Antipeptide antibodies as probes of the recombinant and endogenous murine erythropoietin receptors.
The binding of erythropoietin (Epo) to its plasma membrane receptor activates signal pathways that result in erythroid cell proliferation and differentiation. To elucidate the structural features of the receptor that are important for hormone binding and signaling, we have developed a series of site-specific antibody probes. These antibodies were raised against synthetic peptides homologous to six exoplasmic domains and one cytoplasmic domain of the murine receptor and were affinity-purified by binding to their respective peptide antigen, immobilized on agarose. Western blot analyses demonstrated that the recombinant receptor expressed transiently in COS-7 cells is synthesized as three protein species of 62, 64, and 66 kd, consistent with previous observations. Importantly, probing the endogenous receptor in both virally transformed erythroleukemia cells and normal erythroid cells demonstrated similar 62- to 66-kd receptor species. The affinity-purified antibodies also recognized several antigenically related proteins. An examination of the capacity of the antireceptor antibodies to block receptor activation by Epo revealed that antibodies to five of the six exoplasmic domains blocked the receptor. This was reversed with excess Epo. Inhibition of receptor activation by antibody probes to five discrete hydrophilic domains suggests that receptor function may be critically dependent on the structural integrity (conformation) of the entire exoplasmic portion.
Bailey
S C
SC
New England Deaconess Hospital, Department of Medicine, Harvard Medical School, Boston, MA 02215.
Feldman
L
L
Romanowski
R R
RR
Davis
K L
KL
Sytkowski
A J
AJ
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000032:analysis* / Q000276:immunology / Q000494:pharmacology; D015153:Blotting, Western; D002478:Cells, Cultured; D000939:Epitopes / Q000032:analysis / Q000276:immunology / Q000502:physiology; D015672:Erythroid Precursor Cells / Q000737:chemistry / Q000166:cytology / Q000648:ultrastructure; D005622:Friend murine leukemia virus; D004915:Leukemia, Erythroblastic, Acute / Q000473:pathology; D051379:Mice; D008969:Molecular Sequence Data; D017467:Receptors, Erythropoietin / Q000032:analysis / Q000276:immunology* / Q000502:physiology; D011994:Recombinant Proteins / Q000032:analysis / Q000276:immunology; D015398:Signal Transduction / Q000502:physiology; D014407:Tumor Cells, Cultured
D000906:Antibodies; D000939:Epitopes; D017467:Receptors, Erythropoietin; D011994:Recombinant Proteins