Structure of the murine erythropoietin receptor complex. Characterization of the erythropoietin cross-linked proteins.
The structure of the murine erythropoietin receptor was studied using antibodies against the intracellular part of the cloned erythropoietin receptor chain. These antibodies precipitated erythropoietin-receptor complexes from Triton X-100-solubilized cells. When the complexes were cross-linked by disuccinimidyl suberate, the 85- and 100-kDa erythropoietin-cross-linked proteins previously described were immunoprecipitated. However, these proteins were not precipitated when the complexes were denatured and reduced before immunoprecipitation. Using 1-ethyl 3-(3-dimethylaminopropyl)carbodiimide, we observed erythropoietin cross-linking with a protein of 66 kDa in addition to the 100- and 85-kDa proteins. Only the 66-kDa erythropoietin-cross-linked protein was immunoprecipitated by anti-receptor antibodies after denaturation and reduction of the complex. Thus, our results suggest that the 85- and 100-kDa proteins previously evidenced by cross-linking are associated with the cloned chain of the receptor to form a multimeric complex but these proteins seem immunologically unrelated to the cloned chain. We observed that reducing the length of molecules able to cross-link amino groups decreased the efficiency of cross-linking with the 100-kDa protein and only the 85-kDa protein was cross-linked with erythropoietin using 1,5-difluoro-2,4-dinitrobenzene. These results suggest that the 85- and 100-kDa proteins occupate slightly different positions relative to the erythropoietin molecule bound to the receptor.
Mayeux
P
P
ICGM, Institut National de la Santé et de la Recherche Médicale U152, Hospital Cochin, Paris, France.
Lacombe
C
C
Casadevall
N
N
Chretien
S
S
Dusanter
I
I
Gisselbrecht
S
S
D000818:Animals; D000906:Antibodies; D002478:Cells, Cultured; D003001:Cloning, Molecular; D003432:Cross-Linking Reagents; D004921:Erythropoietin / Q000378:metabolism*; D051379:Mice; D011233:Precipitin Tests; D011489:Protein Denaturation; D011956:Receptors, Cell Surface / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D017467:Receptors, Erythropoietin
D000906:Antibodies; D003432:Cross-Linking Reagents; D011956:Receptors, Cell Surface; D017467:Receptors, Erythropoietin; D004921:Erythropoietin