Cells were lysed on ice in 1% Nonidet P-40 buffer, immunoprecipitated with anti-EpoR antibodies, and analyzed by Western blotting with anti-phosphotyrosine monoclonal antibodies 4G10. tolerate the replacement of its TM domain with that of glycophorin A and retained signaling. We propose a model in which TM domain-induced dimerization maintains unliganded EpoR in an inactive state that can readily be switched to an active state by physiologic levels of Epo. Binding of erythropoietin (Epo) to the Epo receptor (EpoR) is crucial for production of mature red cells. Homodimerizing members of the cytokine receptor superfamily, such as the EpoR and prolactin receptor (PrlR), function as ligand-induced or ligand-stabilized homodimers (1). Ligand binding triggers auto- or trans-phosphorylation of a Janus kinase (JAK) bound to the receptor cytosolic domain, activating JAK kinase activity (2). JAK substrates include the receptors themselves, signal transducers and activators of transcription (STAT) proteins, and a variety (S,R,S)-AHPC-PEG2-NH2 of other cytosolic signaling molecules (3). The relative orientation of the EpoR extracellular (EC) domains in a receptor dimer is directly related to the efficiency of signaling through the cytoplasmic (CT) domain (4C7). The EpoR can be productively activated by several means in additional to binding of Epo, its normal ligand. These include small Epo mimetic peptides (8), bivalent monoclonal antibodies directed to the EpoR (9), and an R129C point mutation in the EC domain that results in a disulfide bond connecting two receptor monomers (1, 10). Dimerization of EC domains is not sufficient for signaling because nonpermissive orientations of the dimerized EC domains have been identified (5). A cornerstone in understanding signaling by EpoR is the oligomerization state of the full-length receptor on the cell surface before ligand binding. One model is that of two monomeric receptors brought together into a dimer after the binding of Epo, with signaling being the result of the close proximity of the two receptor polypeptides. However, the EpoR may be present in the membrane as a preformed dimer or higher oligomer with ligand binding triggering a specific conformational change that activates the receptors. The model of ligand-induced dimerization is in accord with the ability of bivalent monoclonal antibodies, small dimerized peptides, Rabbit Polyclonal to ILK (phospho-Ser246) and the R129C mutation to activate the EpoR (1, 8C10); however, these agents could also shift an already dimeric/oligomeric EpoR from an inactive to an active conformation. The crystal structure of the soluble truncated EC domain of the human EpoR in its unliganded form unexpectedly revealed a preformed dimer with a geometry different from that of the Epo-bound receptor (6, 7). That the unliganded receptor is a dimer was supported by an fragment complementation assay performed on a truncated receptor containing the EC and transmembrane (TM) domains of the mouse EpoR fused to fragments of the dihydrofolate reductase enzyme (11). However, it is not clear whether the dimers observed in the dense crystalline state occur at the much lower receptor densities present on the plasma membrane. The presence of the TM and CT domains may also alter the oligomeric interactions of the intact receptors. Although no dimerization can be detected between EC domains in solution, it was suggested that simple membrane anchorage (not to mention possible interactions between TM domains) might significantly enhance low-affinity (S,R,S)-AHPC-PEG2-NH2 interactions that are undetectable in solution (6), as might be expected on entropic grounds. We therefore studied the oligomerization state of the full-length EpoR situated in its natural environment, the plasma membrane of living (S,R,S)-AHPC-PEG2-NH2 cells. Our findings demonstrate that a high proportion of the murine EpoR expressed at the.