Monoclonal antibodies to chick intestinal receptors for 1,25-dihydroxyvitamin D3. Interaction and effects of binding on receptor function.
Monoclonal antibodies, developed against the chick intestinal receptor for 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), were characterized with respect to their interaction with this protein and for their effects on the polypeptide's hormone-binding and nuclear-binding functions. Antibodies, internally labeled with [35S]methionine, react directly with hormone-labeled receptor, as identified by comigration of both isotopes during sedimentation on hypertonic 10-30% sucrose gradients. Antibodies bound both the unoccupied and occupied forms of the receptor, the latter with equilibrium dissociation constants of 10(-10)-10(-11) M at 4 degrees C. Excess antibody, added to unoccupied receptors prior to incubation with 1,25(OH)2D3, did not affect the receptor's apparent affinity for the hormone (Kd approximately equal to 6 X 10(-11) M). In contrast, all three antibodies, complexed with occupied receptors, significantly reduced the extent of the receptor's association with isolated nuclei (48-64% inhibition). This inhibition most likely represents a general reduction in the affinity of the protein for nuclei under the conditions tested, since the affinity of the occupied 1,25(OH)2D3 receptor for DNA, as well as the ionic strength necessary to elute receptor from both cation and anion exchange resins was significantly reduced by prior incubation with excess antibody. These findings suggest that the epitopes for each of the three monoclonal antibodies may be located in or near the DNA or nuclear binding domain of the 1,25(OH)2D3 receptor. Taken cumulatively, these results indicate that the monoclonal immunoreagents utilized here should prove useful in delineating important biochemical features of this unique sterol hormone receptor.
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D002645:Chickens; D004247:DNA / Q000378:metabolism; D000939:Epitopes / Q000276:immunology; D007413:Intestinal Mucosa / Q000378:metabolism*; D007700:Kinetics; D008715:Methionine / Q000378:metabolism; D051379:Mice; D051381:Rats; D018167:Receptors, Calcitriol; D011987:Receptors, Steroid / Q000276:immunology* / Q000378:metabolism; D013261:Sterols / Q000378:metabolism
D000911:Antibodies, Monoclonal; D000939:Epitopes; D018167:Receptors, Calcitriol; D011987:Receptors, Steroid; D013261:Sterols; D004247:DNA; D008715:Methionine