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Gangliosides inhibit 125I-labeled thyrotropin binding to the thyrotropin receptors on bovine thyroid plasma membranes, on guinea pig retro-orbital tissue plasma membranes, and on human adipocyte membranes. This inhibition by gangliosides is critically altered by the number and location of the sialic acid residues within the ganglioside structure, the efficacy of inhibition having the following order: GD1b greater than GT1 greater than GM1 greater than GM2 = GM3 greater than GD1a. The inhibition results from the interaction of thyrotropin and gangliosides, rather than the interaction of membrane and gangliosides. Fluorescence studies show that the inhibition is associated with a distinct conformational change of the thyrotropin molecule and that the progression from a "noninhibitory conformation" to an "inhibitory conformation" parallels exactly the order of effectiveness in inhibiting 125I-labeled thyrotropin binding. The ganglioside inhibition of 125I-labeled thyrotropin binding appears to be hormonally specific in that it is not affected by albumin, glucagon, insulin, prolactin, follicle-stimulating hormone, growth hormone, or corticotropin. The possibility that a ganglioside or ganglioside-like structure is a component of the thyrotropin receptor is suggested by the finding that gangliosides more complex than N-acetylneuraminylgalactosylglucosylceramide are present in bovine thyroid membranes in much higher quantities than have been previously found in extraneural tissue. The finding that the B component of cholera toxin, which also interacts with gangliosides, has a peptide sequence in common with the beta subunit of thyrotropin, suggests that thyrotropin and cholera toxin may be analogous in their mode of action on the membrane.

Plasma membranes derived from rat thyroid tumor (1-8R) which is unresponsive to thyrotropin but is responsive to dibutyryl adenosine 3':5'-cyclic monophosphate bind less than 20% of the [125I] thyrotropin which can be bound to plasma membranes from normal rat thyroids under conditions which optimize tumor membrane binding relative to normal thyroid membranes. In addition, the binding is different from thyrotropin binding to normal thyroid membranes both in its altered sensitivity to changes in hydrogen ion concentration and in a decreased sensitivity to competition by unlabeled thyrotropin. This reduced capacity to bind [125I] thyrotropin cannot be attributed to degradation of the hormone by membrane-associated proteases. Although the supernatant phase of the thyroid tumor homogenates contains a soluble component which inhibits [125I] thyrotropin binding to thyrotropin receptors on plasma membranes, its level is the same as in homogenates of normal thyroid tissue. Trypsin digestion does not expose thyrotropin receptors in a manner analogous to that seen in normal thyroid tissue. The major ganglioside in the tumor membranes is N-acetylneuraminylgalactosylglucosylceramide and the membranes lack the N-acetylgalactosaminyltransferase required for the synthesis of more complex gangliosides. In contrast, the normal rat thyroid membranes contain more complex gangliosides such as galactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylglucosylceramide and N-acetylneuraminylgalactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylglucosyl ceramide as well as the glycosyltransferase activities required for their syntheses. Galactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylglucosylceramide can also be detected in normal membranes, but not in tumor membranes, by selective labeling with galactose oxidase (D-galactose: oxygen 6-oxidoreductase, EC 1.1.3.9) and [3H] sodium borohydride. These results support the hypothesis that gangliosides are important structural or functional components of thyrotropin receptors on thyroid plasma membranes.

The 1-8 rat thyroid tumor line with a thyrotropin and cholera toxin receptor defect and a deficiency in higher order membrane gangliosides is shown to regain both receptor functions with the in vivo resynthesis or the in vitro reconstitution of higher order gangliosides. Reconstitution was achieved by exposing primary cell cultures of the tumor to preparations of gangliosides from thyroid cells with functional thyrotropin receptor activity.

The micellar properties of mixed, bovine gangliosides and purified galactosyl-N-acetylacetylgalactosaminyl (N-acetylneuraminyl) galactosylglucosylceramide were studied by gel filtration, equilibrium dialysis, and band and boundary centrifugation in sucrose gradients. The dissociation of micelles is very slow (days) in water and required us to approach equilibrium by association of monomers rather than by the dissociation of micelles. The gangliosides were therefore first converted into very low molecular weight aggregates (1-3 molecules) by dissolving them in Me2SO. Galactosyl-N-acetylgalactosaminyl(N-acetylneuraminyl)galactosylglucosylceramide was then diluted into aqueous sucrose gradients and sedimented by the boundary centrifugation technique. This gave a sedimenting micelle and a nonsedimenting monomer concentration of (1-2) x 10-10 M (or less) which corresponds to the critical micelle concentration value. The mixed gangliosides revealed two micellar sizes (i.e., 10 and 4.5 S), the slower sedimenting species being formed from the larger one with time (days). The critical micelle concentration of the mixed gangliosides was found to be approximately 10-8 M by a gel filtration, equilibrium dialysis, and band centrifugation.