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Multiple Forms of Solubilized Gonadotropin Receptors from the Rat Testis

From the ¹ From the Section on Hormonal Regulation, Reproduction Research Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20014

Soluble preparations of the testicular gonadotropin receptor for luteinizing hormone (LH) and human chorionic gonadotropin (hCG) exhibit a spectrum of physical characteristics according to the conditions employed for extraction with detergents. Gonadotropin receptors in particulate testis binding fractions were solubilized before or after equilibration with ¹²⁵I-hCG by detergents, including Triton X-100, Lubrol PX, Lubrol WX, and sodium deoxycholate, and were shown to remain in solution after centrifugation at 350,000 x g. Characterization of such soluble receptors and hormone-receptor complexes by gel filtration and sucrose density gradient centrifugation revealed the presence of several physical forms. In addition to the 6.5 S form of the free receptor extracted by Triton X-100, and the 7.5 S hormone-receptor complex resulting from hCG binding to free receptors extracted with Triton X-100, dialysis of the 7.5 S complex against detergent-free solutions caused reversible conversion to an 8.8 S form of the complex. Extraction of prelabeled testis particles with Triton X-100 gave only the 8.8 S complex, which aggregated reversibly upon dialysis. Lubrol PX extracts of prelabeled particles contained a 7 S complex which also aggregated during dialysis and converted to the 8.8 S form in Triton X-100. The 7 S form of the complex was also observed after extraction of labeled particles with Lubrol WX.

Exposure of the soluble receptors to trypsin and phospholipase A abolished subsequent hormone binding: neuraminidase, RNase, and DNase did not reduce the uptake of ¹²⁵IhCG. Phospholipase A had no effect upon preformed 7.5 S and 8.8 S hormone-receptor complexes, while phospholipase C had no effect on the 7.5 S complex and caused aggregation of the 8.8 S form. The less pronounced effects of phospholipase A on preformed hormone-receptor complexes than on unoccupied particulate or soluble receptors suggest that binding activity is influenced by an essential phospholipid component.

Interpretation of the several forms of the gonadotropin receptors extracted from testis particles is complicated by the differential effects of detergent binding and molecular conformation upon sedimentation velocity during density gradient centrifugation. Because the changes in density of the complexes extracted under various conditions are not sufficient to account for the observed sedimentation behavior of the hormone-receptor complexes, it is likely that changes in the symmetry or degree of association of the complexes are responsible for the multiplicity of forms detected under different conditions of detergent extraction.

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