Photoaffinity labeling of the erythropoietin receptor and its identification in a ligand-free form.
Pure human recombinant erythropoietin (EP) was acylated through a primary amino residue with a cross-linking reagent, N-[[3-[[4-[(p-azido-m-[125I]iodophenyl)azo]benzoyl]amino] propanoyl]oxy]-succinimide (Denny-Jaffe reagent), which is photoreactive and cleavable at the azo residue. The resulting conjugated hormone (DJ-EP) was purified from unmodified EP by reverse-phase high-pressure liquid chromatography and maintained its capacity to bind to receptors for EP on erythroid progenitor cells. The receptor for EP was previously identified as two related proteins of 100 and 85 kDa molecular mass by chemical cross-linking to 125I-EP. Recently, D'Andrea and co-workers [(1989) Cell 57, 277-285] cloned a cDNA that codes for a protein of 55-66 kDa, which is thought to be the EP receptor. In this report, cross-linking to the receptor through the monofunctional DJ-EP labeled the same 140- and 125-kDa molecular mass bands (100- and 85-kDa proteins) cross-linked with 125I-EP and disuccinimidyl suberate. Furthermore, cleavage of the azo bond of the DJ-EP receptor complex by sodium dithionite (80 degrees C, 5 min) demonstrated that proteins of 105 and 90 kDa were labeled in ligand-free form by DJ-EP. This result demonstrates that artifactual cross-linking of multiple proteins or other artifacts of cross-linking do not explain the difference in molecular mass of the EP receptor identified by cross-linking and the receptor identified by expression cloning.
Hosoi
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Department of Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee 37232.
Sawyer
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Krantz
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D000215:Acylation; D000345:Affinity Labels; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D015672:Erythroid Precursor Cells / Q000378:metabolism; D004921:Erythropoietin / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D008024:Ligands; D051379:Mice; D008970:Molecular Weight; D010777:Photochemistry; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000378:metabolism*; D017467:Receptors, Erythropoietin; D011990:Receptors, Transferrin; D011994:Recombinant Proteins
D000345:Affinity Labels; D008024:Ligands; D011956:Receptors, Cell Surface; D017467:Receptors, Erythropoietin; D011990:Receptors, Transferrin; D011994:Recombinant Proteins; D004921:Erythropoietin