chain functionally and physically associates with the EpoR. This suggests that these cytokine receptors exist as a large supercomplex and offers the first molecular explanation for the synergistic effects of IL-3 and GM-CSF with Epo during erythropoiesis.

Colony-stimulating factor 1 (CSF-1) regulates the survival, proliferation, and differentiation of mononuclear phagocytes. The CSF-1 receptor was purified from cell membranes of the J774.2 mouse macrophage cell line by solubilization with Triton X-100, CSF-1 affinity chromatography, and gel filtration. The purified receptor is a protein or glycoprotein of 165 kDa comprising a single polypeptide chain that is not covalently associated, either as a homopolymer, or with any other protein. CSF-1 stimulated autophosphorylation of the purified receptor in tyrosine residues. Casein but not histone was shown to act as a substrate for the tyrosine protein kinase activity of purified receptor. (3,7). Chemical crosslinking studies indicate that the cell surface CSF-1 receptor is a macromolecule of ==165,000 Mr that is not disulfide bonded to any other macromolecule in macrophage membranes (8). The CSF-1 receptor shares physical characteristics with, and is immunologically and functionally related to, the c-fms gene product (9, 10). In this paper, we describe the purification of the CSF-1 receptor and demonstrate that the purified receptor is a CSF-1-dependent tyrosine kinase. MATERIALS AND METHODSPurification of the CSF-1 Receptor. J774.2 mouse macrophage cells (11,12) were seeded (104 cells per ml) into 15 liters of prewarmed 10% (vol/vol) horse serum (Flow Laboratories) in alpha medium (Kansas City Biologicals, Lenexa, KS) and cultured at 37°C in a large spinner flask. After 4 days cells were harvested from log-phase cultures (=z5 x 105 cells per ml, >95% viable) and collected by centrifugation (500 x g for 10 min at 4°C); the cell pellet was resuspended in 5 volumes of ice-cold 4 mM iodoacetic acid in phosphate-buffered saline (0.14 mM NaCl/3 mM KCl/8 mM Na2HPO4/1.5 mM KH2PO4, pH 7.4). All the following procedures were done at 4°C.The methods for the preparation and solubilization of J774.2 cell membranes and the assay of the solubilized receptor have been described in detail (13). Briefly, the postnuclear supernatant fraction from Dounce-homogenized, hypotonically swollen cells was layered over 15% (wt/vol) sucrose and centrifuged at 100,000 x g for 30 min at 40C. The pellet was resuspended in 10-20 volumes of 10% (wt/vol) sucrose/50 mM NaCl/100 mM Tris HCl, pH 7.4, containing 10 ,g of leupeptin (Sigma) per ml, 1 unit of aprotinin (Sigma) per ml, and 1000 units of soybean trypsin inhibitor (type I-S, Sigma) per ml and adjusted to 1% (vol/vol) Triton X-100. After gentle Dounce homogenization with a loose-fitting piston the mixture was diluted with an equal volume of receptor-binding buffer (RBB)-10% (wt/vol) sucrose/50 mM NaCl/0.1% Triton X-100/3 mM NaN3/100 mM Tris HCl, pH 7.4-and centrifuged (100,000 x g for 45 min at 40C), and the supernatant was saved as the solubilized membrane fraction. This fraction (==12 ml) was further diluted with an equal volume of RBB and passed twice through the CSF-1-p-aminobenzamidoethyl (pABAE) Sepharose 4B (see below) column (1.2 x 3.5 cm) at a flow rate of 40-50 ml per hr. The ...

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