Covalent crosslinking of human chorionic gonadotropin to its receptor in rat testes.
The bifunctional crosslinking reagents disuccinimidyl suberate and dithiobis(succinimidyl propionate) were used to attach 125I-labeled human chorionic gonadotropin (125I-hCG) covalently to rat testicular membranes. The extent of crosslinking was dependent on time and concentration; routinely, 30% of the specifically bound hormone was covalently linked to the membranes in the presence of 0.5 mM crosslinking reagent when incubated at 25 degrees C for 15 min. Excess unlabeled hCG blocked the crosslinking of 125I-hCG to the membranes. When solubilized with Triton X-100 and analyzed by sucrose density gradient centrifugation, both the native and the crosslinked hormone-receptor complex sedimented with an apparent Mr of 220,000. Thus, the receptor itself would have Mr 180,000. When the crosslinked complex was analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis, the predominant species had a Mr of 123,000 and appeared to represent the labeled alpha subunit of hCG covalently linked to a membrane component. The Mr of this receptor component would be 100,000, a value approximately half that of the Triton X-100-solubilized receptor. Thus, the membrane receptor for hCG may consist of a dimer of two binding subunits or a binding subunit associated with one or more additional subunits that might play a coupling or regulatory function.
D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D006063:Chorionic Gonadotropin / Q000378:metabolism*; D003432:Cross-Linking Reagents; D003902:Detergents; D006801:Humans; D007158:Immunologic Techniques; D008297:Male; D008565:Membrane Proteins / Q000378:metabolism; D008970:Molecular Weight; D051381:Rats; D011956:Receptors, Cell Surface / Q000378:metabolism*; D011974:Receptors, LH; D013737:Testis / Q000378:metabolism*
D006063:Chorionic Gonadotropin; D003432:Cross-Linking Reagents; D003902:Detergents; D008565:Membrane Proteins; D011956:Receptors, Cell Surface; D011974:Receptors, LH