Mapping on the calf estrogen receptor of the binding domain for an antibody interfering with receptor activation.
The localization on the calf estrogen receptor of the binding domain for B36 (an IgM antibody which prevents and reverses the effects of receptor activation) has been studied by means of controlled proteolysis of the receptor-estradiol complex using trypsin, chymotrypsin, and papain. We successively determined for intact and proteolyzed receptor-estradiol complex (i) the abilities of estradiol-binding species to aggregate in low salt medium, to bind to nonspecific DNA absorbed onto cellulose, and to interact with B36 antibody in sucrose gradients; (ii) the hydrodynamic properties of estradiol-binding species, by gel permeation chromatography and sucrose gradient centrifugation in high salt media and (iii) the molecular weights of B36-reactive species, by immunoblot analysis. Three tryptic receptor fragments of Mr 36,000, 34,000, and 33,000 and two chymotryptic fragments of Mr 36,000 and 33,000 included both the hormone- and B36-binding domains but did not interact with DNA, whereas at least two receptor fragments resulting from the action of chymotrypsin and papain bound estradiol with high affinity but interacted neither with DNA nor with B36. Taking into account these results and assuming that structure of the calf estrogen receptor is similar to those of sequenced estrogen receptors (which show a highly conserved organization with considerable homologies in the functional domains), we propose that the B36-binding domain is located either between the DNA- and hormone-binding domains (model I) or at the C-terminal end of the estrogen receptor (model II). The regions that include the main proteolytic cleavage sites of the receptor are also specified, and the abilities of the two models of the calf estrogen receptor to account for the effect of B36 on receptor activation are discussed.
ORIS-Industrie, Bagnols-sur-Cèze, France.
J
J
Fauque
J
J
Scali
V
V
Cavaillés
J L
JL
Borgna
D000818:Animals; D000906:Antibodies / Q000494:pharmacology*; D001665:Binding Sites; D002417:Cattle; D002918:Chymotrypsin; D004247:DNA / Q000378:metabolism; D004958:Estradiol / Q000378:metabolism; D005260:Female; D015151:Immunoblotting; D007075:Immunoglobulin M / Q000494:pharmacology; D010206:Papain; D010446:Peptide Fragments / Q000378:metabolism; D011960:Receptors, Estrogen / Q000032:analysis* / Q000187:drug effects; D015398:Signal Transduction; D014357:Trypsin; D014599:Uterus / Q000378:metabolism*
D000906:Antibodies; D007075:Immunoglobulin M; D010446:Peptide Fragments; D011960:Receptors, Estrogen; D004958:Estradiol; D004247:DNA; D002918:Chymotrypsin; D014357:Trypsin; D010206:Papain