Properties of follitropin-receptor interaction. Characterization of the interaction of follitropin with receptors in purified membranes isolated from mature rat testes tubules.

We have studied the interaction of biologically active, radiolabeled human follitropin ( Y-hFSH) with plasma membranes prepared from tubules of mature rat testes. Tubules prepared by microdissection and extensive washing still showed considerable specific uptake of radiolabeled human lutropin ( lz51-hLH), presumably reflecting contamination with Leydig cells. Exposure of the washed tubules to hypotonic buffer solution reduced uptake of ““I-hLH to low levels, and of this, 80% was nonspecific. Uptake of Y-hFSH was unaffected by this procedure. Plasma membranes were isolated from this highly purified tubule fraction by sucrose density gradient centrifugation. The effects of temperature, pH, ionic strength, and inorganic ions on follitropin binding to purified tubule membranes were similar to that seen with crude testicular homogenates. However, nucleotide effects on hormone binding, difficult to detect in crude testicular homogenates, could be readily discerned when purified membrane fractions were employed. Specific uptake of ‘? hFSH was reduced by prior or simultaneous addition of 2 mM concentrations of ATP, GTP, UTP, CTP, and GMP. These same nucleotides promoted dissociation of specifically bound hormone, suggesting the apparent inhibition of uptake was actually a consequence of nucleotide-induced dissociation of follitropin from receptor. Nucleotides which did not effect lz51-hFSH binding to receptor did not induce dissociation of hormone from preformed hormone ‘receptor complex. ‘? hFSH unbound to receptor and remaining in the supernatant after the standard 2 hours of incubation did not bind to fresh receptor as well as “‘1-hFSH incubated in buffer alone, suggesting the presence of radioligand uptake-inhibiting factor in the supernatant. Uptake inhibition of this type was much more pronounced in supernatants from tubule homogenates compared to supernatants from purified tubule membranes. This inhibition did not seem associated with gross fragmentation of the unbound lz51-hFSH, as determined by gel filtration studies. Some receptor degradation was noted as a result of the incubation conditions required for maximal specific uptake of ‘? hFSH and this was again more pronounced with tubule homogenates than with purified membranes. The uptake of ‘? hFSH by purified membrane fractions was corrected for uptake inhibition and receptor degradation when utilized for equilibrium and kinetic studies of hormone-receptor interactions. In addition, specific activity of the lz51-hFSH was determined by measurement of hormone mass using radioimmunoassay and Y-hFSH. Scatchard analysis employing such corrected uptake data indicated the tubule membranes to have two classes of binding sites, one with a high affinity (K, about 7 x lo-” M) and low capacity (number of binding sites about 10 x 10-l” mol/mg); the second with a low affinity (K, about 2.6 x lo9 M) and high capacity (number of binding sites about 63 x lo-l4 mol/mg). Similar results were obtained when Scatchard analysis was done by (a) increasing the concentration of radioligand and holding receptor concentration constant or (b) by holding the amount of radioligand and receptor concentration constant, but adding increasing amounts of cold hormone. The rate of dissociation of specifically bound ‘*‘I-hFSH from receptors on purified membranes was temperature-dependent being more rapid at 37” than 4’. Dissociation was not increased by the presence of 10ml M FSH compared to that observed in buffer alone. Therefore, the phenomena of negative cooperativity and site-site interaction, reported to occur in dissociation of other types of protein hormones, does not seem operative in this system. The time course for association of hormone by receptor conformed to second order kinetics, whereas dissociation rates did not conform clearly to either first or second order kinetics, indicating the overall