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.

Monoclonal antibodies specific to the beta and gamma subunits of the Torpedo acetylcholine receptor inhibit single-channel activity

“…This is illustrated in Figure 2. MAb 10, which binds to an extracellular domain of the @ subunit and also cross-reacts with the (Y subunit (LindStrom et al, 198 1 b;Tzartos and Lindstrom, 1980) caused inhibition of the ACh receptor channel when added to the same compartment as ACh ( Fig. 2A, Table 4).…”

“…The mechanism of mAb inhibition of channel function is unknown. Inhibition cannot be accounted for by steric hindrance as introduced by binding the large immunoglobulin molecule on the ACh receptor, since mAbs 35 and 6, which bind to the MIR and crosslink ACh receptors (Conti-Tronconi et al, 198 1;Fairclough et al, 1983;Lindstrom et al, 1983;Wan and Lindstrom, 1985) do not inhibit channel function. The inhibitory mAbs may act by crosslinking sites between adjacent subunits to prevent the sliding or rotation of subunits, thereby leading to an immobilized conformation of the ACh receptor.…”

Characterization of monoclonal antibodies against human low density lipoprotein.

“…Moreover, antigenic determinants tend to be at the surface of the molecule and to occur at bends in the three-dimensional structure. 21 Recently, ContiTronconi et al 24 have used monoclonal antibodies to partially map immunochemically an acetylcholine receptor. They have predicted the relative positions of antigenic determinants from estimations of the composition of antigen-antibody complexes.…”

Determination of amino acids critical to the main immunogenic region of intact acetylcholine receptors by in vitro mutagenesis

“…3, mutations in ~~68, a71 or (r68+71 eliminated the binding of MIR-specific mAbs raised against AChRs from human (mAb 198), rodent (mAb 210), Torpedo (mAb 6), and Electrophor~s (mAbs 22 and 47). mAb 35 is frequently used as the archetypic MIR mAb [5,7]. mAb 35 does not bind to synthetic peptides [12,14].…”

“…More than 60% of the antibodies in sera of MG patients and rats with EAMG are directed at the main immunogenic region (MIR) on the AChR CY subunit [4-61. When injected into rats, monoclonal antibodies (mAbs) to the MIR induce an EAMG response, and when added to cultured muscle cells they cause loss of AChRs [7,8] Torpedo sequence did [ 141. Asparagine cu68 is critical to rnAb binding to the peptides because the human sequence (~68-76 binds MIR mAbs, but ~~69-76 does not [14].…”