Monoclonal antibodies as probes of acetylcholine receptor structure. 2. Binding to native receptor

Abstract

Binding of monoclonal antibodies top Torpedo californica acetylcholine receptor monomers solubilized in Triton X-100 was studied by centrifugation on sucrose gradients. Antibodies to alpha subunits were of two types. One type formed complexes of one antibody and one receptor monomer, independent of antibody/receptor ratio. We conclude that the binding sites for these antibodies are oriented on the two alpha subunits per monomer in such a way that each could be bound by one of the two binding sites of a single immunoglobulin molecule. Most antibodies were of this type. The other type of monoclonal antibody formed complexes of several sizes, including antibody cross-linked receptors, depending on the ratio of antibody to receptor. We conclude that the binding sites for these antibodies are oriented in such a way that the two alpha subunits per monomer could not be cross-linked by a single antibody molecule. A monoclonal antibody of this type raised against Electrophorus electricus receptors was used to show that this receptor also has two alpha subunits per monomer. This antibody cross-reacted with receptor from fetal calf muscle and was able to induce modulation of receptor in muscle cells in culture. This suggests that muscle receptor also has two alpha subunits and that the antibody can cross-link receptor in the plane of the membrane, as it does in solution, and thereby form complexes which enhance endocytosis and increase the rate of receptor destruction. The rate of antigenic modulation decreases at high antibody/receptor ratios, as expected if un-cross-linked complexes of two antibodies and one receptor were not destroyed at a faster rate. Antibodies which cross-link alpha subunits within a receptor monomer are frequent but would not be expected to be able to induce antigenic modulation. This provides one mechanism by which antisera of equivalent antireceptor titer might differ in their ability to induce antigenic modulation. An antibody which binds to denatured delta and gamma subunits forms complexes of only one antibody and one receptor monomer, independent of antibody ratio, as do antibodies thought to cross-link the two alpha subunits in a monomer. It apparently cross-links delta and gamma subunits within the monomer. Some of the monoclonal antibodies to alpha subunits can bind simultaneously to receptor, while the binding of others is mutually exclusive.