Lactogen Receptors in Rat Leydig Cells: Analysis of Their Structure with Bifunctional Cross-Linking Reagents

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Abstract
[125I]Iodohuman GH [growth hormone] was found to bind to receptors with specificity for lactogenic hormones in a Triton X-100 extract from Leydig cell membranes displaying an affinity constant of 3.8 .times. 109 M-1 and a binding capacity of 167 fmol/mg protein. Cross-linking of solubilized [125I]iodohuman GH-receptor complexes with disuccinimidyl suberate followed by analysis by sodium dodecyl sulfate-gel electrophoresis in the presence of .beta.-mercaptoethanol and autoradiography resulted in the appearance of bands with apparent MW of 113,000, 103,000, 59,000 and 53,000. The appearance of these bands was prevented by incubation in the presence of lactogenic hormones. By using a 2-dimensional electrophoresis technique (1st dimension under nonreducing conditions; 2nd dimension under reducing conditions), it was demonstrated that a fraction of the MW 59,000 species can be released from the MW 103,000 species upon cleavage of disulfide bonds. These results suggest the existence of lactogen receptor species with MW of 91,000, 81,000, 37,000, and 31,000 in Triton X-100 extracts from Leydig cell membranes if the contribution of the free hormone (MW 22,000) is subtracted. A fraction of the MW 37,000 subunits appears to be contained within the 81,000 species linked through disulfide bonds.